Advances in Botanical Research, Volume 47

CONTRIBUTORS TO VOLUME 47 SAMIK BHATTACHARYA Plant Molecular & Cellular Genetics, Bose Institute, Kolkata 700054, Indi...

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CONTRIBUTORS TO VOLUME 47

SAMIK BHATTACHARYA Plant Molecular & Cellular Genetics, Bose Institute, Kolkata 700054, India ESTHER CARRERA Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain ´ S Centro de Geno´mica, Instituto Valenciano de MANUEL CERCO Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain HONG‐HWA CHEN Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan and Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan WEN‐HUEI CHEN Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan DONGSU CHOI School of Science and Technology, Kunsan National University, Gunsan 573‐701, Republic of Korea JOSE´ M. COLMENERO‐FLORES Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain MALAY DAS US Environmental Protection Agency, National Health and Environmental EVects Research Laboratory, Western Ecology Division, 200 S.W. 35th Street, Corvallis, Oregon TARCISO S. FILGUEIRAS Reserva Ecolo´gica do IBGE, C.P. 08770, Brasilia‐DF 70312‐970, Brazil YU‐YUN HSIAO Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan CHIA‐CHI HSU Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan DOMINGO J. IGLESIAS Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain JEONG HOE KIM Department of Biology, Kyungpook National University, Daegu 702‐701, Republic of Korea YI LEE Department of Industrial Plant Science and Technology, Chungbuk National University, Cheongju 361‐763, Republic of Korea IGNACIO LLISO Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain

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CONTRIBUTORS

FRANCISCO JAVIER MEDINA Centro de Investigaciones Biolo´gicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain RAPHAE¨L MORILLON Centre de Coope´ration Internationale en Recherche Agronomique, pour le De´velopement (CIRAD), UR 75, Dpt. BIOS, Apdo. Oficial, 46113 Moncada (Valencia), Spain MIGUEL A. NARANJO Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain PATRICK OLLITRAULT Centre de Coope´ration Internationale en Recherche Agronomique, pour le De´velopement (CIRAD), UR 75, Dpt. BIOS, Apdo. Oficial, 46113 Moncada (Valencia), Spain AMITA PAL Plant Molecular & Cellular Genetics, Bose Institute, Kolkata 700054, India ZHAO‐JUN PAN Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan GABINO RIÓS Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain OMAR RUIZ‐RIVERO Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain ´ EZ‐VA ´ SQUEZ Laboratoire Geńome et De´veloppement des JULIO SA Plantes, UMR CNRS 5096, Universite´ de Perpignan, 66860 Perpignan Cedex, France PARAMJIT SINGH Botanical Survey of India, CGO Complex, Salt Lake, Kolkata 700064, India FRANCISCO R. TADEO Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain MANUEL TALON Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain WEN‐CHIEH TSAI Department of Biological Science and Technology, National University of Tainan, Tainan 700, Taiwan YA‐PING YANG Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan

CONTENTS OF VOLUMES 35–46 Series Editor (Volumes 35–44) J.A. CALLOW School of Biosciences, University of Birmingham, Birmingham, United Kingdom

Contents of Volume 35 Recent Advances in the Cell Biology of Chlorophyll Catabolism H. THOMAS, H. OUGHAM and S. HORTENSTEINER The Microspore: A Haploid Multipurpose Cell A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS The Seed Oleosins: Structure Properties and Biological Role J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY Compartmentation of Proteins in the Protein Storage Vacuole: A Compound Organelle in Plant Cells L. JIANG and J. ROGERS Intraspecific Variation in Seaweeds: The Application of New Tools and Approaches C. MAGGS and R. WATTIER Glucosinolates and Their Degradation Products R. F. MITHEN

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CONTENTS OF VOLUMES 35–46

Contents of Volume 36 PLANT VIRUS VECTOR INTERACTIONS Edited by R. Plumb Aphids: Non-Persistent Transmission T. P. PIRONE and K. L. PERRY Persistent Transmission of Luteoviruses by Aphids B. REAVY and M. A. MAYO Fungi M. J. ADAMS Whitefly Transmission of Plant Viruses J. K. BROWN and H. CZOSNEK Beetles R. C. GERGERICH Thrips as Vectors of Tospoviruses D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL, A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN Virus Transmission by Leafhoppers, Planthoppers and Treehoppers (Auchenorrhyncha, Homoptera) E. AMMAR and L. R. NAULT Nematodes S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN Other Vectors R. T. PLUMB


Contents of Volume 37 ANTHOCYANINS IN LEAVES Edited by K. S. Gould and D. W. Lee Anthocyanins in Leaves and Other Vegetative Organs: An Introduction D. W. LEE and K. S. GOULD Le Rouge et le Noir: Are Anthocyanins Plant Melanins? G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD Anthocyanins in Leaves: History, Phylogeny and Development D. W. LEE The Final Steps in Anthocyanin Formation: A Story of Modification and Sequestration C. S. WINEFIELD Molecular Genetics and Control of Anthocyanin Expression B. WINKEL-SHIRLEY Differential Expression and Functional Significance of Anthocyanins in Relation to Phasic Development in Hedera helix L. W. P. HACKETT Do Anthocyanins Function as Osmoregulators in Leaf Tissues? L. CHALKER-SCOTT The Role of Anthocyanins for Photosynthesis of Alaskan Arctic Evergreens During Snowmelt S. F. OBERBAUER and G. STARR Anthocyanins in Autumn Leaf Senescence D. W. LEE A Unified Explanation for Anthocyanins in Leaves? K. S. GOULD, S. O. NEILL and T. C. VOGELMANN

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Contents of Volume 38 An Epidemiological Framework for Disease Management C. A. GILLIGAN Golgi-independent Trafficking of Macromolecules to the Plant Vacuole D. C. BASSHAM Phosphoenolpyruvate Carboxykinase: Structure, Function and Regulation R. P. WALKER and Z.-H. CHEN Developmental Genetics of the Angiosperm Leaf C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE and R. A. MARTIENSSEN A Model for the Evolution and Genesis of the Pseudotetraploid Arabidopsis thaliana Genome Y. HENRY, A. CHAMPION, I. GY, A. PICAUD, A. LECHARNY and M. KREIS

Contents of Volume 39 Cumulative Subject Index Volumes 1–38

Contents of Volume 40 Starch Synthesis in Cereal Grains K. TOMLINSON and K. DENYER The Hyperaccumulation of Metals by Plants M. R. MACNAIR Plant Chromatin — Learning from Similarities and Differences J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI, P. ZIELENKIEWICZ and A. JERZMANOWSKI


The Interface Between the Cell Cycle and Programmed Cell Death in Higher Plants: From Division unto Death D. FRANCIS The Importance of Extracellular Carbohydrate Production by Marine Epipelic Diatoms G. J. C. UNDERWOOD and D. M. PATERSON Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins A. K. CHARNLEY

Contents of Volume 41 Multiple Responses of Rhizobia to Flavonoids During Legume Root Infection JAMES E. COOPER Investigating and Manipulating Lignin Biosynthesis in the Postgenomic Era CLAIRE HALPIN Application of Thermal Imaging and Infrared Sensing in Plant Physiology and Ecophysiology HAMLYN G. JONES Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and Transposable Elements CELIA HANSEN and J. S. HESLOP-HARRISON Role of Plasmodesmata Regulation in Plant Development ARNAUD COMPLAINVILLE and MARTIN CRESPI

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Contents of Volume 42 Chemical Manipulation of Antioxidant Defences in Plants ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON and IAN CUMMINS The Impact of Molecular Data in Fungal Systematics P. D. BRIDGE, B. M. SPOONER and P. J. ROBERTS Cytoskeletal Regulation of the Plane of Cell Division: An Essential Component of Plant Development and Reproduction HILARY J. ROGERS Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol ALYSON K. TOBIN and CAROLINE G. BOWSHER

Contents of Volume 43 Defensive and Sensory Chemical Ecology of Brown Algae CHARLES D. AMSLER and VICTORIA A. FAIRHEAD Regulation of Carbon and Amino Acid Metabolism: Roles of Sucrose Nonfermenting-1-Related Protein Kinase-1 and General Control Nonderepressible-2-Related Protein Kinase NIGEL G. HALFORD Opportunities for the Control of Brassicaceous Weeds of Cropping Systems Using Mycoherbicides AARON MAXWELL and JOHN K. SCOTT Stress Resistance and Disease Resistance in Seaweeds: The Role of Reactive Oxygen Metabolism MATTHEW J. DRING Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus ANNA AMTMANN, JOHN P. HAMMOND, PATRICK ARMENGAUD and PHILIP J. WHITE


Contents of Volume 44 Angiosperm Floral Evolution: Morphological Developmental Framework PETER K. ENDRESS Recent Developments Regarding the Evolutionary Origin of Flowers MICHAEL W. FROHLICH Duplication, Diversification, and Comparative Genetics of Angiosperm MADS-Box Genes VIVIAN F. IRISH Beyond the ABC-Model: Regulation of Floral Homeotic Genes LAURA M. ZAHN, BAOMIN FENG and HONG MA Missing Links: DNA-Binding and Target Gene Specificity of Floral Homeotic Proteins RAINER MELZER, KERSTIN KAUFMANN ¨ NTER THEIßEN and GU Genetics of Floral Development in Petunia ANNEKE RIJPKEMA, TOM GERATS and MICHIEL VANDENBUSSCHE Flower Development: The Antirrhinum Perspective BRENDAN DAVIES, MARIA CARTOLANO and ZSUZSANNA SCHWARZ-SOMMER Floral Developmental Genetics of Gerbera (Asteraceae) TEEMU H. TEERI, MIKA KOTILAINEN, ANNE UIMARI, SATU RUOKOLAINEN, YAN PENG NG, URSULA MALM, ¨ NEN, SUVI BROHOLM, ROOSA LAITINEN, ¨ LLA EIJA PO PAULA ELOMAA and VICTOR A. ALBERT Gene Duplication and Floral Developmental Genetics of Basal Eudicots ELENA M. KRAMER and ELIZABETH A. ZIMMER

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Genetics of Grass Flower Development CLINTON J. WHIPPLE and ROBERT J. SCHMIDT Developmental Gene Evolution and the Origin of Grass Inflorescence Diversity SIMON T. MALCOMBER, JILL C. PRESTON, RENATA REINHEIMER, JESSIE KOSSUTH and ELIZABETH A. KELLOGG Expression of Floral Regulators in Basal Angiosperms and the Origin and Evolution of ABC-Function PAMELA S. SOLTIS, DOUGLAS E. SOLTIS, SANGTAE KIM, ANDRE CHANDERBALI and MATYAS BUZGO The Molecular Evolutionary Ecology of Plant Development: Flowering Time in Arabidopsis thaliana KATHLEEN ENGELMANN and MICHAEL PURUGGANAN A Genomics Approach to the Study of Ancient Polyploidy and Floral Developmental Genetics JAMES H. LEEBENS-MACK, KERR WALL, JILL DUARTE, ZHENGUI ZHENG, DAVID OPPENHEIMER and CLAUDE DEPAMPHILIS Series Editors (Volume 45– ) JEAN-CLAUDE KADER Laboratoire Physiologie Cellulaire et Molećulaire des Plantes, CNRS, Universite´ de Paris, Paris, France MICHEL DELSENY Laboratoire Geńome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France

Contents of Volume 45 RAPESEED BREEDING History, Origin and Evolution S. K. GUPTA and ADITYA PRATAP


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Breeding Methods B. RAI, S. K. GUPTA and ADITYA PRATAP The Chronicles of Oil and Meal Quality Improvement in Oilseed Rape ABHA AGNIHOTRI, DEEPAK PREM and KADAMBARI GUPTA Development and Practical Use of DNA Markers KATARZYNA MIKOLAJCZYK Self-Incompatibility RYO FUJIMOTO and TAKESHI NISHIO Fingerprinting of Oilseed Rape Cultivars ´ ˇ URN and JANA ZˇALUDOVA VLADISLAV C Haploid and Doubled Haploid Technology L. XU, U. NAJEEB, G. X. TANG, H. H. GU, G. Q. ZHANG, Y. HE and W. J. ZHOU Breeding for Apetalous Rape: Inheritance and Yield Physiology LIXI JIANG Breeding Herbicide-Tolerant Oilseed Rape Cultivars PETER B. E. MCVETTY and CARLA D. ZELMER Breeding for Blackleg Resistance: The Biology and Epidemiology W. G. DILANTHA FERNANDO, YU CHEN and KAVEH GHANBARNIA Development of Alloplasmic Rape MICHAL STARZYCKI, ELIGIA STARZYCKI and JAN PSZCZOLA Honeybees and Rapeseed: A Pollinator–Plant Interaction D. P. ABROL

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Genetic Variation and Metabolism of Glucosinolates NATALIA BELLOSTAS, ANNE DORTHE SØRENSEN, JENS CHRISTIAN SØRENSEN and HILMER SØRENSEN Mutagenesis: Generation and Evaluation of Induced Mutations SANJAY J. JAMBHULKAR Rapeseed Biotechnology VINITHA CARDOZA and C. NEAL STEWART, JR. Oilseed Rape: Co-existence and Gene Flow from Wild Species RIKKE BAGGER JØRGENSEN Evaluation, Maintenance, and Conservation of Germplasm RANBIR SINGH and S. K. SHARMA Oil Technology ¨ US BERTRAND MATTHA

Contents of Volume 46 INCORPORATING ADVANCES IN PLANT PATHOLOGY Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features Influencing Root Growth and Development ´ NICA CREUS, MARIÁ CELESTE MOLINA-FAVERO, CECILIA MO LUCIANA LANTERI, NATALIA CORREA-ARAGUNDE, MARIÁ CRISTINA LOMBARDO, CARLOS ALBERTO BARASSI and LORENZO LAMATTINA How the Environment Regulates Root Architecture in Dicots ´ RIE LEFEBVRE, PHILIPPE MARIANA JOVANOVIC, VALE LAPORTE, SILVINA GONZALEZ-RIZZO, CHRISTINE LELANDAIS-BRIE`RE, FLORIAN FRUGIER, CAROLINE HARTMANN and MARTIN CRESPI


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Aquaporins in Plants: From Molecular Structure to Integrated Functions OLIVIER POSTAIRE, LIONEL VERDOUCQ and CHRISTOPHE MAUREL Iron Dynamics in Plants JEAN-FRANC ¸ OIS BRIAT Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the Early Steps of Symbiotic Interactions VIVIENNE GIANINAZZI-PEARSON, NATHALIE SE´JALON-DELMAS, ANDREA GENRE, SYLVAIN JEANDROZ and PAOLA BONFANTE Dynamic Defense of Marine Macroalgae Against Pathogens: From Early Activated to Gene-Regulated Responses AUDREY COSSE, CATHERINE LEBLANC and PHILIPPE POTIN

The Plant Nucleolus

´ EZ‐VA ´ SQUEZ* AND FRANCISCO JAVIER MEDINA{ JULIO SA

*Laboratoire Geńome et De´veloppement des Plantes, UMR CNRS 5096, Universite´ de Perpignan, 66860 Perpignan Cedex, France { Centro de Investigaciones Biolo´gicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain

I. The Structure of the Plant Nucleolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Nucleolar Structural Components...................................... B. The Structural Organization of Functions in the Nucleolus ............. C. Transcription in the Nucleolus and the Identity of FCs.................. D. Epigenetic Regulation of rRNA Gene Expression: Gene Silencing and Nucleolar Dominance ................................. II. The Nucleolus During the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Nucleolus Through Mitosis ............................................. B. The Nucleolus Through Interphase.......................................... III. Nucleolar Protein Factors: Their Role in Ribosome Biogenesis and Plant Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. rRNA Transcription and Processing Factors .............................. B. Nucleolin‐Like Proteins ....................................................... C. Nucleolar Proteins and Plant Development ................................ IV. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Botanical Research, Vol. 47 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

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0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(08)00001-3

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´ EZ‐VA ´ SQUEZ AND F. J. MEDINA J. SA

ABSTRACT The nucleolus is a nuclear substructure that is well known as the ribosome factory of the cell, namely, the site in which the production of mature ribosomal subunits takes place before their export to the cytoplasm. The process involves the transcription of the genes encoding three of the four RNA species of ribosomes (18S, 5.8S and—in plants—25S) as a single pre‐rRNA molecule, and the processing of this molecule up to the assembly of mature rRNA species with specific ribosomal proteins. In this chapter, we review the progress achieved in recent years in understanding the nucleolar structure and organization of nucleolar molecular components in relation to the diVerent functional steps of ribosome biogenesis. Furthermore, the evolution of the nucleolus through interphase and mitosis and its role in cell cycle regulation, as well as the proteinaceous factors which participate in rDNA transcription, pre‐rRNA processing and/or ribosome assembly, are revised. Finally, the role played by the nucleolus and nucleolar factors in plant growth and development is also discussed.

I. THE STRUCTURE OF THE PLANT NUCLEOLUS A. THE NUCLEOLAR STRUCTURAL COMPONENTS

The nucleolus is a nuclear substructure which, to use a very apt expression, ‘‘is formed by the act of building a ribosome’’ (Me´le`se and Xue, 1995). The nucleolus is well known as the ribosome factory of the cell, that is, the site in which the production of mature ribosomal subunits takes place, before their export to the cytoplasm. The process involves the transcription of the genes encoding three of the four RNA species of ribosomes (18S, 5.8S and—in plants—25S) as a single pre‐rRNA molecule, and the processing of this molecule up to the assembly of mature rRNA species with specific ribosomal proteins (RPs) (Fig. 1). The nucleolus is the most outstanding example of functional nuclear compartmentalization. Within the nucleus, there are no membranes or walls capable of delimiting spaces ascribed to particular functions. However, the nucleus does show structural domains in which certain macromolecular components are recruited to perform a precise function. The nucleolus is the most prominent example of this, because the structural organization of the territory devoted to ribosome biogenesis is a nuclear organelle distinguishable at very low magnifications under the microscope. Interestingly, compartmentalization originated to separate ribosome biogenesis from the rest of the nuclear functions including those other nuclear functions which needed to be set apart, at least transitorily. This probably explains the ‘‘plurifunctional nucleolus’’ concept, a term that owes its origin to the fact that factors apparently not playing any role in ribosome biogenesis have been repeatedly

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THE PLANT NUCLEOLUS

Nucleolus Centromere

Knob

NOR 5.8S 18S 25S

rDNA genes

Pre-rRNA

5⬘ETS P site

Transcription RNA Pol l ITS1 ITS2

3⬘ETS

Processing

Mature rRNAs

Fig. 1. Schematic representation of a nucleolar organizer region (NOR), shown as a segment of a specific chromosome (the centromere is indicated). A part of the NOR is structured as a knob of condensed chromatin, whereas the rest forms an extended loop of ribosomal DNA (rDNA) genes from which the nucleolus originates. The NOR is made up of hundreds or thousands (depending on the species) of rDNA genes arrayed in head to tail tandem repeats containing the 18S, the 5.8S and 25S ribosomal RNA (rRNA). The loop contains focal concentrations of rDNA—fibrillar centres— around which transcription is organized. The rDNA gene units are transcribed by the RNA polymerase I (RNA pol I) and a subset of rRNA transcriptional factors as an rRNA precursor (pre‐rRNA) containing external transcribed spacers (5’ETS and 3’ETS) and internal transcribed spacers (ITS1 and ITS2). The mature 18S, 5.8S and 25S rRNA are obtained after a series of pre‐rRNA processing steps including base modification (methylation by C/D snoRNP and pseudouridinylation by H/ACA snoRNP) and endo‐ and exonucleolytic steps to remove the ETS and ITS. Arrows indicate transcription initiation sites. The primary cleavage sites (P) in the 5’ ETS of pre‐rRNA in crucifer plants are indicated.

retrieved from nucleolar extracts (Andersen et al., 2005; Boisvert et al., 2007; Pendle et al., 2005). Despite the emergent and growing discovery of the multiplicity of cellular and nuclear functions which may have their site in the nucleolus, a detailed analysis of the structural subcomponents of the organelle can only explain the functional role of these subcomponents during the diVerent steps in the complex process of ribosome biogenesis. In fact, the work of many diVerent groups, using a full range of biological model systems, throughout an extended period of time, has been needed to reach some conclusions on the precise localization of the diVerent steps of pre‐rRNA synthesis, processing and ribosome assembly, as well as the more or less precise and univocal

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correlation of functional processes with structural components. At present, even though it is agreed that the nucleolus is the site of other functions besides ribosome biogenesis, the structural–functional map of the nucleolus regarding these non‐canonical functions is only in the first steps of its construction. We can mention here two papers reporting the intranucleolar localization of proteins not related to pre‐rRNA synthesis and processing, namely, nucleostemin (Politz et al., 2005) and a reverse transcriptase related to telomere maintenance (Dıéz et al., 2006). Interestingly, both papers show that the localization is not strictly associated with any known subnucleolar component, and suggest that the nucleolus may be more subcompartimentalized than previously thought. Actually, the composition of such map will probably be a long and hard task, since there is a multiplicity of functions and molecular components to localize, not necessarily related to one another, therefore requiring individual studies for each case. The current structural dissection of the nucleolus shows a few permanent (or almost permanent) nucleolar subcomponents, namely, fibrillar centres (FCs), the dense fibrillar component (DFC) and the granular component (GC), which are sometimes accompanied by other structures, such as vacuoles and interstices. (Fig. 2) (Jordan, 1984). The idea supported by the majority of specialists is that all nucleoli in all model systems are built with the same structural components, having the same functional significance in all of them (Shaw and Jordan, 1995). This idea is compatible with the enormous polymorphism exhibited by the nucleolus from one cell type to another and even within a single cell type. In some cases, it is diYcult to distinguish one subnucleolar component from another, and structural transitions can be found between adjacent components. Moreover, the proportion

Fig. 2. Active nucleoli from a root meristematic cell of Allium cepa (A), from a suspension cultured cell of Medicago sativa (B) and from a suspension cultured cell of Arabidopsis thaliana (C). The same nucleolar subcomponents are present in the three species, and their arrangement is also very similar, corresponding to analogous physiological situations. FC: fibrillar centre; DFC: dense fibrillar component; GC: granular component; V: vacuole. Bars indicate 1 m.

THE PLANT NUCLEOLUS

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and relative distribution of basic components may diVer greatly; for instance, an active nucleolus of a plant meristematic cell contains more than 50% of DFC, and only 1% of FCs, the rest being made up of GC and the nucleolar vacuole, whereas the active nucleolus of a mammalian‐cultured cell contains 75% of GC, 17% of DFC and 25% of FCs (Jordan and McGovern, 1981). Even when the proportion of components is similar, one can find diVerences in the spatial arrangement or distribution of them, such as the reticulate pattern versus the compact one, or the segregated versus the intermingled components (Shaw and Jordan, 1995). An important feature of plant FCs which is not usual in animal cell types is the existence of two structural types, namely, homogeneous, similar to those found in animal cells, composed of a fibrous relatively loose material, and heterogeneous, which, in addition to the foregoing, have small condensed chromatin inclusions. Homogeneous FCs are small and numerous and often appear in nucleoli active in ribosome production. Heterogeneous FCs are few and large, and are associated with low rates of nucleolar transcriptional activity (Risuenõ and Medina, 1986). There are several reasons that may account for the high nucleolar structural variability. In most cases, the diVerences are the result of the response either to diVerent functional requirements or to the particular rate at which the process, as a whole or in some of its parts, is performed. This is true, for example, in the case of the varying amounts of GC exhibited by diVerently active nucleoli in the same organism. The explanation is that nucleoli having a high rate of rRNA gene transcription and processing, such as those of highly proliferating cells, produce large quantities of ribosomes, whose structural expression is an increased amount of GC. On the contrary, in diVerentiated cells, ribosome biogenesis proceeds at a rate low enough to support cellular metabolism in a specialized cell whose growth is rather slow. The consequence is a very reduced (and in some cases practically undetectable) GC. In other cases, the structural variability reflects quantitative variations as to the number of ribosomal genes, or the portion of these genes which are active or ready to be active (capable of being transcribed).

B. THE STRUCTURAL ORGANIZATION OF FUNCTIONS IN THE NUCLEOLUS

It is a general feature, observed in all model systems studied, that the molecular components of the nucleolus are organized in a vectorial fashion, from the centre outwards, forming several ultrastructural domains, in which, sequentially, pre‐rRNA synthesis and processing, as well as the ribosome assembly, take place. There may be just one vectorial organization for the whole organelle, or a multiple vectorial organization forming diVerent foci

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which are the result of the organization of the rDNA chromatin. In each of these foci, the same vectorial organization is repeated. The innermost nucleolar components—the ‘‘heart’’ of the nucleolus (Jordan, 1987)—are FCs, and that is why these nucleolar subcomponents have been associated with rDNA chromatin organization and transcription. They can be described as clear areas, when observed in electron microscope images surrounded by a darker component, the DFC. The presence in them of RNA pol I has been reported (Gilbert et al., 1995; Martıń and Medina, 1991; Scheer and Rose, 1984); however, transcriptional activity of rRNA genes, detected after Br‐UTP incorporation, was localized only at the periphery of FCs, in the DFC and, notably, in the transition area FC–DFC (Cmarko et al., 2000; De Ca´rcer and Medina, 1999; Dundr and Raska, 1993). FCs are discrete structures, as revealed after three‐dimensional reconstruction of the nucleolus from ultrathin serial sections (Medina et al., 1983a). Only in the case of quiescent cells, in which ribosome biogenesis is completely inactive in the residual nucleolus, do the individual discrete FCs actually correspond to sections of a continuous channel or cord, extending throughout the nucleolus, from side to side (Risuenõ and Medina, 1986). Depending on the nucleolar structural pattern, they are connected by DFC strands, or immersed in DFC masses. Whatever the case, there is rDNA that extends through the DFC between FCs, and this rDNA is actively transcribing. The FC‐connecting DNA was detected first by using an anti‐DNA antibody (Martıń et al., 1989), and the selective cytochemical staining of DNA using osmium ammine confirmed the existence of these extended DNA filaments (Motte et al., 1991). Three‐dimensional reconstruction from confocal optical sections after transcription in situ revealed the transcriptional activity of these DNA fibres (De Ca´rcer and Medina, 1999). In animal cells, partially disorganized nucleoli after treatment with 5,6 dichloro‐b‐ribofuranosyl benzimidazole (DRB) show necklace‐like structures in whose ‘‘beads’’ FC‐like structures can be identified and transcription detected. The observation that single beads are linked by the DFC (Panse et al., 1999) supports the existence of active DNA connecting FCs. The DFC is the site of pre‐rRNA processing, and several RNAs, intermediaries in the diVerent processing steps, were retrieved from the DFC as reported in a pioneer work (Royal and Simard, 1975). Furthermore, radio‐labelled pre‐rRNA precursors were detected in the DFC by using autoradiographic techniques (Fakan and Puvion, 1980) and, in more recent works, by using Br‐UTP labelling. Modern microscopical methodologies have confirmed these conclusions (Thiry et al., 2000). The same methods have localized late pre‐rRNA processing and preribosome assembly in the GC, constituted by numerous granules, around 15 nm in size.

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7

Finally, the nucleolar vacuole is a feature of those plant nucleoli which show a very high rate of transcriptional and processing activity, typically in proliferating cells at the G2 stage of the cell cycle (Deltour and De Barsy, 1985; Gonza´lez‐Camacho and Medina, 2006; Moreno Dıáz de la Espina et al., 1980). C. TRANSCRIPTION IN THE NUCLEOLUS AND THE IDENTITY OF FCs

The nucleolus results from the expression of a segment of the genome, that is, the set of ribosomal genes, or rRNA genes, or rDNA (Fig. 1). Thus, the nucleolar structure is the result of a sequence of cellular activities which are triggered by the organization and assembly of the transcription complex on rDNA to produce transcription of ribosomal genes by RNA polymerase I. Therefore, an eVective approach to unequivocally identify the nucleolar substructures associated with transcription is to follow the generation of a new nucleolus, starting from situations or conditions in which the nucleolus either does not exist or is totally inactive, up to when it becomes a fully active organelle. This process, generally termed ‘‘nucleologenesis’’, can be found, for example, after mitosis, when the nucleolus is organized in each of the two daughter cells around the segment of chromosomes containing rRNA genes, called the ‘‘nucleolar organizing region’’ or ‘‘nucleolar organizer’’ (NOR). Details of the molecular mechanisms involved in nucleologenesis at the end of mitosis will be extensively considered in the next section; here we will focus on the process of nucleolar structure growth associated with the resumption of nucleolar activity, particularly rDNA transcription. Actually, postmitotic nucleologenesis is a quick process, in which the precise establishment of a sequence of events is a diYcult task, and so the generation pathway of each of the nucleolar subcomponents, and its association with precise nucleolar functions, is nearly impossible to follow. Fortunately, plant cells oVer us a model in which all these events occur at such a slow pace that they are capable of being followed step by step, so that the structural variations in nucleolar subcomponents can be associated with a single functional process. The model referred to is the postmeiotic interphase occurring in the anther. Meiosis in the anther is the first event leading to the formation of the male gametes, and meiosis produces cells called microspores, which undergo an extremely long interphase (5 days or more), before the formation of the pollen grain. In these microspores, nucleolar activity (and, particularly, transcription) is resumed at a very slow rate (Medina et al., 1983b). Structurally, young Allium cepa microspores have a rather small nucleolus formed by two fibrillar balls, whose structure is the same as that of the DFC. These balls of DFC are symmetrically arranged on both sides of the NOR, which, in ultrathin sections, has a cord‐like shape, and

8


Fig. 3. The nucleolus of Allium cepa microspores during postmeiotic reactivation of the nucleolar functions. (A and B): Glutaraldehyde/osmium fixation and uranyl/ lead staining. a: Young microspore. Two balls of dense fibrillar component (DFC) are symmetrically arranged with respect to the nucleolar organizer region (NOR) whose structure is the same as that of the heterogeneous fibrillar centres, and whose activity is kept at very low levels. B: Microspore at a more advanced stage. In addition to a general enlargement, the DFC balls and the NOR show an irregular shape and, within the NOR, the structure is homogeneous, after decondensation of the small inclusions of condensed chromatin (chr). (C and D): EDTA (ethylene diamine tetraacetic acid) regressive staining. The chr is bleached and RNP‐containing structures are preferentially stained. Within the NOR, some contrasted fibrils are seen (small arrows in d) at the periphery of the bleached chr inclusions. E: RNase digestion. The RNP components have been removed, among them the fibrils stained by the EDTA method, located among condensed chr inclusions in the NOR. Unless otherwise expressed, bars indicate 1 m. Courtesy of Dr. Pedro Esponda, CIB‐CSIC, Madrid, Spain.

whose structure totally resembles that of the heterogeneous FCs, that is, it is composed of small inclusions of condensed chromatin, embedded in a fibrous material, lower in electron density (Esponda and Gimeńez‐Martıń, 1974; Medina et al., 1983b) (Fig. 3). Interestingly, when a collection of ultrathin serial sections is examined to get the three‐dimensional perspective, the shape of the NOR appears as a dish, or flattened cylinder (P. Esponda, personal communication). The response of this nucleolar organization, as the rDNA transcription activity develops at an increasing rate, is to enlarge the size of

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9

the DFC balls and to progressively decondense the NOR chromatin inclusions. (Fig. 3). During later stages, the DFC masses are infiltrated with chromatin, with the resulting appearance of FCs in their interior, and the disappearance of the cord‐shaped NOR. Coinciding with the start of the formation of GC, the DFC balls progressively fuse and the nucleolar structure resembles more and more the ‘‘classical’’ structure of a meristematic cell nucleolus (Medina et al., 1983b). The application of the EDTA (ethylene diamine tetraacetic acid) regressive cytochemical staining during the young microspore stage results in the bleaching of condensed chromatin inclusions in the NOR and the preferential staining of RNA fibres (Esponda and Gimeńez‐ Martıń, 1974) (Fig. 3). These fibres are the first evidence of transcription of rRNA genes in the NOR, and they can be found intermingled with the components of the NOR structure resembling the heterogeneous FC. The RNA composition of these fibres is evidenced by their removal with RNase treatment of ultrathin sections (Fig. 3). Important conclusions regarding the nucleolar structures associated with transcription can be reached by re‐evaluating and re‐interpreting the results obtained in this pioneer experiment in the light of our present knowledge of the nucleolus. First of all, these results reveal that transcription actually starts in the NOR, which shows a structure totally identifiable with heterogeneous FCs. Furthermore, it becomes evident that there is a continuity between the chromosomal NOR and nucleolar FCs, and between heterogeneous and homogeneous FCs. If these data are extrapolated to the ‘‘classical’’ interphase nucleolus, a conceivable interpretation would be that transcription actually begins inside FCs, but is not usually detected at these internal locations because DFC generation is such a quick process that transcription can only be visualized together with its product, that is, in the DFC or, on fortunate occasions, in the FC–DFC boundary. Direct research on the localization of transcription in onion meristematic cell nucleoli in interphase (a suitable largely used plant model) began by the quantitative immunolocalization of DNA and RNA pol I in onion nucleoli, which led to the definition of the transition area between FCs and the DFC as a significant nucleolar domain. The interpretation of the immunocytochemical results, which also took into account the former autoradiographic data, strongly pointed to this domain as the site of rDNA transcription (Martıń and Medina, 1991; Martıń et al., 1989). Really, the diVerences between the site in which transcription occurs and the site in which it is detected, due to technical limitations, were pointed out in these papers, where we can read: ‘‘. . .transcription actually begins in fibrillar centres, but, as an immediate result, a dense fibrillar component is gradually generated, giving rise to a structural transition area between the two components’’ (Martıń and Medina, 1991). Further studies, in some cases using the in situ run‐on

10


technique, consistently showed the transcription marker in a restricted nucleolar area made up of the outer periphery of FCs and the inner periphery of the DFC, thus supporting the results obtained in the onion model (Melca´k et al., 1996; Shaw et al., 1995; Testillano et al., 1994). The use of Br‐UTP incorporation in isolated onion cell nuclei, combined with the immunodetection of the nucleolar protein fibrillarin, chosen as a marker for the early processing events, together with observations under both the confocal and the electron microscopes, evidenced a focal arrangement of the transcription sites in the nucleolus. Most of these foci were located at the periphery of FCs, while the core of every FC appeared devoid of labelling, either by the RNA precursor or by fibrillarin. Transcription foci did not totally surround FCs, and so transcription occurred at discrete points in the boundary domain between FCs and the DFC. Regarding colocalization between transcription and fibrillarin, it was only partial in transcription foci, since fibrillarin was not found in the zones of foci immediately bordering FCs. Finally, other transcription foci were found deep in the DFC, in zones located between FCs (De Ca´rcer and Medina, 1999; Medina et al., 2000). At this point, it is worth noting that the identity of FC structures in plant cells was unequivocally established by electron microscopy. Actually, the focal organization of nucleolar transcription had also been shown previously in other plant models (Thompson et al., 1997), but the relationship of these foci with FCs was questioned. Later on, Br‐UTP incorporation was detected in pea nucleoli using pre‐embedding labelling and three‐dimensional electron microscopy of entire nucleoli (Gonza´lez‐Melendi et al., 2001). Labelling, observed as clusters of particles, was claimed to represent single transcription units, or ‘‘Christmas trees’’ located on the DFC, but no indication was given as to where FCs would be situated. The authors argued that FCs would not play any role either in the organization of rDNA or in the structural basis of rDNA transcription. According to their interpretation, actively transcribing rDNA would be a continuous extended loop, emanating from the condensed rDNA chromatin adjacent to the nucleolus, and running through the DFC, without any relationship with FCs (Gonza´lez‐Melendi et al., 2001). The basis for this interpretation, however, was the discrimination of the structures underlying particle clusters; the authors claimed that there was only DFC beneath the particles, though there could also be FCs; actually, in our opinion, the pre‐embedding method used does not provide enough quality of ultrastructural preservation to allow a precise identification. Moreover, we think that many previous results obtained in diVerent plant models are hardly compatible with the model proposed by these authors, among them a highly relevant result obtained in the same laboratory reporting the focal organization of rRNA genes, which, following in situ hybridization,

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were observed structured as intranucleolar spots corresponding to FCs (Rawlins and Shaw, 1990). The nucleolus is indeed organized from a loop of rDNA emanating from the nucleolar organizer chromosome (Fig. 1), as postulated many years ago. Godward (1950), after observing the nucleolar structure of the alga Spyrogyra, called the continuous pathway followed by rDNA through the bulk of the nucleolus the ‘‘nucleolar organizer track’’. According to many research data, this continuous loop is not totally extended, because, in some places, the nucleolus shows focal concentrations of highly folded rDNA, which are FCs (Fig. 1). The concept of FCs as foci containing local concentrations of DNA has been supported by cytochemical experiments of selective staining of DNA (Motte et al., 1991; Risuenõ et al., 1982; Testillano et al., 1991), immunocytochemistry (Martıń et al., 1989) and in situ hybridization (Rawlins and Shaw, 1990). The presence in plants of the so‐called ‘‘heterogeneous FCs’’, containing small inclusions of condensed chromatin (Risuenõ et al., 1982), gives additional support to this concept. Heterogeneous FCs are the same structures that were called ‘‘lacunae’’ by Chouinard (1970) in one of the pioneer works on the localization of nucleolar DNA. Even before the introduction of in situ hybridization methods, there was overwhelming experimental evidence in favour of the presence of rDNA in these structures, based only on their structural identity and continuity with the secondary constriction of chromosomes, the known site of the nucleolar organizer (Medina et al., 1983b,c). There is also experimental evidence to lend support to the conversion of the heterogeneous FCs into homogeneous FCs, as shown previously for microspores (Esponda and Gimeńez‐Martıń, 1974; Medina et al., 1983b), and as reported for the germination of seeds or bulbs (Deltour, 1985). This concept of FCs is compatible with data postulating the existence of a nucleolar matrix, or nucleolar proteinaceous skeleton (Moreno Dıáz de la Espina, 1995), with, among other functions, that of gathering rDNA in FCs, and structurally organizing it, depending on the nucleolar transcriptional activity and cell requirements. Certainly, however, the agreement on these concepts is not total. D. EPIGENETIC REGULATION OF rRNA GENE EXPRESSION: GENE SILENCING AND NUCLEOLAR DOMINANCE

Expression of rRNA genes is regulated at multiple levels. Transcription by RNA pol I, the further transcript processing and the final preribosome assembly and export are regulated by modulating the rate at which each individual process takes place. This regulation is achieved by mechanisms involving the participation of diVerent factors, most of them proteinaceous.

12


The whole Section III of this paper is devoted to reviewing the identity of these factors and their functional role. However, there is a second form of regulation, which acts as an ‘‘on/oV’’ switching mechanism, producing the more or less permanent silencing of large groups of rRNA genes. This mechanism has a structural counterpart, namely, the structure of the rDNA chromatin, and, in particular, its condensation state, so that silenced genes are characterized by the presence of condensed chromatin structures, whereas ‘‘open’’ genes display decondensed chromatin conformations. Many reports, using a wide range of experimental approaches, can be found in the literature dealing with this process. The identification, in plant cells, of the heterogeneous type of FCs, containing small inclusions of condensed chromatin, which has just been discussed in the preceding section, is an example of the visualization of switched‐oV rRNA genes. As indicated above, this mechanism is fully reversible, and inter‐conversions between the condensed and decondensed forms of chromatin inside FCs occur throughout diVerent biological processes, and even throughout the diVerent periods of the cell cycle. The presence of rDNA chromatin under the form of heterochromatin has also been visualized in plant cells at the microscope as the so‐called ‘‘nucleolus‐ associated chromatin’’ (NAC). This NAC usually appears as a large heterochromatic mass, located at the periphery of the nucleolus, showing fibres that interconnect it with the interior of the nucleolar body. The fact that NAC is constituted by rDNA chromatin was unequivocally demonstrated by in situ hybridization (Highett et al., 1993; Rawlins and Shaw, 1990). The same as it was shown for heterogeneous FCs, inter‐conversions between the condensed and the decondensed state of chromatin aVecting NAC were reported, mostly throughout the germination process (Deltour, 1985). Otherwise, the use of psoralen photocrosslinking of rDNA in a wide variety of biological model systems, including plants, led to the conclusion that in intact cells, two distinct types of ribosomal chromatin structures coexist, one that contains nucleosomes and represents the inactive copies and one that lacks a repeating structure and corresponds to the transcribed genes. In particular, in plant cells (tomato) the nucleosome‐containing chromatin is present in the majority (80%) of the rDNA chromatin, only a minor chromatin population (20% of the ribosomal genes) being in an open configuration. Interestingly, the relative amounts of the two types of structures are similar in interphase and metaphase, but their transcriptional activities, evaluated by run‐on, diVer significantly. Also, these relative amounts are similar in stationary and exponentially growing cells. This suggests that the two states of chromatin are maintained independently of the transcriptional process and that they are stably propagated through the

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cell cycle (Conconi et al., 1989, 1992). An exception to this rule was found in yeast, which can modulate the proportion of active (non‐nucleosomal) and inactive (nucleosomal) rRNA gene copies in response to variations in environmental conditions, which suggests that, unlike vertebrate and plant cells, yeast can regulate rRNA synthesis by varying the number of active gene copies (Dammann et al., 1993). Silencing of rDNA chromatin also occurs in an intriguing phenomenon called nucleolar dominance, initially discovered in a classical work performed on plant cells. This phenomenon has received much attention from leading research groups in recent years, and presently concentrates most of the eVorts directed towards discovering the molecular mechanisms supporting the regulation of the expression of rRNA genes by on/oV switching, aVecting rDNA chromatin structure. Nucleolar dominance was discovered in interspecific hybrids of Crepis by Navashin (1934), who called it ‘‘diVerential amphyplasty’’. The finding was that, in hybrids, the secondary constrictions were absent from chromosomes inherited from one species. Navashin observed that the secondary constrictions reappeared in progeny when karyotypes recovered the initial ‘‘pure species’’, indicating the reversibility of the phenomenon and the conservation of the integrity of the apparently absent secondary constrictions. Nucleolar dominance was clearly evidenced by the cytochemical procedure of silver staining of the nucleolar organizer (AgNOR method). Interspecific hybrids, such as Triticale, showed silver staining in those NORs displaying secondary constrictions, whereas the underdominant NORs, lacking secondary constrictions, did not show AgNOR staining (Lacadena et al., 1984). Since AgNOR staining was interpreted in terms of rDNA chromatin structure, as an eYcient and reliable labelling of decondensed forms of this chromatin (Hernandez‐Verdun et al., 1982; Medina et al., 1983c, 1984), the interpretation was that underdominant NORs had repressed their expression by means of a heterochromatinization process (Medina et al., 1984). Actually, the persistence of the decondensed form of chromatin in visible secondary constrictions in chromosomes (either in hybrids or in ‘‘pure species’’), in a phase in which all the rest of chromatin is fully condensed in nucleosomal and supranucleosomal configurations, is caused by the persistence of RNA pol I complex, including transcription factors, associated with rRNA genes during mitosis (see Section II, section A, in this paper). The inactivation of a subset of rRNA genes via heterochromatinization and its reversibility strongly pointed to the consideration that nucleolar dominance was an epigenetic phenomenon, that is, a heritable, but potentially reversible condition of gene activity that is not caused by changes in gene sequence (Preuss and Pikaard, 2007), and highly stimulated the research

14


eVorts looking for the molecular mechanisms responsible for this special form of gene silencing. The first decisive contributions came from the group of R.B. Flavell and their findings on the correlation of rDNA methylation and the transcription potential of rRNA genes; dominant, very active loci have a higher proportion of rRNA genes with unmethylated cytosine residues in comparison with recessive and inactive loci (Flavell et al., 1988). Furthermore, Thompson and Flavell (1988) demonstrated in wheat the existence of a correlation between nucleolar dominance, rDNA undermethylation and decondensed structure of ribosomal chromatin, the latter evaluated by its sensitivity to DNase I. Additionally, direct evidence that cytosine methylation is closely associated with rRNA gene expression and nucleolar dominance was given by experiments of plant growth in the presence of 5‐aza‐20 ‐deoxyciytidine (aza‐dC), a potent inhibitor of cytosine methylation, which caused the silent, underdominant genes to become expressed to high levels (Chen and Pikaard, 1997; Lawrence et al., 2004). However, rDNA methylation was not the unique mechanism responsible for nucleolar dominance. A treatment with histone deacetylase inhibitors, such as sodium butyrate or trichostatin A (TSA), produced similar de‐repressing eVects as those observed upon treatment with aza‐dC (Chen and Pikaard, 1997; Lawrence et al., 2004). Furthermore, other modifications of histones were shown to play a role in nucleolar dominance; in particular, nucleosomes associated with the promoter regions of underdominant genes appeared enriched in histone H3 dimethylated on lysine 9 (H3K9me2), a known mark of heterochromatin, whereas promoter regions of dominant genes associated with H3K4me3, a mark of euchromatin, and also with H3K9me2, supporting the idea that, among dominant rRNA genes, both forms of chromatin (euchromatin and heterochomatin) may coexist. As expected, a close correlation was found between DNA methylation and H3K9me2, on the one side, and between DNA hypomethylation and H3K4me3, on the other side (Lawrence et al., 2004). Therefore, DNA methylation and histone modification states are interdependent and mutually reinforcing. An important question aVecting not only the phenomenon of nucleolar dominance but also the rRNA gene silencing processes in general is whether silencing is regulated taking the full NOR as a single locus, or regulation is exerted on single genes, one by one (or small subsets of genes). Certainly, the choice of which NORs to inactivate is not at random, but it does not depend on the number of rRNA genes or on particularities of a given species. Some experiments with Xenopus using injected plasmid‐encoded rRNA minigenes apparently demonstrated that dominance was due to diVerences in binding aYnity of one or more limiting transcription factors to the enhancers (Reeder and Roan, 1984). However, analogous tests in plants failed to reveal these

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diVerences, since, under natural conditions, transcription factors are not a limiting component for the eYciency of transcription (Preuss and Pikaard, 2007). An interesting study using Arabidopsis thaliana inter‐ecotype hybrids revealed that a certain combination of NORs from the two ecotypes involved was selected, the same in all cases, despite the fact that rRNA gene sequences are >95% identical across A. thaliana ecotypes. These data do not fit into models in which nucleolar dominance is regulated at the level of individual rRNA genes, but they point to larger scale regulation of the NORs (Lewis et al., 2004; Preuss and Pikaard, 2007). Nevertheless, concluding evidence on this question has not yet been provided; further research on this topic will contribute to explain the establishment and maintenance of gene dosage levels through multiple cell generations.

II. THE NUCLEOLUS DURING THE CELL CYCLE A. THE NUCLEOLUS THROUGH MITOSIS

When entering mitosis, during the G2/M transition, the nucleolus is progressively disassembled, more or less at the same time as the nuclear envelope breaks down. Nucleolar disassembly begins with the disaggregation of the GC, and the export of the nucleolar granules to the cytoplasm, and continues with the progressive dispersion of the DFC between the condensed chromatin masses, until the nucleolar structure is lost in prometaphase. Functionally, this process of disassembly is accompanied by the cessation of transcription and processing. At the time of chromosome formation, in metaphase, the chromosomal locus occupied by ribosomal genes can be easily detected in the form of the secondary constriction, a species‐specific zone of one or more chromosomes, cytologically characterized by a negative response to DNA‐staining techniques such as Feulgen and 40 ‐6‐diamidino‐ 2‐phenylindole (DAPI), and by a positive response to staining by a specific silver method known as Ag‐NOR. The absence of staining by DAPI or Feulgen is due to the decondensed state of the chromatin in this chromosomal region, whereas staining by the Ag‐NOR technique is caused by the presence of some specific proteins which remain associated with rDNA chromatin. Apart from the NOR, some other nucleolar remnants can also be detected in metaphase and anaphase as sheath‐like structures located at the chromosome periphery. At the end of mitosis, the nucleolus is reconstituted in the daughter cells. Nucleologenesis includes the formation of prenucleolar bodies (PNBs) from the nucleolar materials carried by the chromosomes at their periphery. The materials forming PNBs are recruited at the NOR and, together with


16

Nu Nu

chr

Prophase

G2 Nu

chr

G1

NOR chr Nu NOR chr

PNBs

Metaphase Fibrillar centres /nucleolar organizer Dense fibrillar component /perichromosomal sheath /prenucleolar bodies Granular component Chromosomes

Telophase

Anaphase

Fig. 4. Schematic view of the evolution of the nucleolar structural components throughout the cell cycle. At the end of interphase, the active nucleolus is made up of territories of dense fibrillar component (DFC) with fibrillar centres (FCs) embedded in them, all of them surrounded by a granular component (GC). Entry in mitosis involves condensation of chromatin to form chromosomes (chr), nuclear envelope breakdown and arrest of nucleolar activity, leading to the dispersion of the GC, followed by that of the DFC, and coalescence of FCs (prophase). In metaphase, the nucleolus is not visible as a diVerentiated organelle. Chromosomes (chr) are at the equatorial plate, ribosomal genes are gathered at the nucleolar organizing region (NOR) and chromosomes appear surrounded by a perichromosomal sheath, whose internal structure resembles the DFC. This organization is maintained at anaphase, when chromosomes migrate to the poles. In telophase, chromosomes (chr) decondense and the material of the perichromosomal sheath forms prenucleolar bodies (PNBs), whose materials, in turn, are recruited at the NOR, which resumes its transcriptional activity. As a result of these two processes, the new nucleolus is progressively enlarged. In G1, in each of the two daughter cells, the nucleolar activity gradually increases, and the GC begins to be observed.

de novo pre‐rRNA synthesis, lead to the formation of the new nucleolus (Fig. 4) (Azum‐Ge´lade et al., 1994; De la Torre and Gimeńez‐Martıń, 1982; Hernandez‐Verdun, 2006; Hernandez‐Verdun and Gautier, 1994; Medina et al., 2000; Moreno Dıáz de la Espina et al., 1976). The components of the RNA pol I transcription complex remain assembled and associated with rDNA chromatin throughout mitosis in the NOR. In mammalian cells, there is experimental evidence of the localization of the following in this chromosomal locus: RNA pol I (Scheer and Rose, 1984), topoisomerase I (Rose et al., 1988), the transcription factors UBF and SL1 (Prieto and McStay, 2005; Roussel et al., 1996) and the termination factor TTF‐1 (Sirri et al., 1999). However, this complex is kept inactive throughout all phases of mitosis, and it is only at the time of nucleologenesis that the transcriptional activity is resumed as one of the factors triggering the formation of the new nucleolus. Therefore, the complex is kept either inactive or

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active as a result of the action of regulatory factors. Among these factors, the CDKA (or CDK1, or cdc2)/cyclin B complex has been shown to play a major role by a mechanism of reversible phosphorylation (Heix et al., 1998; Sirri et al., 2000). This complex, which has a kinase activity, is a central element in the regulation of G2/M transition, as already demonstrated in animals, plants and yeast (Arellano and Moreno, 1997; Doreé and Hunt, 2002; Inze´ and De Veylder, 2006). With regard to the nucleolar‐processing complex, investigations were performed in plant cells to find out whether the components of this complex could remain somehow assembled whilst mitotically inactive and separated from the transcription complex. In fact, the existence of components of this processing complex in the nucleolar remnants located at the periphery of chromosomes in metaphase and anaphase had been proposed (Risuenõ and Medina, 1986). With this in mind, the mitotic course of pre‐rRNA and the nucleolar proteins fibrillarin and nucleolin was traced in onion root cells. The identity of pre‐rRNA was confirmed by in situ hybridization and the time of its production, precisely at the preceding G2, was demonstrated by pulse‐ chase autoradiography on synchronized cells after tritiated uridine incorporation at this point of the cell cycle. Colocalization of this pre‐rRNA and of the nucleolar proteins fibrillarin and nucleolin was detected in the chromosome periphery (perichromosomal sheath) during metaphase and anaphase, in irregular fibrillar masses located among chromosomes in ana‐ telophase, in PNBs during telophase and in the newly formed nucleoli after nucleologenesis, indicating the simultaneous presence of components of processing complexes from the preceding interphase, in all these structures. Therefore, the perichromosomal sheath is the precursor structure of prenucleolar bodies, and this finding shows the continuity of these nucleolar molecular components through successive cell generations (Fig. 4). Colocalization of key elements of the pre‐rRNA‐processing complex in the same transient structures during mitosis strongly suggests that at least a subset of these complexes do not disaggregate during cell division, but remain assembled and become incorporated into the new nucleolus (Medina et al., 1995). In mammalian cells, proteins involved in pre‐rRNA processing, as well as in pre‐rRNA‐processing intermediates, synthesized in the preceding G2, were also localized at the periphery of chromosomes during mitosis, as a result of nucleolar dispersion in prophase, and were then shown to be concentrated in PNBs at telophase (Angelier et al., 2005; Dundr and Olson, 1998; Gautier et al., 1994). Subsequently, a flow of materials is established between PNBs and the NOR, so that PNBs become progressively smaller whereas the new nucleolus emerges and grows at the site of the NOR (Savino et al., 2001). Interestingly, nucleolar protein recruitment at the NOR occurs sequentially,

18


the early processing proteins being recruited first on transcription sites, while late processing proteins remain in PNBs for a longer time, before migrating to the NOR (Savino et al., 2001). The assembly of proteins and RNA in processing complexes in the diVerent structures in which they are localized during mitosis was analyzed in mammalian cells by fluorescence resonance energy transfer (FRET) between fluorochrome‐tagged proteins (Angelier et al., 2005). FRET was not detected at the periphery of chromosomes, but it was registered in PNBs at a progressively higher rate as telophase proceeded. The interpretation was that processing complexes were not assembled in the perichromosomal sheath, but in PNBs, from which they were exported to the NOR (Angelier et al., 2005; Hernandez‐Verdun, 2006). Actually, nothing is known of protein interaction with pre‐rRNA in the diVerent transitory mitotic structures, and the formation of pre‐rRNP complexes, and the possibility exists that they may not be totally identifiable with those found in interphase. In fact, processing complexes, active in interphase, are inactivated in mitosis and become active again during/after nucleologenesis. In any case, all data reported up to now, in diVerent model systems, agree in attributing to PNBs a central and crucial role in postmitotic nucleolar reassembly and reactivation. The synchronization of the postmitotic nucleolar assembly with the resumption of rDNA transcription is again under the control of cell cycle regulators. These events at the NOR occur at the time when degradation of cyclin B1 and a decrease in CDKA activity overcome the mitotic repression of RNA pol I transcription (Clute and Pines, 1999; Sirri et al., 2002). B. THE NUCLEOLUS THROUGH INTERPHASE

It has been experimentally verified that, under normal physiological conditions, cell proliferation is accompanied by an increase in the biosynthesis of ribosomes (Baserga, 2007; Hannan and Rothblum, 1995). The rate of pre‐ rRNA transcription and processing has been shown to change progressively through the successive interphase periods. Thus, whereas in G1 the rate of these processes is kept at moderate levels, G2 is a period characterized, in general, by an intense rate of nucleolar activity (i.e. the rate of pre‐rRNA transcription and transcript processing), although the values reached by the transcription and processing rates are variable in the various cell systems investigated (Klein and Grummt, 1999; Kwiatkowska and Maszewski, 1979; Raska et al., 2004; Risuenõ and Medina, 1986). The precise mechanisms by which transcription of rRNA genes is regulated in a cell cycle‐dependent manner are now beginning to be understood, mostly in mammalian cells and in yeast. Together with the aforementioned role played by CDKA/cyclin B in

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mitosis (Heix et al., 1998; Sirri et al., 2000), CDK/cyclin complexes are also capable of phosphorylating the transcription factor UBF, and this phosphorylation is responsible for the increase in rDNA transcription during G1 progression (Voit et al., 1999). Moreover, there is more and more evident that the correct progression of ribosome biogenesis is a factor influencing cell cycle regulation, in such a way that, in one or more cell cycle checkpoints, the status of ribosome production is sensed and cell cycle progression is allowed or arrested in response to this stimulus. This is true in the case of the nucleolar protein Bop1, a regulator of pre‐rRNA processing, whose alteration induces a rapid cell cycle arrest which is p53 dependent. Thus, p53 could act as a sensor of nucleolar stress caused by defects in ribosome biogenesis (Lapik et al., 2004). It is worth remembering that p53 is a tumor suppressor whose eVect is to induce a G2/M arrest by decreasing intracellular levels of cyclin B1, acting at the level of gene expression (Innocente et al., 1999). In other cases, proteins have been identified that control, simultaneously, ribosome biogenesis and cell cycle progression. This is the case of pescadillo, whose mutation results in cell cycle arrest and inhibition of preribosome maturation (Lerch‐Gaggl et al., 2002), or the nucleolar‐conserved protein SURF‐6, whose depletion causes G1 arrest (Polzikov et al., 2007). Unfortunately, the knowledge we have of all these mechanisms and factors in plant cells is not as high as that of animal cells and yeast. However, little doubt exists that they are conserved through the whole phylogenetic scale, including plants, as has been shown in recent works (Zografidis et al., 2007). The reason why there is a close relationship between cell cycle regulation and ribosome biogenesis regulation can be found in the metabolic requirements of actively proliferating cells. Cell proliferation requires continuous building of cellular materials, particularly proteins, to reach the critical size which will permit cell division (Baserga, 2007; Bernstein et al., 2007). Since the function of ribosomes is the translation of mRNA into proteins, the control of ribosome biogenesis is, necessarily, a key element of proliferation control (Bernstein and Baserga, 2004). The positive correlation between cell proliferation and the activity of pre‐ rRNA transcription and processing causes alterations in the size of the nucleolus and in the distribution of the nucleolar components, correlated with modifications in the proliferative state of the cell. In fact, diVerences in ribosome synthesizing activity are expressed as morphologically detectable changes in the nucleolus (Medina et al., 2000; Shaw and Jordan, 1995; Thiry and Goessens, 1996). Since, as indicated above, each period of the cell cycle is associated with a particular rate of pre‐rRNA synthesis and processing, it is possible that a particular structural pattern of the nucleolus could be ascribed to each interphase period. If this were so, these structural changes could serve


20

G0

G1

S G2

Dense fibrillar component Granular component

Homogeneous fibrillar centres Heterogeneous fibrillar centres

Nucleolar vacuole of G1

Nucleolar vacuole of G2

Fig. 5. Nucleolar models in the diVerent interphase periods. The relative nucleolar size, as well as the distribution of the nucleolar structural subcomponents in each period, is shown. Morphological and morphometrical features correlate to the rate of nucleolar transcriptional and processing activity. From Gonza´lez‐Camacho and Medina (2006). Reproduced with permission of the publisher (Springer).

as an excellent model for the establishment of general patterns of structure– function relationships in the nucleolus, and these nucleolar structural patterns could become excellent markers for identifying cell cycle periods. Despite these possibilities, studies dealing with the variations in nucleolar structure associated with cell cycle periods are really scarce (June´ra et al., 1995; Sacristań‐Ga´rate et al., 1974). A sequential analysis of nucleolus structural variations during the diVerent periods of interphase has been carried out in onion root meristematic cells, synchronized by a treatment with hydroxiurea (HU) followed by growth in a medium from which the drug was removed. Four diVerent structural models were defined, associated, respectively, with G1 (one model), S (one model) and G2 (two models) (Gonza´lez‐Camacho and Medina, 2006) (Fig. 5). In agreement with the well‐known correlation between nucleolar size and activity, an enlargement of the nucleolus was observed from the beginning to the end of interphase, but the nucleolar size remained practically unaltered throughout G1 and S, and it was only in G2 that a dramatic increase was observed, coinciding with the peak of nucleolar activity. Looking at particular nucleolar subcomponents, a correlation of the structure, number and size of FCs with nucleolar activity had been previously defined (Medina et al., 1983a; Risuenõ et al., 1982). In agreement with these data, cell cycle progression was found to be associated with an increase in the number of FCs and the reduction in their size. Structurally, heterogeneous FCs were found to be present in G1, but disappeared progressively in S, so that G2 nucleoli practically contained only homogeneous FCs (Fig. 2). In general, the structure and morphometrical parameters of the diVerent nucleolar subcomponents could be defined in each of the interphase periods, showing a specific arrangement (Fig. 5).

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III. NUCLEOLAR PROTEIN FACTORS: THEIR ROLE IN RIBOSOME BIOGENESIS AND PLANT DEVELOPMENT Genome sequencing programmes in Arabidopsis (The Arabidopsis Genome Initiative, 2000) and other plant species (International Rice Genome Sequencing Project, 2005) and the homology of proteins from yeast and/or animals has permitted the identification of a large number of putative nucleolar protein factors involved in ribosome biogenesis and/or other nucleolar functions. Recently, using a sizeable proteomic approach Pendle and co‐workers carried out a comprehensive analysis of nucleolar proteins in Arabidopsis (Pendle et al., 2005) which led to the identification of 217 nucleolar proteins (Brown et al., 2005). However, this number is relatively low compared with 700 proteins identified in the human nucleolus (Leung et al., 2006), indicating that there are perhaps other stable and/or transitory unknown nucleolar proteins in plants. Nevertheless, the study performed by Pendle et al. revealed that nearly 40% of all nucleolar proteins are ribosomal proteins (RPs), nucleolar factors or C/D and H/ACA small nucleolar ribonucleoproteins (snoRNPs) (Brown et al., 2005), thus confirming that the major function of the nucleolus is ribosome biogenesis. What do we know about the molecular function of these factors? How do they influence growth and plant development? To address these major questions, in the following sections we will describe some of the major advances made concerning nucleolar factors involved in rRNA synthesis and snoRNP biogenesis. A. rRNA TRANSCRIPTION AND PROCESSING FACTORS

As mentioned above, the primary function of the nucleolus is the transcription and processing of pre‐rRNA. These two processes involve a large number of nucleolar protein factors, including an RNA polymerase I enzyme and a subset of transcriptional and pre‐RNA‐processing factors, all directed towards producing mature 18S, 5.8S and 25S rRNA (Fig. 1) (Olson, 2004; Saéz Va´squez and Echeverrıá, 2006). In plants, little is known about the diVerent transcription factors associated with the rRNA transcriptional machinery. For instance, early biochemical and immunological characterization revealed that Brassica oleracea RNA pol I is 600 kDa multimeric enzyme complex of around 12–15 subunits (Guilfoyle and Jendrisak, 1978; Guilfoyle and Malcolm, 1980; Guilfoyle et al., 1976). Since then, many eVorts to identify Pol I transcription factors have relied on gel mobility shift assays to detect proteins interacting with the rDNA promoter, but the identity of these proteins remains

22


unknown (Saéz‐Va´squez and Echeverrıá, 2006). The subsequent description and purification of an RNA polymerase I holoenzyme complex from B. oleracea in the laboratory of C. Pikaard allowed the identification of an associated casein kinase II (CKII) and histone acetyl transferase (HAT) (Saéz‐Va´squez and Pikaard, 1997, 2000; Saéz‐Va´squez et al., 2001). Similar results were observed in RNA pol I holoenzymes from a mouse (Hannan et al., 1999; Seither et al., 1998) and Xenopus (Albert et al., 1999). These results suggest that RNA pol I holoenzyme, both in plants and in animals, is equipped to respond to growth and environmental signals and modify chromatin as necessary to activate transcription. In animals, the CKII and HAT proteins involved in rRNA transcription have already been studied in detail (Halkidou et al., 2004; Voit et al., 1992), in contrast to the CKII and HAT activities in plants that remain much less characterized. However, the existence and functionality of a CKII holoenzyme has been demonstrated both in maize (Riera et al., 2004) and in Arabidopsis (Salinas et al., 2006). The genome of A. thaliana contains four CKII and four CKII subunit genes. Three of the CKII catalytic subunits localize in the nucleus and in the nucleolus (the fourth one in the chloroplast), whereas the CKII regulatory subunits localize in the nucleus, in the cytoplasm or in both compartments (Salinas et al., 2006). A similar situation was observed in maize, where the three CKII subunits distributed over the cytoplasm and nucleus in contrast to the three CKII subunits that remain accumulated in the nucleolus (Riera et al., 2004). Thus, the multiplicity of genes coding for and CKII subunits in plants and diVerential subcellular localization may provoke a high heterogeneity of CKII that may aVect both interaction with substrates and the CKII holoenzyme structure and function (Riera et al., 2004). Thus, we can speculate that distinct CKII holoenzymes are implicated either in transcription and processing of rRNA synthesis and/or other steps of ribosome biogenesis occurring in the nucleolus. In animals and yeast cells, the nucleolar factors UBF/HmoI and TIF‐1A/ RRN3 play a major role in rRNA transcription (Grummt, 1999; Nomura, 2001). UBF/HmoI is an HMG protein which activates rDNA transcription by recruiting RNA pol I to the promoter, and TIF‐1A/RRN3 is a regulatory factor that is associated with the initiation–competent subpopulation of Pol I that senses nutrient and growth factor availability (Grummt, 1999; Nomura, 2001). So far, there is no experimental evidence for a functional TIF‐1A/ RRN3 protein in plants, but antibodies against mammalian UBF have been shown to cross‐react with a single 58 kDa polypeptide and allow localization of an immune‐related protein in the nucleolus of Allium cepa (De Ca´rcer and Medina, 1999; Rodrigo et al., 1992; Tao et al., 2001a,b). Thus, although all

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these data suggest that a UBF‐like protein might also exist in plants, its role in rDNA transcription still has to be established. With the exception of UBF/ HmoI and TIF‐1A/RRN3 genes, there are no genes coding for proteins with detectable homologies to any of the transcription factors such as TAFs, CORE or UAF identified in mammalian and yeast cells (Grummt, 1999; Nomura, 2001). Nevertheless, we can expect that genes encoding functional homologue proteins could have diverged beyond recognition in such an ancestral and UniGene system. In contrast, diVerent components of the nucleolar rRNA‐processing complex can be recognized in plants by their homology with their animal or yeast counterparts. For example, most of the genes encoding proteins that are associated with C/D and H/ACA small nucleolar RNA (snoRNA) can be identified in the Arabidopsis genome (Brown et al., 2003b). In eukaryotic cells, the C/D and H/ACA snoRNP complexes direct rRNA base modifications at a specific position, including 50 methyl ribose methylation and 5‐riboyluracil pseudouridinylation, respectively (Reichow et al., 2007). Although many snoRNAs of the types C/D and H/ACA have been identified in plants (Barneche et al., 2001; Brown et al., 2003a), only the fibrillarin/Nop1 (a C/D snoRNP) and Nap57/Cbf5p (H/ACA snoRNP) have been characterized in any detail in plants. Early studies using antibodies against human fibrillarin detected an immune‐related protein in the nucleolus of onion cells (Medina et al., 1995). More recently, two genes in A. thaliana, AtFIb1 and AtFib2, encoding nearly identical proteins conserved with eukaryotic fibrillarins were reported (Barneche et al., 2000; Pih et al., 2000). Arabidopsis fibrillarin localizes in the nucleolus and the N‐terminal glycine‐ and arginine‐rich region is both necessary and suYcient to target AtFib1 to the nucleolus (Barneche et al., 2000; Pih et al., 2000). Later, it was shown that an Arabidopsis CBF5‐like protein localizes in the nucleolus and Cajal bodies and interacts with an NAF1 protein (Lermontova et al., 2007), which is required for H/ACA snoRNP assembly (Reichow et al., 2007). The Cbf5p/NAP57 homologue is encoded by a single gene in Arabidopsis (Maceluch et al., 2001) and disruption of this gene by T‐DNA insertion is lethal (Lermontova et al., 2007), demonstrating that Arabidopsis CBF5 activity is essential for viability. To our knowledge, plants with mutated or disrupted fibrillarin genes have not been described yet, and consequently how disruption of fibrillarin expression aVects plant growth and development remains to be determined. On the basis of the expected role for AtFib proteins in ribosome biogenesis, we can anticipate that simultaneous inhibition of fibrillarin genes in thaliana would be lethal as well. Consequently, only the analysis of plants with a reduced level of AtFib and AtCBF, by RNAi, for instance, should provide more information on the role of these two proteins in snoRNP and ribosome biogenesis in plants.

24


In the case of Arabidopsis, a protein named Pescadillo (AtPES), with a putative role in pre‐rRNA processing, was recently characterized (Zografidis et al., 2007). This protein AtPES was shown to localize in the plant nucleolus and it was able to rescue the slow growing phenotype of a yeast strain lacking the gene NOP7/YPHI. Although the role of AtPES in the nucleolus remains undefined, the nucleolar localization and experiments in yeast suggest that AtPES might be required for the formation of 25S rRNA. AtPES expression is cell cycle regulated and conforms to the major role of ribosome biogenesis in cell size and proliferation. Over‐expression of AtPES does not aVect plant growth and development (Zografidis et al., 2007); nevertheless we can expect any inhibition or reduction of AtPES gene expression to provoke major changes in the plant phenotype, since a single AtPES gene has been reported in the genome of Arabidopsis. An A. thaliana RNase III‐like protein (AtRTL2) was recently reported to cleave 3’ETS sequences from pre‐rRNA (Comella et al., 2008). AtRTL2 localizes in the nucleus and in the cytoplasm of Arabidopsis cells and consequently it may be involved not only in the early cleavage steps of pre‐rRNA in the nucleolus but also in later processing steps in the cytoplasm. Moreover, the expression of AtTL2 protein is tightly regulated to plant development. In fact, the AtRTL2 protein level decreased during seed formation and became detectable after seed imbibitions. Accumulation of diVerent RNAs, including rRNA precursors and mRNA, in seeds was reported several years ago (Aspart‐Pascot et al., 1976; Aspart et al., 1980). These reports show that whereas some RNAs are degraded during seed formation, others are stored and used as soon as germination starts. Thus, AtRTL2 transcript accumulation in dry seeds could constitute a mechanism to ensure rapid synthesis of RNase III protein for eYcient growth and plant development. Interestingly, analysis of the first generation of homozygous plants, mutant for the AtRTL2 gene, does not display any growth defect or abnormal morphological phenotype. However, plants obtained from self‐pollination of Atrl2 homozygous plants showed growth and developmental defects (dwarf phenotype) that become noteworthy in the third generation (Comella and SaézVa´squez). So far, it cannot be explained how the repeated self‐pollination on the Atrtl2 mutant plants induces dwarf plants, but we would expect that biosynthesis of coding and/or not coding RNA(s) involved directly or indirectly in plant development is being aVected. Finally, two novel nucleolar proteins were identified in maize, which may be part of an RNP complex, MA16 and ZmDRH1 (Gendra et al., 2004). The MA16 protein is an RNA‐binding protein that interacts with a DEAD box RNA helicase (ZmDRH1), which, in turn, interacts with fibrillarin (Gendra et al., 2004). Although this observation suggests that MA16 and ZmDRH1

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play a role in rRNA synthesis, it is also possible that these proteins, together with the fibrillarin complex, might be involved in other nucleolar activities. A major advance, relating characterization of mechanisms controlling both transcription‐ and pre‐rRNA‐processing factors in plants, was the identification of the Nuclear Factor D (Caparros‐Ruiz et al., 1997). The NF D factor is a large U3 snoRNP complex purified from B. oleracea, containing snoRNAs U3 and U14, nucleolin‐like protein and fibrillarin (Saéz‐Va´squez et al., 2004b). Interestingly, although NF D was identified by its specific interaction to 5’ETS rDNA (Caparros‐Ruiz et al., 1997), the purified NF D complex also cleaved pre‐rRNA at the primary cleavage site P in vitro (Saéz‐Va´squez et al., 2004b). Thus, the NF D complex may assemble on the rDNA and subsequently bind and cleave specifically the pre‐rRNA at the primary cleavage site (Fig. 1) (Saéz‐Va´squez et al., 2004a,b). A similar mechanism was also described in yeast, where a large RNP complex that contains the U3 snoRNA and 28 proteins was isolated (Dragon et al., 2002; Gallagher et al., 2004). Seventeen new proteins (Utp1–17) were present of which ten were known components, including C/D snoRNP‐associated proteins (Nop1, Nop56, Nop58 and snu13) and six proteins specific to U3 snoRNP (Dragon et al., 2002). Although the Utp proteins were not reported as belonging to the NF D complex, potential functional homologues were shown to play a major role in Arabidopsis plant development (see below) (GriYth et al., 2007; Shi et al., 2005). In this context, although several nucleolar factors involved in transcription and processing of pre‐rRNA in plants have been identified either (1) throughout the characterization of functional protein complexes or (2) by homology with their animal or yeast counterparts, the functional analysis of these individual factors remains incomplete. However, the last 5 years have been particularly stimulating thanks to the description of several Arabidopsis plant mutants for nucleolar protein genes, including the multifunctional nucleolar protein nucleolin. B. NUCLEOLIN‐LIKE PROTEINS

In eukaryotes cells, nucleolin is one of the most abundant non‐RPs in the nucleolus (Ginisty et al., 1999). Here, nucleolin plays a key role in the diVerent steps involved in ribosome biogenesis, including RNA pol I transcription and processing of pre‐rRNA (Roger et al., 2003), as well as assembly and nucleocytoplasmic transport of ribosome particles (Bouvet et al., 1998). Although nucleolin has been involved essentially in ribosome biogenesis, it has also been implicated in a number of additional processes that take place in the nucleus and in the cytoplasm, including maintenance of chromatin structure (Angelov et al., 2006), RNA pol II transcription regulation

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(Huddleson et al., 2006), DNA replication (Kim et al., 2005), mRNA stability/translation (Takagi et al., 2005) and assembly of RNP complexes (Fouraux et al., 2002; Lefebvre et al., 2002, and references therein). Nucleolin‐like proteins in plants were described early in the 1990s when a nucleolin‐like protein in onion cells was first identified and localized in the nucleolus (Martıń et al., 1992) and in the nucleolar matrix (Mıńguez and Moreno Dıáz de la Espina, 1996) by using an antibody against hamster nucleolin. Other experiments in onion root cells also revealed that nucleolin and fibrillarin‐like proteins colocalize during mitosis, suggesting that at least a subset of RNA‐processing complexes do not disaggregate during cell division, but remain assembled and become incorporated into the new nucleolus (Cerdido and Medina, 1995). Moreover, it was shown that the onion nucleolin‐like protein NopA64 (De Ca´rcer et al., 1997) also colocalized with an immune‐related UBF protein in the DFC surrounding the FCs of the nucleolus from onion cells (De Ca´rcer and Medina, 1999), suggesting that nucleolin‐like protein might also be involved in transcription initiation of pre‐ rRNA in plants. Thus, these observation provided early evidences of a role for nucleolin in controlling both pre‐rRNA transcription and processing. Nucleolin‐like proteins have been cloned and molecularly characterized in alfalfa (Bogre et al., 1996), pea (Tong et al., 1997) and A. thaliana, where two nucleolin‐like genes, AtNUC‐L1 and AtNUC‐L2, were reported (Kojima et al., 2007; Petricka and Nelson, 2007; Pontvianne et al., 2007). The nucleolin‐like proteins of plants have the three distinct structural features of animal nucleolin and yeast Nsr1p protein (Ginisty et al., 1999): (a) a highly charged acidic stretch at the amino terminus with characteristic repeats; (b) two RNA Recognition Motifs (RRMs), each of which contains a highly conserved RiboNucleoProtein‐1 (RNP‐1) octamer motif and a less‐conserved RNP‐2 hexamer motif; and (c) a conserved Gly‐ and Arg‐rich carboxy‐terminal sequence, designated the GAR domain. In both animals and yeast the following three regions were identified: the acidic N‐terminal region, involved in interaction with components of the pre‐rRNA‐processing complex and that may control rDNA transcription (Roger et al., 2003); the central region, which contains two or four RRM and which has been implicated in RNA‐binding specificity and aYnity of pre‐rRNA sequences (Serin et al., 1996), and the C‐terminal region or GAR domain, which interacts with several RPs (Bouvet et al., 1998) and also binds RNA but in a non‐specific manner and with low aYnity (Ghisolfi et al., 1992). In plants, it has been demonstrated that the GAR domain of pea nucleolin‐like protein shows DNA helicase activity (Nasirudin et al., 2005). However, the GAR domain seems to be less conserved in the second nucleolin‐like protein in Arabidopsis, AtNUC‐L2 (Pontvianne et al., 2007), suggesting that AtNUC‐L2 lacks this activity.

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In plants, the abundance of the nucleolin‐like protein is lightly regulated, indicating that phytochrome may regulate the expression of this gene (Tong et al., 1997). Furthermore, a correlation of increased nucleolin‐like protein expression with the cell cycle and cell proliferation was demonstrated in pea (Reichler et al., 2001), alfalfa (Bogre et al., 1996) and onion cells (Gonza´lez‐ Camacho and Medina, 2006). More recently, it was shown that in sugar‐ starved cells, induction of an Arabidopsis nucleolin‐like protein 1 occurred with sucrose and/or glucose (Kojima et al., 2007). The activity of nucleolin‐ like protein in plants also seems to be regulated at a post‐transcriptional level. For instance, antibodies against nucleolin‐like protein cross‐reacted with distinct polypeptides in protein extracts from onion (Gonza´lez‐ Camacho and Medina, 2006), alfalfa (Bogre et al., 1996), pea (Tong et al., 1997) and Arabidopsis (Kojima et al., 2007; Pontvianne et al., 2007), which suggests that these distinct polypeptides might play specific roles in controlling the multiple activities of nucleolin‐like protein in plants. In fact, this is similar to nucleolin which in animals shows aberrant migration on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and is highly susceptible to proteolysis (Bouche et al., 1984). Moreover, it has also been shown that in animals the activity of nucleolin is regulated at various levels (reviewed by Srivastava and Pollard, 1999; Tuteja and Tuteja, 1998), including phosphorylation (Morimoto et al., 2002), dimethylation (Lapeyre et al., 1986) and ADP ribosylation (Leitinger and Wesierska‐Gadek, 1993), glycosylation (Carpentier et al., 2005) and translation level (Bicknell et al., 2005; Kim and Srivastava, 2003). Although several potential phosphorylation sites have been identified in the sequences of nucleolin‐like protein from O. sativa, M. sativa and A. thaliana (Pontvianne F. and Saéz‐Va´squez J., unpublished data), these putative phosphosites have not yet been tested experimentally. Consequently, the role of phosphorylation, protein cleavage and/or other protein modifications of nucleolin‐like protein in plants remains uncertain. An exciting breakthrough regarding the function of nucleolin, not only in plants but also in higher eukaryotic organisms, comes from three recent studies in A. thaliana (Kojima et al., 2007; Petricka and Nelson, 2007; Pontvianne et al., 2007). These reports clearly demonstrate that Arabidopsis nucleolin‐like proteins play a major role in growth and plant development. Analysis of three independent Arabidopsis nucleolin‐like mutants displayed reduced growth rate, a prolonged life cycle, pointed leaves, a defective vascular pattern and aberrant leaf venation (Kojima et al., 2007; Petricka and Nelson, 2007; Pontvianne et al., 2007). In addition, it was suggested that PARL (the name given by the authors to Nucleolin in Arabidopsis) might be involved in auxin‐dependent organ growth and patterning (Petricka and

28


Nelson, 2007), potentially indicating a way in which nucleolin may be controlling plant development. At the molecular level, analysis of these nucleolin‐like mutants revealed that the absence of the AtNUC‐L1/PARL protein induces changes in the amount of unprocessed pre‐rRNA at the primary cleavage site (Kojima et al., 2007; Petricka and Nelson, 2007; Pontvianne et al., 2007) and provokes nucleolar disorganization and nucleolus organizer decondensation (Pontvianne et al., 2007). These findings show that there is a direct link between nucleolin, nucleolus structure and rRNA synthesis. Disruption of nucleolin‐like gene in Arabidopsis does not only aVect synthesis of RNA pol I transcribed genes. Indeed, it was also reported that Atnuc‐L1 plants exhibited a significantly reduced sugar‐induced expression of RP genes, suggesting that AtNUC‐L1 might also control RNA pol II transcription (Kojima et al., 2007), as has been shown for nucleolin in animal cells (Huddleson et al., 2006). Since nutrient availability is a prerequisite for ribosome synthesis, cell growth and division (Bailey‐Serres, 1998; Fromont‐ Racine et al., 2003; Warner, 1999), we can expect AtNUC‐L1 to control growth and plant development by coordinating the expression of diverse factors involved in ribosome synthesis, including RPs and rRNA. In this context, an interesting observation in Atnucl‐L1 plants is the induction of the homologous AtNUC‐L2 gene expression, which is usually repressed under normal growing conditions (Pontvianne et al., 2007). Plants, in contrast to animals, contain at least two genes encoding nucleolin‐like proteins, suggesting a putative specialization of this multifunctional protein in response to growth or developmental conditions. AtNUC‐L2 localizes in the nucleolus of Atnucl‐L1 and might rescue, at least partially, the loss of AtNUC‐L1 (Pontvianne et al., 2007). Consequently, activation of AtNUC‐L2 in Arabidopsis mutant plants raises questions about possible regulating mechanisms and the cellular role of AtNUC‐L1 and AtNUC‐L2. Answering these questions should be the next challenge in understanding the role of nucleolin‐like proteins in plants. C. NUCLEOLAR PROTEINS AND PLANT DEVELOPMENT

The earliest reports linking nucleolar activity with plant development come from studies of Arabidopsis plants with mutated genes that encode RPs of the small ribosome subunit. Pointed leaves and growth retardation were observed in plants mutated at the RP loci, RPS13B (Ito et al., 2000) and RPS18A (Van Lijsebettens et al., 1994), encoding two proteins belonging to the class of early ribosome‐associated proteins that assemble on the pre‐ rRNA in the nucleolus (Kressler et al., 1999). Interestingly, pointed leaves

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were also observed in Arabidopsis nucleolin mutants (Kojima et al., 2007; Petricka and Nelson, 2007; Pontvianne et al., 2007), suggesting the existence of a functional relationship between nucleolin and these RPs with early events in ribosome biogenesis. In agreement with this possibility, RPS13 and RPS18 proteins were detected in the proteome of the nucleolus (Pendle et al., 2005) and copurified with nucleolin‐like protein in the NF D complex fraction (Saéz‐Va´squez and Echeverrıá, unpublished results). Although mutations of the RPS13B and RPS18A genes do not seem to have any eVect on plant survival, most likely because RP genes are encoded by only a small family of genes (Barakat et al., 2001), several other nucleolar factors have been shown to be essential in Arabidopsis for gametogenesis (Jiang et al., 2007; Shi et al., 2005), embryogenesis (Fleurdepine et al., 2007; GriYth et al., 2007; Lahmy et al., 2004) and central cell and endosperm development (Portereiko et al., 2006). The best characterized genes are the SLOW WALKER1 (SWA1) and TORMOZ (TOZ) genes, both of which encode WD40 proteins (GriYth et al., 2007; Shi et al., 2005). The closest homologues of the SWA1 and the TOZ proteins are the yeast Utp15 and Utp13. These two yeast proteins were identified as being part of a large nucleolar U3 ribonucleoprotein complex required for 18S rRNA synthesis (Dragon et al., 2002). The role of these factors in processing 18S ribosomal rRNA synthesis was confirmed at least for SWA1 (Shi et al., 2005). The SYN3 gene, which encodes for a nucleolar cohesin protein, is also essential for megagametogenesis in Arabidopsis (Jiang et al., 2007). Even though its role in the nucleolus needs to be functionally demonstrated, SYN3 protein may be involved in rDNA chromatin structure, transcription and/or pre‐rRNA processing (Jiang et al., 2007). Another factor involved in plant development is the Arabidopsis La protein 1. Interestingly, the AtLa1 protein localizes in the nucleolus only in cells displaying nucleolar cavities or vacuoles. Thus, AtLa1 is the first protein known so far that accumulates in this subnucleolar compartment that seems to be plant specific. AtLa1 it is not exclusively a nucleolar protein; it localizes predominantly in the plant nucleoplasm but is excluded from the Cajal bodies (Fleurdepine et al., 2007—S. Fleurde´pine and C. Bousquet‐Antonelli, unpublished results). In yeast, the AtLa1 protein was shown to be able to fulfil the functions of genuine La protein in RNA pol III primary transcript stabilization and processing, as well as in maturation and assembly of some non‐coding pol II precursors such as sn‐ or snoRNAs. Furthermore, the AtLa1 protein has been demonstrated to bind RNA Pol III primary transcripts in planta. The AtLa1 defective homozygous plants display an early embryonic lethal arrest together with nucleolar hypertrophy (Fleurdepine et al., 2007). The reason for this observed altered nucleolus size in plants with

30


a deficient AtLa1 function remains unaddressed, but it is tempting to speculate that it may be linked with rRNA synthesis. Indeed, the human La (hLa) protein was shown to interact with nucleolin in the DFC, suggesting a role of human La in the early phase of rRNA biogenesis (Intine et al., 2004). However, additional experimental analysis will be required to determine whether or not AtLa1 interacts with AtNUC‐L1 in the plant nucleolus and if the AtLa1 defective homozygous plants display changes in the rate of transcription and processing of pre‐rRNA. An association between plant development and nucleolus hypertrophy was also observed in plants containing a T‐DNA insertion in the DOMINO locus of Arabidopsis (Lahmy et al., 2004). DOMINO is a nuclear protein containing some features of nucleolar protein including an acidic region and a Gly/ Arg‐rich C‐terminal extension of a GAR‐like domain. The dom1 mutant plants display a larger size and a compacted nucleolus composed mainly of DFC (Lahmy et al., 2004). The role of DOMINO has not been demonstrated yet; however, DOMINO is very similar to a chloroplast DCL protein of tomato which is required for chloroplast rRNA processing and correct ribosome assembly (Bellaoui and Gruissem, 2004). Enlarged or reduced nucleoli were also observed in titan (Liu Cm et al., 2002), pilz (Steinborn et al., 2002) or fem111 (Portereiko et al., 2006) mutants. TITAN and PILZ genes encode condensin and cohesin proteins involved in chromosome formation (Liu Cm et al., 2002) and in the tubulin‐folding cofactor required for the organization of microtubules during cell division (Steinborn et al., 2002). The fem111 mutant, on the contrary, has an insertion in the Agamous‐like80 (AGL80) gene which encodes a type 1 MADS domain containing protein that is required for central cell development and function. Thus, all three of these proteins can be linked in one way or another to nucleolus activity since ribosome synthesis is dependent on cell development and/or division (Dez and Tollervey, 2004). Finally, an analysis of the nucleolar proteome (http://bioinf.scri.sari.ac.uk/ cgi-bin/atnopdb/home) and the seedgenes project (www.seedgenes.org/) of Arabidopsis reveals that at least nine embryo defective mutants correspond to gene mutations encoding nucleolar proteins. From this analysis, it is important to mention that three genes encode for RPs of the large ribosome subunit: RPL19 (EMB 2386), RPL3 (EMB 2207) and RPL8 (EMB 2296). In contrast to RPS13B and RPS18A plant mutants which are viable, in all three EMB mutants, embryo development was arrested at a globular embryonic stage as described for the aml1 (Arabidopsis Minute like‐1) mutant (Weijers et al., 2001). The aml1 mutant has an insertion in the RPS5A gene, one of the two genes encoding for the RP subunit RPS5, which causes semi‐ dominant development phenotypes in the heterozygous mutant. However, in

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the homozygous mutant, development is completely arrested at an early stage of development (Weijers et al., 2001). RPS5A and RPS5B are diVerentially expressed during embryogenesis and in the diVerent tissues and organs these expressions do not overlap. Consequently, one functional gene copy per diploid genome might be insuYcient for complete gene function. This closely resembles the situation in Drosophila Minute mutants, where for each RP gene only one functional gene is present in the genome. The RPL1, RPL3 and RPL8 are also encoded by a small family of genes, but it remains to be determined whether or not these genes are also expressed diVerentially. However, it is also possible that these RP proteins (and probably other nucleolar proteins belonging to small gene families) may possess additional functions to that of being a structural component of the ribosome, and consequently disruption of the gene may aVect a specific and essential nucleolar cellular function. A summary of the genes from A. thaliana which have been mentioned above as encoding nuclear and/or nucleolar proteins potentially participating in pre‐rRNA synthesis and processing and ribosome assembly is shown in Table I.

IV. PERSPECTIVES The history of nucleolus research is already long and it contains many outstanding milestones in the form of cytological, genetic and molecular findings of undoubted relevance, not only for basic life sciences but also for applied sciences such as medicine, agriculture and food technology. However, the complexity of the organelle and of the cellular functions located in it is high enough to conclude that there is still much that we do not know and that many lessons still remain hidden in the nucleolus to be uncovered and learned on fundamental aspects of gene expression and its regulation, macromolecular interactions, expression of molecular events into cellular discernible structures, relationships between cellular growth and cell proliferation and many others. We could conclude this chapter indicating some of the problems that, in our opinion, could be more relevant for nucleolar research in the coming years. Certainly, the list is neither exhaustive nor unquestionable. Whereas the discovery of nucleolar functions not linked to ribosome biogenesis (the plurifunctional nucleolus) has attracted the attention of many researchers, little is still known on the nucleolar structures associated with these novel functions, in other words, on the structural organization of these functions, actually relevant for the cellular physiology. An exciting new

TABLE I Reported Arabidopsis Thaliana Genes Encoding Nuclear and/or Nucleolar Proteins with Potential Function in rRNA Synthesis or Ribosome Biogenesis Protein gene

AGI number

Similarity

Function

AtFib1

At5g52470

Fibrillarin/Nop1p

rRNA methylation

AtFib2 AtNAP57 AtNAF1 AtPes AtNUC‐L1/PARL1

At4g25630 At3g57150 At1g03530 At5g14520 At1g48920

Fibrillarin/Nop1p CBF5/NAP57 NAF1 Pescadillo/Nop7p Nucleolin/Nsr1p

rRNA methylation Pseudourydilation H/ACA assembly 25S rRNA synthesis Ribosome biogenesis

AtNUC‐L2

At3g18610

Nucleolin/Nsr1p

Ribosome biogenesis

TOZ SWA1 EDA14/UTP11 AtRTL2 AtLa1 DOMINO1 TITAN/AtSM2 PILZ/POR AGLM80/FEM111

At5g16750 At2g47990 At3g60360 At3g20420 At4g32720 At5g62440 At3g47460 At4g39920 At5g48670

Utp15‐like protein Utp13‐like protein Utp11‐like protein RNaseIII La‐like proteins DCL Condensin‐like protein Tubulin‐folding cofactor MADS domain protein

18S rRNA synthesis 18S rRNA synthesis 18S rRNA synthesis 30 ET pre‐rRNA processing RNA synthesis Nucleolus size Nucleolus size Nucleolus size Nucleolus size

References Barneche et al., 2000; Pih et al., 2000 Barneche et al., 2000 Maceluch et al., 2001 Lermontova et al., 2007 Zografidis et al., 2007 Pontvianne et al., 2007; Kojima et al., 2007; Petricka and Nelson, 2007 Pontvianne et al., 2007; Kojima et al., 2007 GriYth et al., 2007 Shi et al., 2005 Pagnussat et al., 2005 Comella et al., 2008 Fleurdepine et al., 2007 Lahmy et al., 2004 Liu Cm et al., 2002 Steinborn et al., 2002 Portereiko et al., 2006

SYN3

At3g59550

Cohesin‐like protein

PFL1

At1g22780

RPS18A

PFL2

At4g00100

RPS13B

AtRPS5/AML1

At3g11940

RPS5A

AtA

At5g67380

CK2 subunit

AtB

At3g50000

CK2 subunit

AtC At1 At2 At3

At2g23080 At5g47080 At4g17640 At3g60250

CK2 subunit CK2 subunit CK2 subunit CK2 subunit

Chromatin structure, synthesis of rRNA? Small subunit ribosomal protein Small subunit ribosomal protein Small subunit ribosomal protein Casein kinase 2 Phosphorylation of transcription and pre‐RNA processing factors?

Jiang et al., 2007 Van Lijsebettens et al., 1994 Ito et al., 2000 Weijers et al., 2001 Saéz‐Va´squez et al., 2001; Salinas et al., 2006

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finding is that at least some of these functions might lay in novel nucleolar structural domains. The unequivocal relationship of ribosome biogenesis with cell growth and proliferation makes this organelle a very eYcient marker of these processes and opens the possibility of exploitation of this potentiality, mostly in studies of cellular stress caused by either biotic agents or abiotic agents. Although it aVects mammalian cells and not plant cells, it would be worth mentioning here the findings on the involvement of the nucleolus in the p53 pathways. Specifically in plants, novel lines of research have shown the relationship of the nucleolus with the gravitropic response and the gravitational stress, in the context of the eVects of this stress on cell growth and proliferation. The intriguing behaviour of the nucleolus during mitosis has still many unresolved points, such as the mechanisms of interaction of proteins with pre‐rRNA in mitotic structures. Gene silencing processes aVecting rDNA are not yet well understood, mainly concerning the selection criteria of particular NORs or subsets of rRNA genes to be silenced. Finally, regarding nucleolar proteins, the discrimination of the nucleolar proteome of Arabidopsis, a fundamental milestone in the research field, is not totally resolved as to the functional roles played by the identified proteins there are several proteins with still unknown functions either in Arabidopsis or other eukaryotes. What is the role of these proteins in ribosome biogenesis, or in plant growth and development? Characterization studies should provide insights into the function of these proteins not only in Arabidopsis nucleolus but also in animals. Indeed, the study of molecular mechanisms involved in ribosome biogenesis in plant systems has already proven to be an excellent system in higher eukaryotic organisms. The large number of Arabidopsis plant mutants, the development of RNAi technologies to silence one or more homologous genes and the availability of both in vitro transcription and in the pre‐rRNA‐processing system should certainly provide new insight into the nucleolar function in plants in the coming years.

ACKNOWLEDGMENTS The courtesy of Dr. Pedro Esponda (CIB‐CSIC, Madrid, Spain) in providing the electron microscopical images of Fig. 3 is gratefully acknowledged. Thanks to Dr. M. Devic and Dr. Cećile Bousquet‐Antonelli (LGDP, Perpignan, France) for critical reading of this manuscript, and Dr. T. Roscoe and Mr. R. V. Chiverton for checking the English style. Works performed in

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the authors’ laboratories were supported by the Centre National de la Recherche Scientifique (CNRS) by a grant from PAI ‘‘Programme d’Actions Integreés franco‐espagnol’’, Picasso 11398VF (French team) and HF 2005‐ 0209 (Spanish team) and by the Spanish ‘‘Plan Nacional de IþDþi’’ (ESP2006‐13600‐C02‐02).

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Expansins in Plant Development

DONGSU CHOI,*,1 JEONG HOE KIM,{,1 AND YI LEE{

*School of Science and Technology, Kunsan National University, Gunsan 573‐701, Republic of Korea { Department of Biology, Kyungpook National University, Daegu 702‐701, Republic of Korea { Department of Industrial Plant Science and Technology, Chungbuk National University, Cheongju 361‐763, Republic of Korea [email protected]

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The Expansin Superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Nomenclature of Expansin Genes ......................................... B. Common Structural Features of Expansin Protein ..................... C. Expansin A .................................................................... D. Expansin B .................................................................... E. Expansin‐Like A.............................................................. F. Expansin‐Like B .............................................................. G. Expansin‐Like X.............................................................. III. Evolution of Expansin Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Origin of Expansin Genes ................................................... B. Expansin Gene Evolution ................................................... IV. Biochemical and Biophysical Properties of Expansins. . . . . . . . . . . . . . . . . . . . A. Cellular Localization......................................................... B. Three‐Dimensional Structure ............................................... C. Action Mechanism ........................................................... D. Cell Wall‐Binding Properties ............................................... E. The Controversial Hydrolytic Activity of the Pollen‐Type EXPBs... V. Expansins in Vegetative Growth and Development . . . . . . . . . . . . . . . . . . . . . . A. Seedling Growth ..............................................................

48 50 50 51 51 52 55 55 56 57 57 57 59 59 59 61 61 62 63 63

1

Equal contribution.


0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(08)00002-5

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B. C. D. E. F.

Leaf Development ............................................................ Elongation of Stems and Petioles .......................................... Root Growth .................................................................. Root Hair Development..................................................... Limited Correlation of Expansin Action with Plant Vegetative Growth ........................................................... G. Differentiation of Xylem Tissues........................................... H. Growth‐Related Environmental Changes ................................ I. Seed Germination ............................................................ VI. Expansins in Reproductive Growth and Development . . . . . . . . . . . . . . . . . . . A. Male Gametophytic Development ......................................... B. Seed Formation and Embryogenesis ...................................... C. Floral Development .......................................................... VII. Expansins in Fruit Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Fruits and Cell Wall Proteins............................................... B. Expansins in Fruit Development........................................... C. Expansins and Hormones Other than Ethylene ......................... D. Differential Expression of Expansin Genes in Fruit Development ... VIII. Future Prospects: Further Understanding of Expansin Function and Agricultural Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Expansins are a class of cell wall proteins that mediate pH‐dependent wall loosening, probably by disrupting hydrogen bonds between cellulose and matrix glycans. Expansins are distributed primarily over the expanding cell wall and bind to the cell wall. Expansin proteins share the common structural features of protein: a signal peptide for secretion to the cell wall and two distinct domains. Domain 1 contains the conserved Cys residues and HFD (His‐Phe‐Asp) motif while domain 2 has the conserved Trp residues. Crystal structure of an expansin B (EXPB) protein suggests that the latter domain may participate in binding to polysaccharides. Expansins are encoded by the four gene families in vascular plants: expansin A (EXPA), expansin B (EXPB), expansin‐like A (EXLA), and expansin‐like B (EXLB). It appears that the moss Physcomitrella patens lacks expansin genes that belong to EXLA and EXLB families. Based on intron position and gene clusters, all the families appear to originate from a common ancestor. Biological functions of expansins are considered in diverse aspects of plant growth and development: shoot and root development, shoot and root elongation, leaf morphogenesis, floral organ development, fruit development, embryogenesis, pollen development, and pollen tube growth. Expression of expansin genes in the processes is regulated by hormones, partly contributing to adaptation of plants in response to environmental stimuli and partly to morphological development of plants.

I. INTRODUCTION Although strength and rigidity of the plant cell wall is important for mechanical support of plant tissues, it also constrains expansion of the cell it encloses. To accommodate cell expansion and, thus, induce plant growth,

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cells should overcome the constraints imposed by the rigid cell wall. This means that plant cell expansion should be preceded by changes in cell wall extensibility. Robert Cleland and his colleagues found that the extensibility of cell walls from growing tissues was sustained in low pH buVers ranging from 5.5 to 4.0, but not in neutral or alkaline solution; these findings led them to formulate the ‘acid growth theory’ of plant cells (Rayle et al., 1970). Later, by using the ‘‘creep’’ test, Cosgrove (1989) demonstrated that the factor(s) involved in acid‐induced wall extension would be proteinaceous because the creep activity induced by acid buVer is completely abolished by heat inactivation and protease treatment. Proteins responsible for acid‐induced wall extension were discovered in cell walls from cucumber and named expansins (McQueen‐Mason et al., 1992). The first or EXPA proteins from cucumber hypocotyls, CsEXP1 and CsEXP2, met the criteria to be the cell wall‐loosening factor, as predicted. First, the proteins were able to restore the extensibility of the heat‐inactivated wall specimen, and their optimal pH for the action was acidic, lower than pH 5.5, which is compatible with the acid growth theory. Second, the cucumber expansin proteins were also active in inducing the creep of heat‐inactivated wall specimens from diverse plants, such as tomato, radish, and pea, indicating that their action is ubiquitous and conserved in plants, as is the acid growth. However, it should be mentioned that although expansins are necessary and suYcient for in vitro wall extensibility, they alone may not be suYcient for sustaining cell wall extension in growing plant tissues. For instance, cell walls should recruit new wall materials to accommodate expansion, making cell wall biosynthetic enzymes indispensable to plant growth. Hydrolytic enzymes in the wall, such as xylose endotransglycosylases and pectin esterases, are also important to modify wall polymers, and thus reconstruct the wall for growth. Nevertheless, these enzymes are not responsible for acid‐induced wall extension (McQueen‐Mason et al., 1993; McQueen‐Mason and Cosgrove, 1995). To date, only expansin proteins are known to meet the requirements for acid‐induced cell wall extension, as formulated in the cell expansion model of McQueen‐Mason et al. (2007). Expansins have been found in all the plants examined, and they are encoded by multiple genes that make up the expansin superfamily. In this review, we first discuss expansin classification, origin, and evolution, including the recent findings of expansin genes in the basal land plant, the moss (Physcomitrella patens). We also discuss the action mode of expansins, which still remains speculative, but the recent determination of crystal structure of an EXPB protein gives some important insights into its binding site to glycans and into the debated hydrolytic activity. During the 15 years since the first discovery of expansins, numerous reports have presented evidence

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that expansins are also important in regulating many aspects of plant growth and development. This chapter will be comprehensive especially with respect to those various biological roles of expansins, highlighting recent progress. The recent review article by McQueen‐Mason et al. (2007) can be read for an excellent briefing of the earlier history in this field as well as for a concise summary of biological roles of expansins. Readers can also consult other recent reviews for extensive analysis of the phylogenetic relationship between expansins (Sampedro and Cosgrove, 2005) and for expansins in hormones action (Cho and Cosgrove, 2004).

II. THE EXPANSIN SUPERFAMILY Expansins were first identified as a cell wall‐loosening factor that promotes acid‐induced cell wall extension (Li et al., 1993; McQueen‐Mason et al., 1992), and were subsequently found to be encoded by a number of genes that were classified as the ‐expansin family (Shcherban et al., 1995). Based on amino acid sequence similarity to the ‐expansins, another group of genes was defined and later shown to have cell wall‐loosening activity. This second group of expansins, the ‐expansins, includes genes for group‐1 pollen allergens. Recently, the ‐ and ‐expansin families have been renamed expansin A (EXPA) and expansin B (EXPB), respectively (Kende et al., 2004). The availability of the full genomes of Arabidopsis and rice enabled the identification of another two gene families, the expansin‐like A (EXLA) and expansin-like B (EXLB). Their deduced amino-acid sequences are related to those of EXPA and EXPB. Until now, however, no biological or biochemical function has yet been assigned to EXLA and EXLB (McQueen‐Mason et al., 2007). In addition, other expansin‐like genes that are remotely related to expansin genes were found both inside and outside the plant kingdom, and, according to the current nomenclature (Kende et al., 2004), we categorize them in the expansin‐like X (EXLX) family. A. NOMENCLATURE OF EXPANSIN GENES

Before 2004, there were no guidelines set for naming expansin genes. Researchers named in their own way the expansin genes they found, such that some genes were referred to by diVerent names. To prevent confusion, the expansin community needed an agreement on the rules for naming expansin genes and proteins. The chosen system basically follows the conventions established by Meinke and Koornneef (1997) for Arabidopsis (Kende et al., 2004). The website http://www.bio.psu.edu/expansins/,


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currently maintained by Daniel Cosgrove, contains information about names of expansin genes. In this chapter, we follow the current nomenclature, but we use the names endowed by individual researchers before 2004 as long as there is no confusion.

B. COMMON STRUCTURAL FEATURES OF EXPANSIN PROTEIN

Expansins are small proteins consisting of 250–270 amino acid residues and can be divided into three regions (Fig. 1). First, the N‐terminal signal peptide is composed of 20–30 amino acid residues that direct proteins to the cell wall. When analysed by the PSORT program (Nakai and Kanehisa, 1992), all the expansins tested have a signal peptide in the N‐terminus, indicating that they are to be secreted to the cell wall (Lee and Kende, 2002). Second, domain 1, located next to the signal peptide, consists of 120–135 amino acid residues and contains a series of the conserved Cys residues and HFD (His‐Phe‐Asp) motif, the hallmark of expansin. This domain is also related to the catalytic domain of glycoside hydrolase family 45 (GH45). Third, domain 2 is composed of 90–120 amino acid residues and contains the conserved Trp residues, forming a putative cellulose‐binding domain (Sampedro and Cosgrove, 2005). After truncation of the signal peptide, the molecular mass of the mature expansin protein is about 25–28 kD (Cosgrove et al., 2002).

C. EXPANSIN A

This group of proteins was the first to be recognized as exhibiting the so‐called creep activity of the cell wall specimen secured by an extensometer (Cho and Kende, 1997a; McQueen‐Mason et al., 1992). EXPA has served as a prototype in studies for biochemical activities and structural features of diVerent types of expansins. EXPA has all the common structural features, as mentioned above, some of which, for example, the HFD motif, are missing in other families (see below). EXPAs form the largest family in the expansin superfamily in most plants, and most of the studies on expansins have focused on EXPAs (Table I; Choi et al., 2006; Li et al., 2002; Sampedro et al., 2005). It is notable that unlike other families of expansins, the sizes of EXPA gene families present across various taxa are comparable to each other, which may be a manifestation of a vital role of the gene family size in expansin function (Carey and Cosgrove, 2007).

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Fig. 1. Comparisons of the deduced amino acid sequences of expansin superfamily genes from Arabidopsis and rice. The amino acid sequences deduced from cDNA or genomic DNA sequences are aligned. Positions of the highly conserved sequences, cysteins, HFD (His‐Phe‐Asp) domain, and tryptophans are indicated. Signal peptide sequences were determined by the PSORT program (Nakai and Kanehisa, 1992) and are represented in lower case. Conserved intron positions are indicated by arrows. D. EXPANSIN B

Cosgrove et al. (1997) recognized structural similarities between EXPA and the group‐1 allergens of grass pollen, and demonstrated that maize pollen extract induces extension and stress relaxation of maize silk and wheat coleoptile cell walls. The group‐1 pollen allergens have been placed in the EXPB family. This new type of expansin barely increases the extensibility of

TABLE I Number of Expansin Genes from Plant Species Species Arabidopsis thaliana Oryza sativa Populus tricocarpa Physcomitrella patens

EXPA

EXPB

EXLA

EXLB

Total

26 33 27 27

6 19 3 7

3 4 2 0

1 1 4 0

36 57 36 34

EXPB, expansin B; EXPA, expansin A; EXLA, expansin‐like A; EXLB, expansin‐like B.

Data source

Reference

Complete genome Complete genome Incomplete genome Incomplete genome

Sampedro et al., 2005 Sampedro et al., 2005 Sampedro et al., 2006 Carey and Cosgrove, 2007

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cucumber hypocotyl cell walls, thus leading to the notion that EXPB may act selectively in monocot cell walls, whereas EXPA is active in dicot cell walls (Cho and Kende, 1997a; McQueen‐Mason et al., 1992). Many EXPB cDNAs have been abundantly catalogued in the Expressed Sequence Tag (EST) collections established from the monocot plants, maize and rice (Cosgrove et al., 1997; Cosgrove, 2000a). Because grass cell walls, unlike those of dicots, are notably rich in glucuronoarabinoxylans and ‐(1!3),(1!4)‐D‐glucans (Carpita, 1996), these polysaccharides might be potential binding targets for EXPBs (Yennawar et al., 2006). It is notable that most EXPBs are predicted to have N‐linked glycosylation sites near the amino and carboxyl termini whereas EXPAs do not (Fig. 2A; Downes et al., 2001; Lee and Kende, 2001; Wu et al., 2001). The EXPB family is composed of 6 genes from Arabidopsis, 19 from rice, 3 from Populus, and 7 from Physcomitrella (Table I; Carey and Cosgrove, 2007; Choi et al., 2006; Li et al., 2002; Sampedro et al., 2005), suggesting that the EXPB family is usually larger in grasses than in other groups of plants. EXPBs could be further separated into group‐1 pollen allergens and vegetative EXPBs. The group‐1 pollen allergens are specifically expressed in pollen and probably have a unique wall‐loosening activity

Fig. 2. Schematic structures and phylogenetic tree of expansin families. (A) Schematic structures of expansin genes and proteins. Positions of the conserved amino acid residues, C (Cys), W (Trp), HFD (His‐Phe‐Asp) motif, and N (N‐linked glycosylation site) are indicated. The positions and names of conserved introns are indicated by upward arrows. (B) Brief phylogenetic relationship between the four expansin families and EXLX (expansin‐like X).


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associated with pollen function, whereas the vegetative EXPB genes are widely expressed during the growth and development of vegetative tissues (Kerim et al., 2003; Lee and Kende, 2001; Schipper et al., 2002; Wu et al., 2001). The role of EXPBs in vegetative tissues is not clear yet. It has been shown that the expression of some EXPB genes is rapidly induced during Gibberellic Acid‐induced internodal growth in deepwater rice and is dramatically increased by wound treatment (Lee and Kende, 2001). E. EXPANSIN‐LIKE A

This gene family was identified only from the amino acid sequence similarity with EXPA and EXPB proteins (Lee et al., 2001; Li et al., 2002). EXLA proteins have a very high amino acid identity among one another (average 84% and 73% from Arabidopsis and rice, respectively). EXLAs have conserved Cys and Trp residues in the N‐ and C‐terminal regions, respectively. Unlike the EXPAs and EXPBs, however, EXLAs have no HFD motif in domain 1, and the positions of the conserved Trp residues of domain 2 are diVerent from those of EXPAs and EXPBs. Interestingly, EXLAs have additional conserved Trp residues in the C‐terminal end and additional conserved Cys residues in both N‐ and C‐terminal ends (Fig. 1; Fig. 2A; Choi et al., 2006). The EXLA family is composed of three genes in Arabidopsis, four in rice, and two in Populus, but no gene member was found in Physcomitrella (Table I). The amino acid sequences of rice EXLA genes show significant amino acid identity with EXPA and EXPB, although some of the characteristic motifs of EXPA and EXPB are missing. Gene expression patterns of EXLA are very diVerent from those of EXPA and EXPB of rice. Usually, EXPA and EXPB genes are highly expressed from the growing regions of internodes and leaves, and are induced by GA treatment. On the contrary, OsEXLA1 shows highest expression in the non‐growing regions of the internodes and leaves, and expression of OsEXLA1 and OsEXLA2 genes was reduced by GA treatment (Lee and Kende, 2002). These results indicate that the EXLA proteins do not act in GA‐regulated stem elongation of rice and that their biological function in the tissue may be diVerent from that of EXPA and EXPB proteins (Lee and Kende, 2002). F. EXPANSIN‐LIKE B

This gene family was also identified only from the amino acid sequence similarity to EXPA and EXPB proteins (Lee et al., 2001; Li et al., 2002). EXLBs have conserved Cys residues in the N‐terminus but only one

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conserved Trp residue in domain 2. They also lack the HFD motif in the domain 1, as do EXLAs (Fig. 1; Choi et al., 2006). Only one EXLB gene was found in rice and Arabidopsis, four in Populus, and none in Physcomitrella (Table I). There has been no evidence to date that EXLB genes are actively expressed in plants. Lee and Kende (2002) tested whether or not the only EXLB gene in rice, OsEXLB1, is expressed in various tissues, such as internodes, leaves, coleoptiles, and roots; they confirmed the lack of expression signal by RNA gel blot analysis. G. EXPANSIN‐LIKE X

A group of expansin‐like proteins, later classified as the EXLX family, was found in a slime mould, Dictyostelium discoideum. Molecular phylogenetic analysis suggests that D. discoideum is situated somewhere at the foot of the division between Animalia and Fungi (Baldauf et al., 2000). This slime mould has a cellulosic cell wall similar to that of plants. Although cellulose is synthesized by a range of organisms, only limited taxa, including algae, land plants, and D. discoideum, use cellulose to construct a strong cell wall. The EXLX family of D. discoidium (DdEXLX), which comprises at least six members, shows significant similarities to plant expansins (e.g., 19% amino acid sequence identity and 30% amino acid sequence similarity between DdEXPL6 and CsEXP1). The deduced amino acid sequence of DdEXLX genes shares the common characteristic features of plant expansins, such as a signal peptide, conserved Cys residues in the N‐terminal region, the expansin signature motif HFD, as well as several conserved aromatic residues in the C‐terminal half, some of which have been proposed to form a cellulose‐binding domain (Darley et al., 2003). Expansin‐like X proteins were also found in the parasitic potato cyst nematode Globodera rostochiensis (Kudla et al., 2005; Qin et al., 2004). GrEXP1 encodes a protein of 271 amino acids that has a signal peptide in the N‐terminus. Similarity searches revealed that the mature EXLX proteins contain two distinct regions: domain 1 from amino acid residues 26 to 118 shows significant identity with the carbohydrate‐binding module of various nematode species, and domain 2 from 150 to 271 with EXPBs of tobacco and Arabidopsis. Local alignment of domain 2 with those EXPBs reveals that GrEXP1 shares the conserved Cys residues, HFD motif, and other conserved residues with the plant EXPB proteins (Qin et al., 2004). Strictly speaking, these proteins cannot be called expansin proteins, even though they exhibit plant cell wall‐loosening activity; expansin research society decided not to classify a non‐plant protein as an expansin simply because it has cell wall‐loosening activity and shares sequence similarity with expansins (Kende et al., 2004).


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In addition to the EXLX proteins mentioned above, numerous proteins that have sequence similarity to expansin were found in many non‐plant organisms, such as bacteria (Nembaware et al., 2004), fungi (Li et al., 2002; Saloheimo et al., 2002), and mussels (Xu et al., 2001). All those genes could be designated EXLX (Kende et al., 2004).

III. EVOLUTION OF EXPANSIN GENES A. ORIGIN OF EXPANSIN GENES

In plants, there are secreted proteins that have a region homologous only to the domain 1 or domain 2 of expansins. Group‐2 pollen allergens have the signal peptide for secretion and show similarity to domain 2. They appear to have evolved from a truncated EXPB because they show about 35–45% amino acid identity with the domain 2 of the closest related EXPB (Sampedro and Cosgrove, 2005). Plant natriuretic peptide (PNP), which is sometimes called p12 and abundant in xylem of blighted citrus trees (Ceccardi et al., 1998), have signal peptide for secretion and show similarity to the domain 1, and also have the introns A and B that are conserved in expansin genes (Sampedro and Cosgrove, 2005). Another distantly related protein is the barley wound‐ induced (bawin) protein. This protein has a signal peptide for secretion, similarity to the domain 1, and the conserved intron C of expansin (Sampedro and Cosgrove, 2005). The sequence similarities and conserved gene structure suggest that all of these proteins may have evolved from the same ancestor as expansins. The function of these proteins in plants is unknown. Expansin genes might have originated from an ancestral gene that encodes a signal peptide for secretion, a GH45‐like hydrolytic domain, and cellulose‐ binding domains. After losing the capability of hydrolytic activity, the ancestral gene may have evolved into two groups of genes, one encoding both domains as in the case of expansins and the other encoding only one of the two domains (Ludidi et al., 2002). Each domain of expansin may have an important role in plant survival. Understanding the exact functions of expansin proteins may enable us to appreciate the biological role of each domain in the plant kingdom. B. EXPANSIN GENE EVOLUTION

Expansin genes have been found in all the land plants examined. All the four expansin families, EXPA, EXPB, EXLA, and EXLB, are present in the gymnosperm Pinus taeda (Sampedro and Cosgrove, 2005). EXPA genes

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have been cloned in the semi‐aquatic ferns Marsilea quadrifolia and Regnelidium diphyllum (Kim et al., 2000). Furthermore, the moss P. patens has a number of EXPA and EXPB genes, but no EXLA or EXLB (Table I; Carey and Cosgrove, 2007; Schipper et al., 2002). However, the attempt to find any expansin gene from Chara has not been successful (Lee et al., 2001). We could not find any expansin genes from the fully sequenced genome of Chlamydomonas reinhardtii and Ostreococcus lucimarinus. Therefore, P. patens may be the phylogenetically lowest plant species that has expansin genes at this point. The deduced amino acid sequence of the expansin genes from the ferns and mosses already shows a high degree of identity with those of seed plants, indicating that expansin genes have been highly conserved during the evolution of vascular plants. Therefore, it is not now possible to define when expansins appeared in the evolutionary course of the plant kingdom. We only know that the EXPA and EXPB families already existed by the time the vascular plants and mosses diverged, and that the EXLA and EXLB families can be traced back only to the common ancestor of angiosperms and gymnosperms (Li et al., 2002; Sampedro and Cosgrove, 2005; Schipper et al., 2002). Angiosperm expansin genes have been continuously duplicated independently since the divergence of dicot and monocot plants, resulting in independent gene clusters in each subdivision. Sampedro et al. (2005) reconstructed the lineage of expansin gene families by gene structure and sequence alignment. Both dicot (including Arabidopsis) and monocot (including rice) plants share 17 independent common clades. This means that there might be at least 17 diVerent ancestral groups of genes before the diversification of those two subdivisions of Magnoliophyte. Evidence for the event of recent gene duplication can be easily recognized from the tandemly repeated expansin genes in rice and Arabidopsis genomes. The tandemly arrayed genes have the same gene orientation in the genome and the same gene structure with respect to intron distribution, and they are phylogenetically very close (Sampedro et al., 2005). As the EXPB families comprise 19 genes in rice and 6 genes in Arabidopsis (Table I), it is conceivable that the EXPB family grew significantly faster in grasses than in dicots. Comparative analysis of gene structure helped define the evolutionary relationship between gene families as well as between individual genes (Lee et al., 2001; Li et al., 2002; Sampedro et al., 2005). Expansin genes have conserved the four introns, that is, A, B, C, and F. The conserved introns are located in exactly the same position as the aligned amino acid sequences, and have the same intron phases (the intron phase represents the position of the intron within the codon; Fig. 2A; Lee et al., 2001; Sampedro et al., 2005). Although the EXPA family genes contain only introns A and B, the EXPB,


59

EXLA, and EXLB family genes have all four conserved introns. Together with the phylogenetic analysis of amino acid sequence, the analysis of gene structure strongly supports the hypothesis that the four expansin families have evolved from a common ancestor (Fig. 2B; Lee et al., 2001; Li et al., 2002; Sampedro et al., 2005).

IV. BIOCHEMICAL AND BIOPHYSICAL PROPERTIES OF EXPANSINS A. CELLULAR LOCALIZATION

Expansin proteins have a signal peptide sequence and, thus, are predicted to be targeted to the cell wall (Lee et al., 2001; Lee and Kende, 2002). By employing immunogold electron microscopy with anti‐cucumber EXPA antibodies, Cosgrove et al. (2002) found that, indeed, EXPA proteins were mainly localized in cell walls of epidermal cells of cucumber hypocotyls and maize coleoptiles. Balestrini et al. (2005) showed that EXPA proteins are colocalized with cellulose and diVerentially localized to the cell walls during the accommodation of root cells to an arbuscular mycorrhizal fungus. Immunohistochemistry and immunogold electron microscopy studies showed that OsEXPB3, an EXPB of rice, was also localized to the cell walls from the elongating region of roots, specifically to the primary cell walls (Fig. 3; Lee and Choi, 2005). They also indicated that the anti‐ OsEXPB3 serum binds to OsEXPB3 protein specifically. The antibody did not recognize other members of the same subfamily (Lee and Choi, 2005). These results confirm that both EXPA and EXPB are secreted into the cell wall, as predicted. B. THREE‐DIMENSIONAL STRUCTURE

The low solubility, low abundance, and unsuccessful production of recombinant expansins have hampered the study of three‐dimensional structure of expansin proteins by X‐ray crystallography. Recently, however, the protein structure of ZmEXPB1 (Zea m 1), a group‐1 pollen allergen of maize, has been resolved (Yennawar et al., 2006). This breakthrough was possible because ZmEXPB1 proteins were produced in copious amounts by grass pollen so that they could be easily extracted, purified, and concentrated to high levels without further precipitation. According to the crystal structure of ZmEXPB1, its two major domains 1 and 2 closely packed and aligned so as to form a long, shallow groove between them, shaping a potential binding

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Fig. 3. Immunocytochemical localization of OsEXPB3 in rice seedlings by electron microscopy and immunohistochemical localization by light microscopy. (A) Coleoptile section probed with antibody against OsEXPB3 (1:500); (B) coleoptile section probed with pre‐immune serum (1:500); (C) root section probed with antibody against OsEXPB3 (1:500); (D) root section probed with pre‐immune serum (1:500). All sections were subsequently probed with gold‐conjugated goat anti‐rabbit


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site to glycan. The structure of domain 1 resembles that of the family‐45 glycoside hydrolase, and domain 2 is an immunoglobulin‐like ‐sandwich with aromatic and polar residues that form a potential surface for polysaccharide binding (Yennawar et al., 2006). C. ACTION MECHANISM

McQueen‐Mason and Cosgrove (1995) proposed that expansin proteins might exert a cell wall‐loosening eVect by binding to cellulose microfibrils and thus disrupting the hydrogen bonds between the cellulose microfibrils and hemicellulose. The hypothesis is supported by the observations of several researchers (McQueen‐Mason et al., 1992, 1993; McQueen‐Mason and Cosgrove, 1994, 1995; Yennawar et al., 2006). Expansins exert no direct action on chemical composition of cell wall materials, such as cellulose, hemicellulose, and pectins. No hydrolytic release of the wall polymers has been detected. Expansins immediately induce relaxation of stress on the wall, followed by wall extension with no requirement for ATP or another chemical energy source. The wall continues to extend as long as the wall specimen bears tension and expansins are present. Expansins are able to weaken pure cellulose paper where the cellulose fibres are interconnected with hydrogen bonds. Expansins aid the hydrolysis of cellulose by cellulases. Disruption of hydrogen bonds by urea treatment increases the eVect of expansin (Sharova, 2007). However, the exact biochemical mechanism of expansin action still remains to be experimentally confirmed. D. CELL WALL‐BINDING PROPERTIES

Expansins are believed to bind to cellulose because the protein has a domain (2) that resembles the cellulose‐binding domain of cellulase (Cosgrove, 2000b). ZmEXPB1 proteins bound to isolated maize cell wall and preferentially bound to xylans and xyloglucan with negligible binding to ‐(1!3), secondary antibodies; (E) cross section of a coleoptile probed with antibody against OsEXPB3 (1:2,500); (F) cross section of coleoptile probed with pre‐immune serum (1:2,500); (G) cross section of root probed with antibody against OsEXPB3 (1:2,500); and (H) cross section of root probed with pre‐immune serum (1:2,500). Sclerenchymatous tissue gave a colour reaction that was not due to an immune response. All sections were subsequently probed with alkaline phosphatase‐conjugated goat anti‐ rabbit secondary antibodies (1:2,500), and alkaline phosphatase activity was visualized. Bars: 500 nm for A, B, C, and D and 0.2 mm for E, F, G, and H. c, Cytoplasm; m, mitochondrion; p, proplastid; v, vacuole; w, cell wall. Arrow heads in A and C indicate putative vesicle‐like structures. Adapted from Lee and Choi (2005).

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(1!4)‐D‐glucan and glucomannan (Yennawar et al., 2006). The crystal structure of ZmEXPB1 suggests that binding to glycans may be aided via the two aromatic residues Trp194 and Tyr160 of domain 2, the putative cellulose‐binding domain (Yennawar et al., 2006). It should be noted, however, that domain 2 alone of the pollen‐type EXPB or even together with its domain 1 might result in only weak binding to target polysaccharides, as it is well known that the ZmEXPB1 proteins are easily extracted with aqueous solution of low ionic strength (Cosgrove et al., 1997). On the contrary, OsEXPB3, a vegetative form of EXPB in deepwater rice, could not be extracted without Sodium Dodecyl Sulfate (SDS) treatment, indicating that the EXPBs were tightly bound to the cell wall (Lee and Choi, 2005). Purified recombinant OsEXPB3, produced from tobacco BY2 cells, also bound to native cell walls and SDS‐washed cell walls, as well as to pure cellulose. Once bound to those specimens, the EXPB could not be extracted without SDS treatment (Lee and Choi, 2005), and this recalcitrance to extraction holds true for native OsEXPB3 proteins as well (Y. Lee and H. Kende, unpublished data). Therefore, the tight binding property of OsEXPB3 indicates that the vegetative EXPB may have a diVerent function from that of the pollen‐type EXPBs and of at least some EXPAs that have been extracted from the cell wall with 1M NaCl (Lee and Choi, 2005). E. THE CONTROVERSIAL HYDROLYTIC ACTIVITY OF THE POLLEN‐TYPE EXPBS

Cosgrove and his colleagues have convincingly demonstrated that expansin action does not involve hydrolytic activity (McQueen‐Mason et al., 1992, 1993; McQueen‐Mason and Cosgrove, 1994, 1995; Yennawar et al., 2006). Nevertheless, two research groups hotly debated the hydrolytic activity of the pollen types of EXPB proteins. Grobe et al. (1999, 2002) repeatedly presented evidence that the EXPB has proteolytic activity, as in the case of cathepsin B. However, Li and Cosgrove (2001) found no hydrolytic activity in the purified group‐1 pollen allergens, such as Lol p 1, Phl p 1, and Zea m 1 (ZmEXPB1), using several methods of analysis: hydrolysis of N-alpha-benzoyl-arginine-p-nitroanalide (BAPNA) and Chromozyme PL, digestion of bovine serum albumin and ovalbumin in solution, SDS‐Polyacrylamide Gel Electrophoresis zymogram, native PAGE zymogram, and detection of proteolysis from agarose gel. They could not find any cell wall‐ loosening activity in any of several kinds of proteases. Moreover, they found no inhibitory eVect of protease inhibitors on the EXPB’s creep activity. Crystallographic studies on the structure of ZmEXPB1 show that the residue corresponding to the active site of the protease papain is inaccessible and is


63

nowhere near the conserved surface (Yennawar et al., 2006), supporting Li and Cosgrove’s failure to detect hydrolytic activity.

V. EXPANSINS IN VEGETATIVE GROWTH AND DEVELOPMENT A. SEEDLING GROWTH

Expansins were first isolated from the growing region of cucumber hypocotyls and shown to induce the extension of isolated cell walls from diverse plant species (McQueen‐Mason et al., 1992). Because the first expansins were identified in cucumber hypocotyls, early studies mainly focused on seedling growth and successfully demonstrated a high correlation between the level of expansin transcripts or proteins and seedling growth. Cosgrove and Li (1993) analysed spatial and temporal growth patterns of oat (Avena sativa L.) coleoptiles with four diVerent cell wall properties or criteria: (1) extensibility of cell walls in an acidic environment, (2) ability of heat‐inactivated walls to regain extensibility by exogenously applied expansin proteins, (3) the quantity of expansin proteins in cell wall extracts that can be measured by immunoblot analysis, and (4) the amount of expansin activity in cell wall extracts. These properties have been adopted as important parameters to study expansin‐related plant growth, such as in growing leaves of tomato (Keller and Cosgrove, 1995), elongating internodes of deepwater rice (Cho and Kende, 1997a,b,c; Choi et al., 2003), and elongating petioles of Rumex palustris (Vreeburg et al., 2005). In rice seedling growth, coleoptiles and mesocotyls showed increased rates of growth in submergence or hypoxic environments, accompanied by an increase in the level of EXPA mRNAs, indicating that rice expansins are necessary for the growing tissue of rice seedlings (Huang et al., 2000). Choi et al. (2003) provided positive in vivo evidence in line with the hypothesis that expansins induce cell enlargement, leading to growth. They prepared transgenic rice plants overexpressing OsEXPA4 in sense or antisense orientation under the control of an inducible promoter. The growth of mesocotyls and coleoptiles of the sense transgenic rice was significantly enhanced. Immunoblot analysis with anti‐OsEXPA4 polyclonal antiserum showed that the level of OsEXPA4 proteins increased in sense transgenic seedlings, but decreased in antisense seedlings in the presence of the inducer N‐(aminocarbonyl)‐2‐chlorobenzenesulfonamide (2‐CBSU), showing a close correlation between the growth response and the induced expression of OsEXPA4. The authors also showed that mesocotyl growth in transgenic rice seedlings was altered because of cell enlargement but not cell number; the average cell length increased by up to 58% in sense

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transgenic lines, but decreased by 22% in antisense lines. Cell wall extensibility was also greatly aVected by the altered level of expansins: cell wall extensibility of sense transgenic lines increased by up to 32%, whereas that of antisense transgenic lines decreased by up to 20%. These results confirm that promoted or reduced growth of transgenic rice seedlings is closely correlated with the OsEXPA4 protein level, altered cell size, and cell wall extensibility, thus fulfilling the criteria for expansin action. Unfortunately, it should be noted in the following sections that most, but not all, studies on the biological role of expansin in various physiological and developmental processes do not satisfy those criteria fully, with many cases showing only a strong correlation at best. B. LEAF DEVELOPMENT

Leaf development begins with formation of leaf primordia on the flanking region of the shoot apical meristem (SAM). Developing leaves expand or enlarge, which gives plants maximal capability to get energy for photosynthesis. In temperate zones, annual or deciduous plants prepare for the winter by shedding their leaves. Even in tropical zones, plants drop their leaves for various reasons. Thus, leaf abscission also has a part in the leaf development process. Expansins seem to have diverse roles during the entire process of leaf development. Expansins in leaves were first reported from tomato plants as a potent factor responsible for the acid growth property of leaves (Keller and Cosgrove, 1995). Formation of leaf primordia depends on the change in the wall extensibility of the outermost cell layers in the SAM tissues. To investigate the role of expansins from the SAM in leaf formation, Fleming et al. (1997, 1999) prepared beads loaded with expansins purified from cucumber hypocotyls and applied them to the apical meristem. They found that the expansin proteins induced bulging‐out of leaf primordium‐like structures from the epidermal tissue, or L1 layer expansins, although leaf development was not completed at all, indicating that expansins play important roles in leaf initiation. Another eYcient way of proving expansin’s role in leaf initiation was demonstrated successfully in the opposite way (Pien et al., 2001). In this study, transgenic tobacco plants were generated to express CsEXP1, a cucumber EXPA gene, under the control of a tetracycline‐inducible system. To induce expansins locally, the authors applied anhydrotetracycline‐ containing lanolin paste to the apical meristem, forcing production of leaf primordia. After the expansin‐induced formation of leaf primordia, the leaf initials grew further to form intact leaf structures. The newly formed leaves displayed complete histological structures in the vasculature and lamina, as


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did normal leaves. These results demonstrate that expansins are truly capable of inducing leaf primordia, resulting in a complete leaf (Pien et al., 2001). In addition, the authors found that transient local induction of expansins on the flanks of young primordia resulted in increased local growth of lamina, which is indicative of the reversal of phyllotaxis. Taken together, these results demonstrate that induction of expansins in several cell layers of the meristem can initiate leaf formation, and expansin‐induced leaves can influence subsequent phyllotaxis of the plant. In transgenic rice, overexpression of OsEXPA4 produced at least two additional leaves, indicating that expansins regulate not only location of leaf formation but also number of leaf primordia (Choi et al., 2003). Newly formed leaves undergo cell division and enlargement to become full‐sized leaves. Correlation between activity or gene expression patterns of expansins and leaf enlargement has been well documented. Since Keller and Cosgrove (1995) demonstrated that expansins extracted from the growing leaves of tomato induced cell wall extension in cucumber seedlings, numerous reports have shown that expansins are positively related to leaf enlargement. Cho and Cosgrove (2000) presented in vivo evidence that altered EXPA activity modulated the leaf size of transgenic Arabidopsis plants. The AtEXP10 gene, which is preferentially expressed in the growing leaves and the basal part of the pedicel, was introduced into Arabidopsis plants to make sense and antisense transgenic plants. Compared to control plants, the sense plants overexpressing AtEXP10 had larger leaf blades, whereas the antisense plants had smaller ones. Some expansin genes in maize were identified as leaf growth‐related genes: 19 expansin genes were isolated in the leaf elongation zone and were classified into three diVerent groups related to cell division, leaf expansion, and cell wall diVerentiation (Muller et al., 2007). In the study, the authors proposed the hypothesis that distinct expansins might play diVerent roles in leaf widening and elongation in a concerted manner. Abscission is regulated by the susceptibility of tissues or organs to ethylene and by interaction between auxin and ethylene. In other words, depletion of auxin lends ethylene sensitivity to the cells in the abscission zone. Then, some cell wall‐modifying proteins under the influence of hormone interaction participate in cell wall degradation, leading to abscission. Thus, as cell wall‐loosening factors, some expansins are thought be involved in abscission. In common elderberry (Sambucus nigra), expression of SniEXP2 and SniEXP4 genes was specifically detected in the ethylene‐treated leaf abscission zone (Belfield et al., 2005). Cell wall proteins from the ethylene‐treated samples showed a sevenfold increase in expansin activity on cellulose/xyloglucan composite strips, compared to those from untreated ones, implicating the role of expansins in abscission. The sense AtEXP10 transgenic Arabidopsis plants enhanced abscission at the base of the pedicel, whereas the

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antisense plants reduced abscission (Cho and Cosgrove, 2000). However, observations of an EXPB gene from Mirabilis jalapa do not support these observations because no expansin gene was identified in abscission‐ competent petioles after auxin depletion, whereas the same gene was downregulated in those petioles (Meir et al., 2006). Summarizing these findings, it is probable that some expansins take part in the leaf abscission process by loosening the walls of abscission zone cells. C. ELONGATION OF STEMS AND PETIOLES

Many expansin genes are expressed in elongating internodes of deepwater rice (Table II). Cho and Kende (1997c) found that OsEXPA4 was preferentially expressed in the elongation zone of deepwater rice internodes. Overexpression and downregulation of the OsEXPA4 gene resulted in alteration of internode growth in transgenic rice (Choi et al., 2003). Further studies with deepwater rice demonstrated that at least six more expansin genes (OsEXPA6, OsEXPA7, OsEXPB3, OsEXPB4, OsEXPB6, and OsEXPB11) were expressed mainly in the intercalary meristem and elongation zone, areas that are responsible for intermodal elongation (Lee and Kende, 2001, 2002). Submergence or ethylene treatment induces petiole elongation in Rumex palustris, and its petiole elongation is correlated with the enhanced gene expression of RpEXPA1 (Vreeburg et al., 2005). Stem growth of chick pea (Cicer arietinum) is also accompanied by a high level of CaEXPA2 transcripts in response to treatment of brassinolides and Indole Acetic Acid (IAA) (Sańchez et al., 2004). D. ROOT GROWTH

The root is a good model system for studies of plant cell elongation because it contains a distinguishable zone just behind the root tip that is dedicated to the rapid elongation process (Dolan and Davies, 2004). Another region that starts from the end of the elongation zone is defined as the diVerentiation zone, where epidermal cells develop into hair cells that are specialized for the absorption of water and nutrients as well as for interaction with soil microbes. In this and following sections, we discuss the role of expansins in cell wall loosening with respect to rapid growth of cells in the elongation zone, as well as root hair cells in the diVerentiation zone. Consistent with the acid growth hypothesis (Rayle et al., 1970), the elongation zone of maize roots is mostly acidic, which is, in general, a prerequisite for expansin action to allow cell wall extension and, thus, subsequent growth (Peters and Felle, 1999). Over the past decade, a number of expression studies

TABLE II Expansin Genes Expressed in Plant Organs and Developmental Stages Plant organs and developmental stages Root

Plant species

Genes

Oryza sativa (rice)

Most EXPAs

Oryza sativa (rice)

OsEXLA1 (AY100693), OsEXLA3 (AY100694) OsEXPA3 (U30479)

Rumex palustris (marsh dock) Rumex palustris (marsh dock) Zea mays (maize)

Glycine max (soybean)

Most EXPAs Some EXPAs Collective EXPAs ZmEXPA1 (AAK56119) ZmEXPA5 (AAK56123) ZmEXPB2 (AF332175) ZmEXPB8 (AF332181) GmEXPA1 (AF516880)

Lycopersicon esculentum (tomato)

LeEXP8 (AF184232)

Arabidopsis thaliana

AtEXP6 (U30480) AtEXLA3 (NM_114465)

Proposed action Specifically or preferentially in root tip (0.5 cm): elongation Particularly in the epidermis, vascular cylinder, and around the pericycle: elongation Upper tap root, root tip: elongation

References Cho and Kende, 1997c Lee and Kende, 2002 Shin et al., 2005 Cho and Kende, 1997c, 1998 Colmer et al., 2004

Stele and cortex: elongation

Colmer et al., 2004

Growing region: elongation, increase in response to water deficit

Wu et al., 2001

Elongation zone: enhancement of root growth Initiation and perhaps continued elongation of the radicle Repressed in root elongation mutants

Lee et al., 2003 Chen et al., 2001 Lou et al., 2007

(continues)

TABLE II Plant organs and developmental stages Root hair

Leaf

Stem

Plant species

(continued)

Genes

Proposed action

References

Cucumis sativus (cucumber) Arabidopsis thaliana

CsEXPA1 proteins

Root hair swelling

Cosgrove et al., 2002

AtEXP7 (AC025416)

Cho and Cosgrove, 2002

Hordeum vulgare (barley)

AtEXP18 (AF332444) HvEXPB1 (AY351786)

Root hair cell files: hair cell development

Arabidopsis thaliana

AtEXPA10 (AF229437)

Sambucus nigra (common elderberry)

SniEXP2 (AY299690)

Repressed in rhl1.a mutant: hair cell initiation Leaf growth, pedicel abscission Leaf abscission

Kwasniewski and Szarejko, 2006 Cho and Cosgrove, 2000 Belfield et al., 2005

Stem elongation

Lee and Kende, 2001 Lee and Kende, 2002

Stem elongation

Sańchez et al., 2004

Oryza sativa (rice)

Cicer arietinum (chick pea)

SniEXP4 (AY299692) OsEXPA4 (U85246) OsEXPA6 (AF247163) OsEXPA7 (AF247164) OsEXPB3 (AF261271) OsEXPB4 (AF261272) OsEXPB6 (AF261274) OsEXPB11 (AY046917) CaEXPA2 (AJ291817)

Petiole

Rumex palustris

RpEXPA1 (AF167360)

Xylem

Populus tremulaPopulus tremuloides (hybrid aspen)

PttEXP1 (AY435099)

Zinnia elegans Fruit

Fragariaananassa (strawberry) Ficus carica (fig)

PttEXP5 (BI131728)a ZeEXP1 (AF230331) ZeEXP2 (AF230332) ZeEXP3 (AF230333) FaEXP1 (AF163812) FaEXP2 (AF159563) FaEXP5 (AF226702) FcEXP1 (AY487313)

Petiole growth under submergence Xylem diVerentiation

Vreeburg et al., 2005 Gray‐Mitsumune et al., 2004

Xylem diVerentiation

Im et al., 2000

Fruit softening

Dotto et al., 2006

Fruit expansion, ripening onset Fruit softening

Owino et al., 2004

Lycopersicon esculentum (tomato)

LeEXP1 (U82123)

Lycopersicon esculentum (tomato) Lycopersicon esculentum (tomato)

LeEXP2 (AF096776)

Fruit expansion

LeEXP3 (AF059487)

Fruit expansion, ripening onset

Rose et al., 1997 Powell et al., 2003 Kalamaki et al., 2003a,b Catala et al., 2000 Brummell et al., 1999 (continues)

TABLE II Plant organs and developmental stages

Plant species Lycopersicon esculentum (tomato)

Musa acuminata (banana) Musa acuminata (banana) Mangifera indica (mango) Olea europaea (olive) Prunus armeniaca (apricot) Prunus armeniaca (apricot) Fragaria ananassa (strawberry) Pyrus communis (pear)

(continued)

Genes

Proposed action

References

LeEXP4 (AF059488)

Fruit expansion

Brummell et al., 1999

LeEXP5 (AF059489) LeEXP6 (AF059490) LeEXP7 (AF059491) MaEXP1 (AY083168)

Fruit ripening

MaEXP4 (EF213102)

Fruit expansion

Trivedi and Nath, 2004 Wang et al., 2006b Asha et al., 2007 Asha et al., 2007

MiEXPA1 (AY600964)

Fruit ripening

Sane et al., 2005

OeEXP1 (AF384051) PaEXP1 (U93167)

Fruit ripening Fruit ripening

PaEXP2 (AF038815)

Fruit growth, ripening

FaEXP3 (AF226700)

Fruit expansion

Ferrante et al., 2004 Mbe´guie´‐A‐Mbe´guie´ et al., 2002 Mbe´guie´‐A‐Mbe´guie´ et al., 2002 Harrison et al., 2001

FaEXP4 (AF226701) PcEXP1 (AB093028) PcEXP4 (AB093031)

Fruit expansion

MaEXP2 (AF539540)

Hiwasa et al., 2003 Fonseca et al., 2005

Fruit softening

Hiwasa et al., 2003 Fonseca et al., 2005

Prunus mume (mume) Prunus persica (peach)

PcEXP6 (AB093033) PcEXP7 (AB093034) PcEXP2 (AB093029) PcEXP3 (AB093030) PcEXP5 (AB093032) Pm68 (AB218787) PpEXP1 (AB029083)

Fruit ripening Fruit ripening

Prunus persica (peach)

PpEXP2 (AB047518)

Fruit growth

Prunus persica (peach)

PpEXP3 (AB047519)

Fruit ripening, softening

Vitis labruscana cv. Kyoho (grape)

VlEXP1 (AB104442)

Fruit expansion, ripening

Mita et al., 2006 Hayama et al., 2003 Obenland et al., 2003 Hayama et al., 2001 Hayama et al., 2003 Hayama et al., 2001 Trainotti et al., 2003 Ishimaru et al., 2007

VlEXP2 (AB104443) VlEXP3 (AB104445)

Fruit softening

Ishimaru et al., 2007

Coleorhiza in developing seeds

Huang et al., 2000

Seeds in immature fruit: embryonic growth Seed developing

Chen et al., 2001

Somatic embryogenesis and in male cones

Bishop‐Hurley et al., 2003

Pyrus communis (pear)

Embryogenesis

Vitis labruscana cv. Kyoho (grape) Oryza sativa (rice) Lycopersicon esculentum (tomato) Triticum aestivum (wheat) Pinus taeda (pine)

OsEXP1 (Y07782) OsEXP2 (U30477) LeEXP10 (AF184233) Most EXPAs or EXPBs Collective EXPA

Lin et al., 2005

(continues)

TABLE II Plant organs and developmental stages

Plant species

(continued)

Genes

Proposed action

References

Seed

Datura ferox

DfEXPA1 (AF442773)

Seed germination

LeEXP4 (AF059488)

Seed germination

Anther or pollen

Lycopersicon esculentum (tomato) Zea mays (maize)

Zea m 1 (ZmEXPB1) and its isoform proteins

Successful fertilization

Mella et al., 2004 Arana et al., 2006 Chen and Bradford, 2000 Wang et al., 2006a

Lol p 1 proteins (EXPB)

Vegetative cell of pollen

Valdivia et al., 2007a,b Taylor et al., 1994

Phl p 1 (EXPB)

Exine and cytoplasm of pollen Intine of pollen

Behrendt et al., 1999

Promoter activities in germinating and mature pollen

Xu et al., 1999

Lolium perenne (perennial ryegrass) Phleum pretense (timothy grass) Phleum pretense (timothy grass) Oryza sativa (rice)

Triticum aestivum (wheat) Ovary

Nicotiana tabacum (tobacco) Oryza sativa (rice)

Phl p 5 (EXPB) Ory s 1 (OsEXPB1); OsEXPB9 (AC020666)b OsEXPB13 (AC107224)c TaEXPB1 (AY533101) TaEXPB2 (AY533102) PPAL (EXPB; AF333386) OsEXPB9 (AF261277) OsEXPB13 (AF391106)

Behrendt et al., 1999

Bhalla et al., 1999

Microspores but not in mature pollen

Jin et al., 2006

Stigma, placenta

Pezzotti et al., 2002

Pistil

Yoshida et al., 2005

Flower

Mirabilis jalapa Mirabilis jalapa Petunia hybrida

Most EXPAs MjEXPB1 (AY147412) PhEXP1 (AY487167)

Lycopersicon esculentum (tomato) Lycopersicon esculentum (tomato)

LeEXP18 (AJ004997, AJ270960) LeEXP2 (AF096776)

Flower tube expanding Flower bud development Expanding petal limbs: wall loosening and deposition of crystalline cellulose Flower primordium initiation

Gookin et al., 2003 Gookin et al., 2003 Zenoni et al., 2004

Flower bud expanding

Reinhardt et al., 1998

EST sequence registered in GenBank EST database. GenBank accession numbers for genomic clones. The numbers in parentheses designate GenBank accession number. EXPB, expansin B; EXPA, expansin A; EXLA, expansin‐like A; PPAL, pistil pollen allergen‐like.

a

b,c

Reinhardt et al., 1998

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showed a tight positive correlation between root growth and the level of expansin mRNA or expansin activity in the elongation zone. One of the earliest examples showing this correlation can be found in deepwater rice: the four EXPA genes, OsEXP1 through OsEXP4, were expressed specifically or preferentially in the most rapidly growing region of the root (Cho and Kende, 1997c). In situ hybridization results with antisense probes for OsEXP3 showed abundant transcripts in the root tip region, particularly in the epidermis and vascular cylinder, and around the pericycle, even in the upper region (Cho and Kende, 1998). Subsequently, almost all rice EXPA genes that are expressed in the root showed a similar pattern of expression in the rice root (Lee and Kende, 2002; Shin et al., 2005). The high levels of expansin mRNA were observed to be localized in adventitious root tips of Rumex palustris, especially in the stele and cortex tissues (Colmer et al., 2004), indicating that expansins are involved in mediating cell elongation in the root. In maize, two EXPA genes (ZmEXPA1 and ZmEXPA5) and two EXPB genes (ZmEXPB2 and ZmEXPB8) were actively expressed in the apical 1‐cm region of the maize root that corresponded to the elongation zone (Wu et al., 2001). A soybean EXPA, GmEXP1, was specifically expressed in the elongation zone of both primary and secondary roots (Lee et al., 2003). Overexpression of GmEXP1 in tobacco plants considerably enhanced root growth, which resulted from the increased length of root epidermal cells. Tomato LeEXP8 mRNA was most abundant in the cortical tissue of the root elongation zone during germination, which indicates that LeEXP8 is likely involved in initial and perhaps continued elongation of the radicle (Chen et al., 2001). Even in a gymnosperm, the loblolly pine, some expansins may be involved in root development, as the excised hypocotyl tissues that had already ceased their elongating growth induced expression of an EXPA gene(s) and adventitious roots in response to exogenous treatment of auxin (indole‐3‐butyric acid; Hutchison et al., 1999). Arabidopsis mutants that have T‐DNA insertions in a phosphatidylinositol monophosphate 5‐kinase (PIP5K9) and a cytosolic invertase (CINV1) are defective in root cell elongation, resulting in short roots (Lou et al., 2007). Interestingly, the root elongation mutants, PIP5K9 and CINV1, showed repressed expression of an EXPA gene, At‐EXP6, and an EXLA gene, AtEXPL3, respectively, together with genes for many other cell wall‐modifying enzymes (Lou et al., 2007). Despite these ample observations, however, it still remains to be established whether there is a causal link between expression of those root‐ specific expansin genes and the root elongation process. Up to now, the only evidence for the causal relationship is that overexpression of GmEXP1 in tobacco plants considerably increases the length of root epidermal cells, leading to enhanced root growth (Lee et al., 2003).


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It is believed that local changes in wall extensibility by expansins may play an important regulatory role that enables the root tissue to eVectively respond to biotic or abiotic stimuli, ultimately enhancing plant adaptability to environmental changes. In maize roots that bend downward in response to a gravitropic stimulus, the upper half of the root accumulated more expansin proteins than the lower half did, and both gravitropism and the asymmetrical distribution of expansins in the root were disturbed by naphthylphthalamic acid (NPA) (Zhang and Hasenstein, 2000). When the maize roots were subjected to water stress, the apical part of the root elongation zone maintained its growing capacity, which was accompanied by increases in wall extensibility (Wu et al., 2001). Maintenance of active growth was accompanied by increased levels of expansin proteins and transcripts in the region. Among several expansin genes expressed in the root, the mRNA levels of one EXPB and two EXPA genes were rapidly enhanced in the growing region during water stress (Wu et al., 2001). Several EXPA genes from the semi‐ aquatic Rumex palustris were expressed diVerentially in response to oxygen deficiency (Colmer et al., 2004), again supporting the notion that some expansin genes of the superfamily may be relevant to adaptation to adverse environmental factors, such as water deficit or water logging. E. ROOT HAIR DEVELOPMENT

Root hairs are specialized root epidermal cells that play important roles in the absorption of water and nutrients as well as in interaction with soil microbes. Development of a root hair starts with a localized projection in the lateral wall of an epidermal trichoblast cell, resulting in a bulge; the bulge keeps elongating by tip growth (Grierson and Schiefelbein, 2002). Bibikova et al. (1998) observed, using confocal imaging, that the apoplastic pH in the initiation site was lowered when the bulge started to form and that prevention of wall acidification by pH buVers caused the cessation of tip growth, demonstrating that localized apoplastic acidification is required for root hair elongation. This observation is compatible with the findings that EXPA require low pH for optimal activity and that the treatment of cucumber root hairs with EXPA CsEXP1 proteins results in swelling and, in the long run, bursting of the hair cell tip (Cosgrove et al., 2002). Expansin proteins also highly accumulate in outgrowing bulges of maize trichoblasts (Baluska et al., 2000), suggesting that expansins have a role in root hair cell expansion. EXPA genes in Arabidopsis, AtEXP7 and AtEXP18, are expressed solely in root hair cells before hair initiation and remain actively expressed thereafter (Cho and Cosgrove, 2002). The specific expression pattern is driven primarily by the proximal region of promoters that have root hair‐specific cis‐elements (RHEs; Kim et al., 2006).

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The authors demonstrated by using mutational analysis of the RHEs that the cis‐elements are both necessary and enough to induce the root hair‐specific expression pattern of AtEXP7 and AtEXP18. Furthermore, a group of monophyletic EXPA genes from a variety of angiosperm species was found to have functional RHEs in their promoters and to be expressed specifically in the root hair (Kim et al., 2006). These results are indicative not only of the role of EXPA in cell wall loosening during hair cell development but also of the importance of regulatory cis‐elements in determining a specific biological role of expansin genes. It should be noted, however, that lack of loss‐of‐function mutants for AtEXP7 and AtEXP18 leaves their direct causal link to the in vivo function to be demonstrated, although their expression level is clearly related to root hair formation (Kim et al., 2006). Although Kim et al. (2006) focused on EXPAs in root hair initiation, Kwasniewski and Szarejko (2006) found a barley EXPB gene, HvEXPB1, that may also be involved in hair cell initiation. They isolated the EXPB gene by employing a transcriptome subtractive hybridization method using cDNAs from wild type and the barley hairless mutant rhl1.a. Wild‐type roots actively expressed HvEXPB1 before diVerentiation of epidermal cells and maintained it in the subsequent root growth, whereas the rhl1.a and its allelic mutant brb completely lacked any sign of hair cell formation and HvEXPB1 expression. On the contrary, the level of HvEXPB1 transcripts was not aVected in another type of root hair mutants, rhp1.a and rhs1.a, which produced slightly elongated hair bulges and very short hair cells, respectively. These results indicate that the barley EXPB gene may play a role in hair cell initiation, but not in elongation. The rhl1.a and brb mutants had no mutation in either coding region or promoter of the HvEXPB1 gene, indicating that the EXPB gene was not mutated in those mutants. Although HvEXPB1 exhibits a root‐specific expression pattern, it remains yet to be better resolved whether it is expressed specifically in hair cells or in epidermal cells that will diVerentiate into hair cells. Nevertheless, it would be interesting to examine whether EXPB genes, including HvEXPB1, have RHEs in their promoters. According to Kim et al. (2006), HvEXPA7, a barley EXPA gene, belongs to the monophyletic group that comprises EXPA genes having RHEs, indicating that root hair initiation and elongation in barley may require both EXPA and EXPB. F. LIMITED CORRELATION OF EXPANSIN ACTION WITH PLANT VEGETATIVE GROWTH

Expression of expansin genes and proteins is not always correlated with elongation. Early in the history of expansin research, Cosgrove and Li (1993) already suggested that, in some cases, susceptibility of the wall to expansin action might be more important than the level of expansin activity itself.


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Expansins had been believed to play a positive role in plant growth only by promoting cell enlargement until Caderas et al. (2000) and Reidy et al. (2001) reported unexpected results from studies using tomato seedlings and fescue leaves, respectively. Caderas et al. (2000) reported limited correlation between expansin gene expression and tomato seedling growth. They proposed that under some unique physiological conditions such as hypocotyl growth in the dark, where they failed to find a correlation between the observed rate of extension growth and the levels of expansin proteins and gene expression, other factors may be involved. Expression patterns of EXPAs and EXPBs in fescue leaves were not correlated with leaf elongation, whereas expression of xyloglucan endotransglycosylase was closely correlated with the rate of leaf growth (Reidy et al., 2001). To investigate expansin action in vivo, Rochange et al. (2001) generated transgenic tomato plants expressing CsEXP1 under the control of the CaMV35S promoter. This was one of the first attempts to address the biological function of expansins. However, the transgenic plants displayed paradoxical phenotypes opposing the historical concept of expansin action. The heights of adult plants and dark‐grown seedlings were mostly reduced compared to those of non‐transgenic plants. In addition, leaf length was also frequently reduced. These results proved to be caused by a reduction in cell wall extensibility in each transgenic plant, thus leading to the hypothesis that excess amounts of expansins may disturb organization and mechanical properties of plant cell walls (Rochange et al., 2001). Choi et al. (2003) have provided similar results, where highly overexpressed expansins may cause stunted growth in a portion of transgenic rice plants. These results, taken together, indicate that increased levels of expansin gene expression do not always induce plant growth. There are at least two possible explanations for the unexpected behaviour of expansins. First, susceptibility of plant cell walls to expansin action may be a major factor. Reduced susceptibility of walls to expansins may be caused by changes in cell wall composition or shortage in supply of cell wall materials during cell growth after expansin‐induced wall loosening (Caderas et al., 2000; Rochange et al., 2001). Second, diVerent organs may depend on diVerent threshold levels of expansins (Choi et al., 2003). Choi et al. (2003) ruled out the possibility of co‐suppression or gene silencing because all the sense transgenic lines, including the stunted plants, accumulated high level of OsEXPA4 transcripts. G. DIFFERENTIATION OF XYLEM TISSUES

In Zinnia elegans, sub‐cellular localization of expansin genes reveals that they are preferentially localized in apical or basal tip regions of xylem cells, indicating that xylem‐specific expansins in Zinnia are possibly involved in

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xylem diVerentiation (Im et al., 2000). In woody plants, diVerentiation of xylem tissue requires enlargement of cambial cells before development of a secondary wall, which is crucial for xylem function. In hybrid aspen (Populus tremula Populus tremuloides Michx.), three EXPA genes (PttEXP1, PttEXP2, and PttEXP8) and an EXPB gene (PttEXPB1) were identified in a cambial region (Gray‐Mitsumune et al., 2004). The authors found that PttEXP1 and PttEXP5 were expressed abundantly in diVerentiating xylem tissues. PttEXP1 transcripts were present in the tips of the fusiform cells and throughout the cells in Populus, whereas ZeEXP genes in Zinnia are strictly localized to the apical ends. These results indicate that the PttEXP1 protein may play a role in radial xylem cell expansion and intrusive tip growth. H. GROWTH‐RELATED ENVIRONMENTAL CHANGES

Expansins seem to be involved in regulating the growth rate of plants in response to changes in specific environmental factors, such as hormones, osmotic stress, light, anoxia, submergence, and carbon dioxide. Hormones are a major factor aVecting gene expression of expansins. A number of expansin genes from rice are regulated by gibberellin (Cho and Kende, 1997c; Lee and Kende, 2001, 2002). CaEXPA2, an expansin gene from chick pea (Cicer arietinum), is tightly regulated by brassinolides and IAA (Sańchez et al., 2004). In Rumex and Sambucus, expansin genes are regulated by ethylene (Belfield et al., 2005; Vreeburg et al., 2005). Shoot elongation from arrowhead (Sagittaria pygmaea Miq.) tubers is enhanced by anoxia, ethylene, and carbon dioxide and is correlated with increased expression of four expansin genes (SpEXPA1, SpEXPA2, SpEXPA3, and SpEXPA4), although each of these genes responds to diVerent factors (Ookawara et al., 2005). Some expansin genes are expressed in plants under drought conditions. Under severe water stress, growth‐related expansin genes in maize are downregulated, indicating that water deficit‐induced reduction in leaf expansion is correlated with reduction in expansin gene expression (Muller et al., 2007). Germination and seedling growth of rice can be achieved in hypoxia or anoxia. Huang et al. (2000) showed that OsEXPA2 and OsEXPA4 genes responded to submergence (hypoxia), leading to promoted seedling growth above water level. I. SEED GERMINATION

Seeds contain the next generation of plants as embryos. To establish a new plant, the embryo has to emerge from the seed, which is defined as germination. Germination is triggered by water uptake by the dry seed, followed by


79

the resumption of metabolic activities. The process is complete when a part of the embryo, the radicle, extends to protrude through the seed tissues surrounding it (Bewley, 1997). For the extension process to occur in many dicot seeds, the radicle axis should overcome the mechanical barrier imposed by the surrounding endosperm in the micropylar region. Therefore, protrusion of the radicle requires a weakening of the micropylar endosperm, which must include disassembly and/or modification of the cell wall materials. Breakdown of the micropylar endosperm cell wall is closely associated with the activities of many cell wall hydrolases, including endo‐ ‐mannanase, ‐mannosidase, and xyloglucan endotransglycosylase (Bradford et al., 2000). Interestingly, expression of DfEXPA1 was detected in the micropylar endosperm and up‐regulated by phytochrome and gibberellin signallings in Datura ferox, as were the wall‐degrading and ‐modifying enzymes (Arana et al., 2006; Mella et al., 2004). A tomato EXPA gene, LeEXP4, was also expressed specifically in the micropylar endosperm region, with a correlation to the weakening process during germination (Chen and Bradford, 2000). These results suggest that the wall loosening of the endosperm tissue by those EXPA may play a role in facilitating protrusion of the radicle, breaking the coat‐imposed dormancy. It has been hypothesized that diVerent expansin proteins may interact with distinct cell wall substrates, probably resulting in tissue‐dependent wall‐loosening activity (Cosgrove et al., 1997). Those specific EXPA found in the micropylar endosperm may prefer as a substrate the endosperm cell wall that is highly rich in ‐(1, 4)‐mannan (Chen et al., 2001).

VI. EXPANSINS IN REPRODUCTIVE GROWTH AND DEVELOPMENT A. MALE GAMETOPHYTIC DEVELOPMENT

As mentioned earlier, a subset of EXPB proteins, originally recognized as group‐1 allergens, is present specifically and abundantly in pollen. Zea m 1, which was the first member identified as a EXPB, was specifically localized in the exine and cytoplasm of pollen (Wang et al., 2006a). Lol p 1 proteins from rye grass were predominantly present in the vegetative cell of pollen (Taylor et al., 1994). Phl p 1 and Phl p 5 were the major group‐1 allergens in timothy grass pollen: the former was localized to the exine and cytoplasm and the latter to the intine (Behrendt et al., 1999). The presence of group‐1 allergens in the pollen coat and wall is well in line with the notion that they may help pollen tubes germinate on the stigma and penetrate into the maternal tissues (Cosgrove et al., 1997).

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Although their localization in pollen was not defined yet, many EXPB genes encoding group‐1 allergens and their close homologues are predominantly expressed in mature pollen. Comparative analysis of maize pollen proteins revealed the existence of several isoforms of Zea m 1 allergen (Li et al., 2003; Wang et al., 2004), and recently Valdivia et al. (2007a) reported that the maize genome contained at least 15 pollen‐type EXPB genes whose expression levels were high in mature pollen, indicating that the Zea m 1 allergen is a mixture of distinctive gene products. Ory s 1 from rice (Oryza sativa), later renamed OsEXPB1, belongs to the group‐1 allergens (Cosgrove et al., 1997), and its promoter activities are high in mature and germinating pollen but not in other floral and vegetative tissues (Bhalla et al., 1999; Xu et al., 1999). Proteomic analysis of rice pollen proteins also revealed the presence of three isoforms of the Ory s 1 allergen (OsEXPB1) but no single EXPA (Kerim et al., 2003). These results suggest that both the pollen‐ type EXPB and the group‐1 allergens may play a potential role in pollen invasion into and through the stigma and style by loosening the cell wall structure of those tissues (Cosgrove et al., 1997). However, it should be noted that, with exceptions of the collective Zea m 1 allergen and one of its isoforms, Zea m 1d (Cosgrove et al., 1997; Li et al., 2003), most of the EXPB from pollen or even vegetative tissues have not been experimentally tested for cell wall‐loosening activities (McQueen‐Mason et al., 2007). Direct in planta evidence that expansins aid in pollen tube invasion have been scarce. Although Wang et al. (2004) showed that the sterility of the maize gaMS‐2 mutant was correlated with a reduced level of Zea m 1, they did not provide any evidence for a causal relationship between those two factors. Recently, however, Valdivia et al. (2007b) were able to assess in vivo function of the Zea m 1 allergen by isolating a mutant for ZmEXPB1, the most abundant isoform of Zea m 1. The mutation was caused by a transposon insertion in the third intron and reduced the level of Zea m 1 allergen by 31% compared to wild type. When pollen grains from the heterozygous EXPB1/expb1 plants were deposited on wild‐type silks of maize plants, the mutant gene expb1 was significantly underrepresented in the progeny, which is indicative of a reduction in the ability to achieve fertilization. They also found that silks pollinated with expb1 pollen contained a significantly smaller number of pollen tubes than did silks pollinated with wild‐type pollen, indicating that the expb1 mutant has a defect in in vivo pollen tube growth. This is the first in vivo evidence that the pollen‐type EXPB genes are, indeed, required for pollen tube growth to achieve successful fertilization. On the contrary, in vitro pollen viability and pollen tube growth were not aVected at all, suggesting that Zea m 1 is not directly involved in determining those pollen properties per se, but it is an important regulatory factor in the context


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of interaction with the female reproductive organ. Further detailed studies employing such genetic manipulation will provide not only new insights into the biological role of other pollen‐type EXPB but also an important engineering tool for developing hypoallergenic grass plants (Bhalla et al., 1999). We should note that not all pollen‐type EXPB genes are expressed in the mature stage of pollen. For instance, two EXPB genes isolated from wheat anthers were found to be expressed only in microspores until the stage of mid‐bicellular pollen, but not in mature pollen (Jin et al., 2006). Some of maize pollen‐type EXPB genes are expressed before the first pollen mitosis (Valdivia et al., 2007a). Those genes in immature pollen may play an additional role in male gametophytic development, such as microspore enlargement and tetrad separation, rather than in pollen tube growth (Jin et al., 2006). Apart from the pollen‐type EXPB genes, Pezzotti et al. (2002) found an interesting gene in Nicotiana tabacum, Pistil Pollen Allergen‐Like (PPAL), which was a close homologue of pollen‐type EXPB genes, but whose transcripts and proteins were expressed exclusively in the stigmatic secretory zone and the epidermal layer of the placenta. In contrast to the maize allergen expansins, however, PPAL did not enhance cell wall loosening (Nieuwland et al., 2005), leaving its function open to question in regard to pollen–stigma interaction. By performing cDNA microarray analysis, Yoshida et al. (2005) found that OsEXPB9 and OsEXPB13 are specifically and highly represented in mRNAs harvested from pistil tissues after anthesis, although detailed studies are needed to determine their possible implication. B. SEED FORMATION AND EMBRYOGENESIS

Many expansin genes are expressed with various spatiotemporal patterns during seed development. Two rice EXPA genes, OsEXP1 and OsEXP2, exhibited a relatively strong expression at coleorhizae in developing seeds but not in the early stage of seedlings (Huang et al., 2000), indicating that they may be involved in embryonic development of coleorhizae that is thought to protect the seminal root during germination. In tomato, the level of LeEXP10 mRNA was highest at early stages in germination of seeds harvested from immature fruits, which is temporally consistent with active cell division and cell enlargement in developing seed. Thereafter, LeEXP10 mRNA was maintained at a low level, being expressed throughout the entire mature embryo before germination and preserved even in dry seed, indicating that it may play a general role in embryonic growth (Chen et al., 2001). It has been reported that 16 of the 18 EXPA or EXPB genes in wheat are expressed in developing seeds and in leaves and roots, with variable

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patterns of expression from days 2 through 12 after pollination (Lin et al., 2005). Bishop‐Hurley et al. (2003) reported that a gymnosperm (Pinus radiate) expansin gene that is more closely related to vegetative EXPB than to the pollen type was expressed during somatic embryogenesis and in male cones, that is, at low levels in pre‐meiotic cones and at much higher levels after meiosis. In spite of the ample studies on expression patterns, however, no further or functional studies have followed, making it diYcult to determine a possible role of those genes in seed formation. The major obstacle to the functional studies is that the expansin genes seem to act in a functionally redundant manner (Cosgrove et al., 2002; Li et al., 2003), which suggests that one may need to genetically and collectively manipulate gene expression of a group of redundant expansin genes, for example, by employing RNA interference technology. C. FLORAL DEVELOPMENT

Several EXPA genes isolated from Mirabilis jalapa flowers are, in general, highly expressed during maximal elongation of the calyx but expressed at low levels in the small bud stage, which is compatible with the general role of EXPA in rapidly growing tissues (Gookin et al., 2003). Interestingly, however, EXPB transcripts were most abundant at the small flower bud stage, after which the transcripts were sharply decreased to very low or undetectable levels throughout the remaining stages of flower development, suggesting that the EXPB may not be directly relevant to rapid elongation of the flower tube. Zenoni et al. (2004) also found an EXPA gene from Petunia hybrida, PhEXP1, whose maximal expression matched the onset of cell expansion of petal limbs. PhEXP1 antisense plants developed smaller petal limbs, resulting from a reduction in cell size rather than cell number. The transgenic plants also showed thinner cell walls because they had a decreased deposition of crystalline cellulose. These results not only indicate the role of the expansins in cell wall loosening in petal tissue but also suggest a novel mechanism by which expansins may regulate cell expansion by aVecting cellulose deposition in such a way that permits a battery of wall‐modifying enzymes to act properly for cell wall assembly. It seems, however, that the role of expansins in flower development may not be confined to cell expansion processes. Some expansins may also play a role in the formation of flower and leaf primordia. Expression of a tomato EXPA, LeEXP18, was high in early stages of floral meristem formation; then, it was partitioned into emerging flower organs such as sepals, petals, and anthers. At the same time, expression of another expansin gene, LeEXP2, was commonly associated in expanding tissues, including flower buds (Reinhardt et al, 1998). The authors proposed that


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initiation of lateral organs (leaf and flower) from the meristem may be triggered by a local up‐regulation of expansin expression, which leads to a localized expansion of the meristematic tissue and, thus, to activation of subsequent genetic processes involved in organogenesis.

VII. EXPANSINS IN FRUIT DEVELOPMENT A. FRUITS AND CELL WALL PROTEINS

Fruits are initiated from various floral structures and undergo extremely complex developmental processes until maturity (Adams‐Phillips et al., 2004). From the anatomical point of view, fruits, including attached floral parts, can be defined as modified ovaries or mature carpels (Giovannoni, 2001; White, 2002). According to water content and maturation process, fruits are identified as either fleshy or dry. Even though dry fruits are dominant among plant species, studies on fruit development have been mainly focused on fleshy fruits because of their nutritional value to mankind (Giovannoni, 2001, 2004). Fleshy fruit development consists of three distinct stages: growth, maturation, and ripening. As the growth stage is closely related to cell division and enlargement, cell wall loosening resulting in cell size increase and cell division plays an important role in the growth and maturation stage. During fruit maturation and ripening, however, changes in cell wall properties and structure, so‐called fruit softening, are more important. Physical properties of the cell wall, such as cell wall integrity, are determined by combinatorial structures of cellulose, glycan, and pectin polymers. Thus, to understand the mechanism of fruit cell wall softening, it is necessary to consider the overall networks of wall polymers (Vincente et al., 2007). From this point of view, wall disassembly in tomato fruit can be explained by coordinated action of expansins, endo‐ ‐1,4‐D‐glucanases, xyloglucan endotransglycosylases/ hydrolases, and glycosidases in a cellulose–xyloglucan network (Rose and Bennett, 1999). Among those, we are going to discuss the role of expansins.

B. EXPANSINS IN FRUIT DEVELOPMENT

1. Expansins in various fruit developmental stages In the followings, we first focus on expansins mainly involved in the ripening process of climacteric and non‐climacteric fruits because ripening is most well‐studied process, and, then, we deal with expansins that are expressed

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diVerentially, depending on developmental stages, such as growth and maturation as well as the ripening stage. Since Rose et al. (1997) first showed that a fruit‐specific expansin gene in tomato, LeEXP1, was highly expressed specifically in ripening fruits where cell expansion had reached a plateau, a number of reports have accumulated to show a correlation between expansin expression and fruit development. In general, the process of fruit development can be divided into three or four stages. The first stage is characterized by active cell division, which contributes mainly to rapid growth of green fruits. During the second stage, cell expansion leads to an increase in fruit size. Several expansin genes, including LeEXP3, LeEXP4, and LeEXP5, were identified as specific contributors in the green stage of tomato fruit (Brummell et al., 1999). The third stage, the ripening stage, is represented by diverse physiological and biochemical changes in fruit tissue. These changes are the result of very complicated and highly coordinated events and occur only in a short time period. The final stage is abscission, when plants naturally shed their ripe fruits. Abscission occurs as a consequence of the breakdown of the attachment between the specific cells located at the site of organ shedding (Roberts et al., 2000, 2002). During organ shedding, specific expansin genes accumulate specifically in the abscission zone tissue and are controlled by ethylene (Belfield et al., 2005). 2. Expansins in climacteric ripening Tomato is considered a primary model plant to study the molecular biology of fruit development and climacteric ripening because it has great advantages both in agricultural and scientific aspects (Adams‐Phillips et al., 2004; Giovannoni, 2001, 2004). Tomato has a relatively small genome size and short generation time, and a wide array of genetic and genomic resources are available for this plant. Ethylene is known as the most influential hormone in fruit ripening and softening. An elevated level of ethylene switches on ripening‐related genes in climacteric fruits, leading to cell wall disassembly and, thus, fruit softening (Giovannoni, 2004). Ethylene causes ripening fruits to enter into senescence by promoting climacteric respiration and enhanced expression of the genes encoding hydrolytic enzymes in fruit tissue. As ethylene is a major modulator of fruit ripening, most expansins related to ripening are regulated by ethylene. There are a number of reports regarding expansin genes and their unique behaviour in climacteric ripening. During ripening, expansins seem to play a diVerent role that is not related to cell expansion. Powell et al. (2003) proposed a secondary role of expansins: to loosen cell walls, thus allowing


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various hydrolytic enzymes to access their substrates in the cell wall. Overexpression of LeEXP1 promotes fruit softening, while softening is inhibited in antisense transgenic tomato (Brummell et al., 1999). In addition to LeEXP1, a number of ripening‐specific expansin genes have been cloned and characterized: MiEXPA1 from mango fruits, Pm68 from mume (Prunus mume), PpEXP3 from peach, PaEXP1 and PaEXP2 from apricot, and OeEXP1 from olive (Ferrante et al., 2004; Hayama et al., 2003, 2006; Mbe´guie´‐A‐Mbe´guie´ et al., 2002; Mita et al., 2006; Sane et al., 2005). Interestingly, expression of most of these genes is regulated by ethylene. MaEXPA2 and MaEXPA4, expansin genes from banana fruits, are also specifically expressed in ripening banana fruit, and gene expression of MaEXPA2 is regulated by ethylene (Asha et al., 2007; Trivedi and Nath, 2004; Wang et al., 2006b). 3. Expansins in non‐climacteric ripening Non‐climacteric ripening usually does not accompany an increase in ethylene biosynthesis. Strawberry is one of the best model plants to study non‐climacteric fruit ripening (Carbone et al., 2006; Giovannoni, 2004). Dotto et al. (2006) observed expression patterns of three expansin genes that were closely correlated to fruit firmness in strawberry. The authors found that FaEXP1, FaEXP2, and FaEXP5 genes were more highly expressed in the softest cultivar than in firmer cultivars, implying that these genes may be involved in enhancing fruit softening by promoting cell wall disassembly. Grape berry (Vitis vinifera L.) is another non‐climacteric fruit that undergoes a double sigmoid pattern of growth. The first phase consists of cell division and growth. The second phase coincides with the ripening process and provokes significant changes in fruit structure and texture. Grimplet et al. (2007) reported that several expansin genes were specifically expressed in grape skin, whereas others were preferentially expressed in the pulp or seed. A significant reduction in berry size under water deficit was accompanied by reduction in mRNA level for genes encoding cell wall metabolism proteins such as polygalacturonase, pectin methylesterase, and two expansins. These results demonstrated that expansins may play a role in the ripening process of non‐climacteric fruits as well. C. EXPANSINS AND HORMONES OTHER THAN ETHYLENE

Hormones other than ethylene do not seem to have a direct relationship with fruit development. It is possible, however, that some hormones may be active in the earlier stages of fruit development, considering the nature of biological

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function exerted by individual hormones. Brassinolides, cytokinins, and auxins may play a role in the fruit growth stage by activating cell division. Gibberellins may participate in fruit enlargement by promoting cell expansion. There is evidence that the fate of a fruit is somehow determined by the viability of the seeds it contains. Usually, fruits having seeds with reduced vitality do not complete maturation. In other words, fruit maturation requires communication between fruits and viable seeds inside. This is the case in pea pericarp development. Together, auxins and gibberellins residing in pea seeds regulate pericarp growth by enhancing cell division and elongation (Ozga et al., 2002; Ozga and Reinecke, 2003). According to recent reports showing hormonal regulation of expansin action, as mentioned above, it is probable that expansins are involved in hormone‐mediated fruit growth, although there is no supportive evidence provided (Cho and Cosgrove, 2004). D. DIFFERENTIAL EXPRESSION OF EXPANSIN GENES IN FRUIT DEVELOPMENT

Considering fruit development with respect to changes in cell wall architecture, the process of fruit development can be divided into two phases: cell expansion leads to fruit growth, and cell wall disassembly promotes fruit softening. Either process can be achieved by expansin action. In other words, a group of expansins may be involved in mediating cell expansion leading to fruit growth while others may act to promote cell wall disassembly resulting in fruit softening. Among tomato fruit‐specific expansin genes, LeEXP3 is detected throughout fruit growth and ripening stages; LeEXP2 and LeEXP4 are expressed only in expanding fruits; LeEXP5, LeEXP6, and LeEXP7 are detected in expanding or mature green fruits (Brummell et al., 1999; Catala et al., 2000). In peach fruit, PpEXP2 and PpEXP3 showed diVerential expression pattern: PpEXP2 was expressed only in the fruit growth phase, whereas PpEXP3 was expressed only in the ripening phase (Hayama et al., 2001; Trainotti et al., 2003). Among five expansin genes in banana fruit, MaEXPA4 was expressed both in fruit growth and ripening stages and may be related to expansion (Asha et al., 2007). A study showed that the expression patterns of PaEXP1 and PaEXP2 from apricot fruits were positively correlated with increase in fruit size before the ripening stage, suggesting involvement of expansins in fruit expansion (Mbe´guie´‐A‐Mbe´guie´ et al., 2002). Expansin activity was also detected highly in the fruit set stage of apple (Goulao et al., 2007). In pear fruit, PcEXP2, PcEXP3, PcEXP5, and PcEXP6 were preferentially expressed in the softening phase, but their expression was reduced during the over‐ripe phase. PcEXP7 was exclusively expressed in young growing fruits. PcEXP4, PcEXP5, and PcEXP6 were also


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highly expressed in the young fruit stage, the latter two being expressed in the ripening stage as well, showing an overlapping pattern (Hiwasa et al., 2003). Goulao et al. (2007) observed the highest expansin activity at the fruit set stage of apple. These results show typical diVerential expression of individual expansin genes in a broad range of plant species, implying diVerent roles of individual expansins in diVerent stages of fruit development. In other words, in addition to the role in fruit ripening, some expansins active in the fruit growth stage may act to loose cell wall for cell expansion as do expansins in other organs, for example, in leaf. However, it is also recognized that, sometimes, the same genes active in the early stages of fruit development are activated again in the later stage of fruit softening.

VIII. FUTURE PROSPECTS: FURTHER UNDERSTANDING OF EXPANSIN FUNCTION AND AGRICULTURAL APPLICATION As discussed above, there have been numerous reports showing correlations between gene expression patterns and growth properties of various plant organs. Unfortunately, however, there have been only a handful of studies showing a specific causal relationship between them. One of major limiting factors for that is that most plants examined are not amenable to genetic manipulations such as overexpression or downregulation techniques. Second, there is another limiting factor even for those plants subject to genetic modifications, including Arabidopsis and rice. In other words, functional redundancy between expansin genes in the superfamily hinders the eVort to understand what genes exert specific eVects on a given growth and developmental process. To overcome the obstacle, it is necessary to genetically manipulate a group of expansin genes that share a common pattern of expression by using the RNA interference technique. It is also important to select and investigate genes that are expressed in a specific manner. To decide what a specific gene or a specific group of genes can be investigated, it would be greatly helpful to extract relevant information from the global expression data in microarray databases. We found that except three EXPA genes, all the expansin genes of the Arabidopsis are represented in the AYmetrix GeneChip ATH1 (ftp://ftp.arabidopsis.org/home/tair/Microarrays/Datasets/ AtGenExpress). Majority of those expansin genes are highly expressed in many diVerent growth and developmental stages of various organs in an overlapping manner, again substantiating the diYculties expansin researches encounter, as mentioned above. Interestingly enough, however, some of them share a similar expression pattern. In addition, some genes are not

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expressed in almost all the tissues but with an exceptionally high level in a specific tissue. We have also compiled the microarray data retrieved from a plethora of global expression studies (Genevestigator, http://www. genevestigator.ethz.ch) and found overlapping or specific expression patterns between expansin gene members, depending on the nature of treatments and experimentations. Although, because of the size and complexity of the compiled microarray data, detailed profiling and analysis of those expression patterns is beyond the scope of our present review (will be described elsewhere), it would provide a valuable base for further research approaches to understand what genes are specific or general in plant growth and development. To improve fruit quality and to prolong post‐harvest life of fruit, a number of eVorts have been applied. For that purpose, a transgenic approach is a better choice among diverse strategies, as shown in several studies using transgenic tomatoes (Brummell et al., 1999; Kalamaki et al., 2003a,b; Powell et al., 2003). In some crop plants, increased biomass would be a good agricultural trait to which expansins can contribute. Green tea plants with larger leaves have been obtained by treatment of functional fragments of a harpin protein from Xanthomonas oryzae pv. oryzicola (Wu et al., 2007). Three expansin genes were among the genes upregulated by the protein treatment, indicating that those genes may play a role in increasing the productivity of tea plants in the presence of the harpin protein. This result raises the intriguing possibility that a specific inducer of expansin activity could be applied to crop plants to promote increased productivity and thus avoid using a worrisome transgenic approach. Expansins can be used to generate horticulturally valuable ornamental plants with bizarre shapes and unusual size. Zenoni et al. (2004) generated transgenic petunias with flowers of reduced size using an antisense PhEXP1 transgene. Pien et al. (2001) showed that local induction of expansin caused mitten‐shaped leaf margins in tobacco. These results demonstrate that one can modulate the shape or size of plant organs by modulating the expression of expansin genes. To expand the agricultural usefulness of expansins, it is important to understand the exact action mechanism of expansin. In spite of numerous studies, there has been no report of the exact biochemical action mechanism of expansins. This failure is primarily due to the unavailability of a mass production system for biochemically active expansin proteins, truly a bottleneck to expansin research. Therefore, for the next step of expansin research, it is necessary and urgent to develop such a mass production system for active expansins.


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ACKNOWLEDGMENTS This study was carried out with the support of Forest Science & Technology Projects (Project No. S110707L0501D01) provided by Korea Forest Service (Y. L.) and supported partly by KRF‐2006–C00264 (J. H. K) and research funds of Kunsan National University (D.C.).

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Molecular Biology of Orchid Flowers: With Emphasis on Phalaenopsis

WEN‐CHIEH TSAI,* YU‐YUN HSIAO,{ ZHAO‐JUN PAN,{ CHIA‐CHI HSU,{ YA‐PING YANG,{ WEN‐HUEI CHEN,{ AND HONG‐HWA CHEN{,}

*Department of Biological Sciences and Technology, National University of Tainan, Tainan 700, Taiwan { Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan { Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan } Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan

I. Introduction to Orchid Flower Biology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Evolutionary Trends in the Orchid Flower................................ B. Orchid Pollination Strategies ................................................ II. Recent Progress in Orchid Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Genome‐Size Estimation for Various Orchids ............................ B. Karyotype Determination for Phalaenopsis Orchids..................... C. Chloroplast Genome of Phalaenopsis aphrodite SUBSP. Formosana............................................................ D. Expressed Sequence Tags..................................................... E. Bacterial Artificial Chromosome Libraries ................................ F. Molecular Markers ............................................................ G. Microarray Analysis for Orchid Floral Biology .......................... H. Virus‐Induced Gene Silencing for Functional Genomics Study of Orchid Floral Genes .......................................................... I. Genetic Transformation System for Various Orchids ................... Advances in Botanical Research, Vol. 47 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

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0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(08)00003-7

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III. Molecular Biology of Orchid Floral Development . . . . . . . . . . . . . . . . . . . . . . . . A. A‐Class Genes in Orchids .................................................... B. B‐Class Genes in Orchids .................................................... C. C‐ and D‐Class Genes in Orchids ........................................... D. E‐Class Genes in Orchids .................................................... IV. Regulation of Orchid Flowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. External and Internal Regulation of Flowering .......................... B. Gene Regulation of Floral Development in Orchids..................... V. Molecular Biology of Orchid Scent Production . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Significance of the Orchid Floral Scent .................................... B. Investigation of Orchid Scent Components ............................... C. Difficulties in Orchid Scent Research ...................................... D. Molecular Aspects of Floral Scent.......................................... E. Molecular Research in Scent Metabolism of P. bellina.................. VI. Molecular Biology of Orchid Flower Colour Presentation . . . . . . . . . . . . . . . . A. Orchid Flower Colour ........................................................ B. Orchid Flower Colour Biosynthesis Genes ................................ VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Orchidaceae constitutes one of the largest families in angiosperms. The versatility and specialization in orchid floral morphology, scent and colour patterns endear orchidologists and plant biologists to orchid plants. Moreover, the co‐evolution of the sophisticated orchid floral presentation and pollinators leads to the ingenious device of the orchid flower. Because of market needs and the current level of breeding technologies, the industry for biotech seedling products of Phalaenopsis spp. in Taiwan is currently focussed on the development of orchid species. Research into the molecular regulatory mechanism of floral development, scent production and colour presentation can undoubtedly enhance the understanding of orchid floral biology. Owing to advances in genomics and functional genomics, such as karyotypes, expressed sequence tags, bacterial artificial chromosomes and molecular markers, the isolation and identification of orchid floral genes has been increased rapidly. Orchid floral development was revealed to involve MADS‐box‐containing transcriptional regulators. The modified ‘ABCDE model’ of duplication and diversification of MADS‐box genes has been proposed as a major driving force behind orchid floral organ identities. The scent biosynthesis pathway in the Phalaenopsis bellina flower was unravelled and found to be controlled by geraniol and linalool metabolism. Further interest has been promoted by the recent expansion of studies of orchid floral molecular biology. This information will provide broad scope for study of orchid floral development and serves as a starting point for uncovering the mystery of orchid evolution.

I. INTRODUCTION TO ORCHID FLOWER BIOLOGY A. EVOLUTIONARY TRENDS IN THE ORCHID FLOWER

With more than 270,000 known species, angiosperms are by far the most diverse and widespread group of plants. The ancestry of angiosperms is still uncertain. Fossil records show that the angiosperms appeared at the early

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Cretaceous, about 130 million years ago. By the end of the Cretaceous, 65 million years ago, angiosperms radiated and became the dominant plants on earth, as they are today. The origin and diversification of angiosperms— what Charles Darwin characterized as ‘an abominable mystery’—has been the subject of much speculation for the last 100 years (Cronquist, 1988; Darwin, 1903; Doyle, 1994). The rapid explosion in diversity that followed their origin in the early Cretaceous may be linked to modularity within their new structure, the flower (Carroll, 2001). The flower is the defining reproductive adaptation of angiosperms and is the predominant source of characters for angiosperm taxonomy and phylogeny reconstruction (Doyle, 1994). Like all other living organisms, present‐day orchids have evolved from ancestral forms as a result of selection pressure and adaptation. They show a wide diversity of epiphytic and terrestrial growth forms and have successfully colonized almost every habitat on earth. The radiation of the orchid family has probably taken place in a comparatively short period in comparison to that of most flowering plant families, which had already started to diversify in the mid‐Cretaceous (Crane et al., 1995). The time of origin of orchids is in dispute, although Dressler suggests that they originated 80–40 million years ago (late Cretaceous to late Eocene). Perhaps the only general statement that can be made about the origin of orchids is that most extant groups are probably very young. Recently, dating the origin of the Orchidaceae was achieved with a fossil orchid and its pollinator. The authors showed that the most recent common ancestor of extant orchids lived in the late Cretaceous (76–84 Mya) (Ramirez et al., 2007). Containing more than 20,000 species, the Orchidaceae family, classified in class Liliopsida, order Asparagales, is one of the largest angiosperm families. According to molecular phylogenetic studies, Orchidaceae comprise five subfamilies, including Apostasioideae, Cypripedioideae, Vanilloideae, Orchidoideae and Epidendroideae. Associated with this enormous size is an extraordinary floral diversity. Orchids are extremely rich in species, and speciation rates are presumed to be exceptionally high (Gill, 1989), which suggests that orchids are still actively evolving. Although this spectacular diversification has been linked to the intimate and sometimes bizarre interaction of many species with their pollinators (Darwin, 1885), we still face the challenge of explaining how these mechanisms work and the driving forces behind their evolution. Although staggering variation in orchid floral form has long attracted the interest of evolutionary biologists, Orchidaceae represent an unusually coherent group among monocots, possessing several reliable floral morphological synapomorphies, including a gynostemium, or column, fused by the style and at least part of the androecium; a highly evolved petal, the labellum and resupination caused by 1808 torsion of the pedicel (Rudall and Bateman, 2002).

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In addition to floral morphological synapomorphies, several unique reproductive strategies contribute to the success of the orchid family. These include mature pollen grains packaged as pollinia, pollination‐regulated ovary/ovule development, synchronized timing of micro‐ and mega‐ gametogenesis for eVective fertilization, and the release of thousands or millions of immature embryos (seeds without endosperm) in mature pods (Yu and Goh, 2001). Despite their unique developmental reproductive biology, as well as specialized pollination and ecological strategies, orchids remain under‐represented in molecular studies relative to other species‐rich plant families (Peakall, 2007). Among the five subfamilies of Orchidaceae, Epidendroideae is the main subfamily studied at the molecular level. Because only a few species are being studied, so far the main studies deal with genomics, functional genomics, floral morphology, flower fragrance and colour. B. ORCHID POLLINATION STRATEGIES

The ability to attract animals to their flowers can be an important component of fitness for plants that rely on biotic pollination. Colour, shape, size and fragrance are the flower signals that attract pollinators. Pollinators take these signals as indicators of quality or quantity of reward. The spectacular floral diversity of orchids has often been attributed to their intricate and intimate interactions with their pollinators, and pollinator specificity has been considered as the main ethological, prezygotic reproductive isolation mechanism among orchid species (Dressler, 1993; Gill, 1989). The orchid family is renowned for its enormous diversity of pollination mechanisms and unusually high occurrence of non‐rewarding flowers as compared with other plant families (Jersa´kova´ et al., 2006). Arguably, the most fascinating aspect of the orchid pollination strategy is deceit, which was discovered by Sprengel (Cozzolino and Widmer, 2005): to attract pollinators, orchids advertise general floral signals that are typical rewarding features such as inflorescence and/or floral shape, flower colour, scent, nectar guides, spurs and pollen‐like papillae (Galizia et al., 2005; Gumbert and Kunze, 2001). For example, orchids with food‐deceptive floral mimicry attract pollinators that use mainly colour, rather than scent, as their primary foraging cue (Anderson et al., 2005; Johnson et al., 2003). Some orchids mimic oviposition sites by presenting a brownish or dull reddish floral colour and foul odours to attract flies for pollination (Borba and Semir, 2001; Proctor et al., 1996). Orchid flowers that use sexual deception to attract male insects possess not only odours as a long‐range signal but also visual (labellum shape) and tactile cues (Bergstro¨m, 1978; Schiestl et al., 1999). Most orchids are allogamous (cross‐pollinated) and animals are the vectors, and therefore, the orchid


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genome is highly heterozygous. Orchid floral presentation is under developmental control and is commonly linked to fertility status. Revealing the molecular biology of the orchid flower will lead the way to understanding orchid evolution.

II. RECENT PROGRESS IN ORCHID GENOMICS Orchidaceae have diverse, specialized pollination and ecological strategies and provide a rich subject for investigating evolutionary relationships and developmental biology. In addition, the Phalaenopsis orchid is one of the most popular ornamental flowers exported worldwide and has been targeted by Taiwan government for delicate agricultural development. With the availing of genomics and functional genomics tools, the study of genomics and other aspects of orchids, with an emphasis on Phalaenopsis, has progressed profoundly (Chen and Chen, 2007). A. GENOME‐SIZE ESTIMATION FOR VARIOUS ORCHIDS

An accurate determination of genome size provides basic information for breeders and molecular geneticists. Interspecific comparison of nuclear DNA amounts is also useful in cytotaxonomic and evolutionary studies. The state‐ of‐the‐art technique to estimate genome size is the use of DNA‐specific fluorescent dyes in flow cytometry analysis. DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathways and estimation of absolute DNA amount or genome size (Dolezel and Bartos, 2005). Lin et al., using this technique to estimate nucleic DNA content, found within 18 Phalaenopsis species a 6.07‐ fold variation in genome size ranging from 2.74 pg/2C (1322 bp/haploid) for P. sanderiana to 16.61 pg/2C (8014 Mbp/haploid) for P. parishii (Lin and Lee, 2007; Lin et al., 2001). The two native species of Phalaenopsis in Taiwan, P. aphrodite subsp. formosana and P. equestris, have a genome size of 2.80 pg/2C (1351 Mbp/haploid) and 3.37 pg/2C (1626 Mbp/haploid), respectively, that is, about three‐ to fourfold larger than the rice genome size (430 Mbp/haploid) (Lin and Lee, 2007; Lin et al., 2001). Interestingly, while estimating genome size for Phalaenopsis orchids, Lin et al. found widespread endoreduplication in orchid plants. Especially, floral buds and opened flowers in an inflorescence showed diVerent tendencies of endoreduplication. The proportion of endoploidy in cells increased with increasing size of a floral bud and in the fully opened flowers.

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The authors examined this phenomenon nicely and designed a model to describe the relation between endoreduplication and cell growth and suggested that endoreduplication in Phalaenopsis is a contributing factor to cell growth (Lee et al., 2004). The genome size of 37 Dendrobium species and 33 species from 25 other genera, including some important commercially valuable genera such as Cattleya, Cypripedium, Encyclia, Epidendrum, Oncidium, Vanda and Vanilla, has been estimated (Jones et al., 1998). The Dendrobium genome ranges from 1.53 to 4.23 pg/2C (738–2041 Mbp); Catellya and Vanilla have a relatively large genome size, ranging from 3.29 to 9.29 pg/2C (1587–4482 Mbp) and 14.45–15.19 pg/2C (6972–7329 Mbp), respectively. Recently, 54 genome sizes for species in the subtribe Oncidiinae underwent phylogenetic analysis to evaluate the correlation between genome size and life history traits (Chase et al., 2005). The authors’ results supported the hypothesis of a ‘large genome constraint’ that suggests that a large genome may play a role in both ecological and evolutionary constraints (Bennett and Leitch, 2005).

B. KARYOTYPE DETERMINATION FOR PHALAENOPSIS ORCHIDS

Karyotypes of native Phalaenopsis species were analysed by use of Feulgen‐ stained somatic metaphase chromosomes prepared from root tips (Kao et al., 2001, 2007). The authors found that all nine native species examined were diploids, with a chromosome number of 2n ¼ 38, but varied in chromosome size and centromere position. The chromosomes of five species P. aphrodite, P. stuartiana, P. equestris, P. cornu‐cervi and P. lueddemanniana are small and uniform in size (1–2.5 m), and all are metacentric or submetacentric. In contrast, two species, P. venosa and P. amboinensis, possess small, medium and large chromosomes, and most of these are subtelocentric or acrocentric. The karyotype of P. violacea is 6 small, 6 medium and 26 large chromosomes and that of P. mannii is comparatively more symmetrical; most of its chromosomes are metacentric or submetacentric. The amount of constitutive heterochromatin (CH) in the cell cycle from interphase was estimated and compared in Phalaenopsis to understand the causes of karyotype variation in these species. The value measured as the percentage of the total area of CH in the interphase nucleus showed a range of 15‐fold variation, from 1.77% in P. stuartiana to 27.17% in P. violacea (Kao et al., 2001). In addition, the amount of CH was positively correlated with the nuclear DNA content reported by Lin et al. (2001), and thus the authors suggested that diVerential accumulation of CH may be a major cause for karyotype variation in Phalaenopsis orchids (Kao et al., 2001).


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Several research groups have made significant progress in Phalaenopsis genomics during the last few years. However, high heterozygosity of Phalaenopsis genome might be a problem for a future genome projects. One can create double‐haploid Phalaenopsis species by using pollinia as materials in a well‐established tissue culture system for mapping and establishing an orchid genome project. In addition, orchid species, such as Phius tankervilleae and Cattleya aurantiana, are also noteworthy because of their autogamy.

C. CHLOROPLAST GENOME OF PHALAENOPSIS APHRODITE SUBSP. FORMOSANA

Although the chloroplast genomes are conservative in plants, comparative genomic studies of chloroplast genomes are useful for phylogenetic studies (Chang et al., 2006) and provide useful information for designing expression cassettes for chloroplast genetic manipulation and elucidating functional genomics (Burrows et al., 1998; Daniell et al., 2002; Drescher et al., 2000). The entire circular double‐stranded chloroplast genome of P. aphrodite subsp. formosana is 148,964 bp separated into two regions by a pair of inverted‐repeat regions (IR) of 25,732 bp (Chang et al., 2006). The chloroplast genome encodes 110 scattered genes (not including genes duplicated in the IR), including 76 protein‐coding genes, 4 ribosomal RNA genes and 30 transfer RNA genes. In addition, 24 open reading frames (ORFs) were identified to have a threshold of 225 bp. The chloroplast genome of Phalaenopsis completely lost the ndhA, ndhH and ndhF genes, and the other eight ndh genes show various degrees of nucleotide insertion/deletion and are all frame‐shifted. This characteristic is quite diVerent from that of other chloroplast genomes of photosynthetic angiosperms, which have a complete set of genes for the 11 subunits of NADH dehydrogenase. This loss results in the shortest SSC region in the chloroplast genome of Phalaenopsis among known photosynthetic angiosperms (Chang et al., 2006). On systematic examination of RNA‐editing sites in transcripts of 74 known protein‐coding genes in the chloroplasts of P. aphrodite subsp. formosana, 44 editing sites were identified in 24 transcripts, the highest reported in seed plants (Zeng et al., 2007). Twenty‐one of the editing sites are unique to Phalaenopsis orchids as compared with other seed plants. All editing is C‐to‐U conversion, and 42 editing sites bring about changes in amino acids. One of the remaining two editing sites occurs in the transcripts of the ndhB pseudogene and the other is in the 50 untranslated region of psbH transcripts (Zeng et al., 2007).

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Functional genomics studies of non‐model organisms to reveal functions of genes have been a big challenge in biology (Costa et al., 2005; Gewin, 2005). Few molecular studies of orchid biology exist because of the large genome size, ineYcient transformation system, long regeneration time and long life cycle (Kao et al., 2001; Lin et al., 2001). Recently, expressed sequence tags (EST) have appeared as a powerful tool for genomics research. Over 5 million plant EST sequences are now publicly available, in collections of more than 5000 sequences for more than 60 plant species (van de Peer, 2004). However, the genomic resources available for studying plant biology are concentrated mainly among the major crops and their experimental models (such as Arabidopsis). ESTs generated by large‐scale single‐pass cDNA sequencing have been valuable for discovering novel genes in specific metabolic pathways. More recently, two Phalaenopsis orchid floral bud EST databases derived from a scented species, P. bellina, and a scentless species, P. equestris, were constructed (Hsiao et al., 2006; Tsai et al., 2006; Table I). The relative EST frequencies based on functional categories among floral tissues of five species, including P. equestris, Acorus americanus, Asparagus oYcinalis, Oryza sativa and Arabidopsis thaliana, were found to be very similar across these species. These findings suggest that the basic floral transcriptomes are parallel among the flowering plants from basal monocots to monocots and eudicots (Tsai et al., 2006). ESTs from P. bellina and P. equestris were also used to compare gene expression profiles for deduction of the monoterpene biosynthesis pathway in orchids (Hsiao et al., 2006; Tsai et al., 2006). In addition, 1080 subtractive ESTs derived from Oncidium leaves and psuedobulbs were obtained to dissect the genes related to metabolism in pseudobulbs (Tan et al., 2005;

TABLE I Expressed Sequence Tags from Orchidaceae Subfamily

Species

Organ

Epidendroideae Epidendroideae

Sedirea japonica Oncidium cv. ‘Gower Ramsey’

15 1080

Kim et al., 2002 Tan et al., 2005

Epidendroideae

Phalaenopsis equestris Phalaenopsis bellina

Flower Pseudobulb and its upper leaf Floral bud

5604

Tsai et al., 2005

Floral bud

2359

Hsiao et al., 2006

Epidendroideae

Number

Reference


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Table I). Multidimensional analysis of EST data from various plant species can provide further insight into not only identification and annotation of anonymous genes but also identification of novel regulatory elements and representative genes involved in metabolic pathways. E. BACTERIAL ARTIFICIAL CHROMOSOME LIBRARIES

To facilitate the isolation and characterization of functional genes important to orchids, two large‐insert (BAC) libraries have been constructed from genomic DNA of the Taiwan native diploid orchid species P. equestris. From cell flow cytometry, this species shows a relatively small genome size among the various orchids tested and is widely used in breeding programmes for its colourful flower. One of the libraries was constructed from DNA partially digested with Hin dIII and consists of 100,992 clones with an average insert size of 100 kb. The other library was constructed from DNA partially digested with Bam HI and consists of 33,428 clones with an average insert size of 111 kb. These two libraries together represent approximately 8.4 equivalents of the wild Phalaenopsis orchid haploid genome. Both libraries were evaluated for contamination with organellar DNA sequences and were shown to have a very low percentage of clones (0.61%; 0.13%) derived from the chloroplast genome. To estimate the possibility of isolating specific clones, high‐density filters prepared from the two libraries were screened with single or low‐copy gene‐specific probes. Initial screening of the libraries revealed an average of 5.87 positive BAC clones per gene for the Hin d III library. The results agree with the library’s predicted extent of coverage of the orchid genome and indicate that the libraries are useful resources for the molecular isolation of economically important genes in wild orchid species (Kuo et al., 2008). F. MOLECULAR MARKERS

Molecular markers have been developed in abundance for important crops such as O. sativa, Triticim aestivum, Zea mays and Hordeum vulgare. With the available molecular marker techniques, fingerprinting for specific species, genetic mapping for linkage‐targeted phenotypes and for genetic diversity for species, and resources are abundant. In many plant breeding strategies, existing varieties are often used to breed new varieties. However, diVerent cultivars cannot easily be distinguished by the characteristics visible early during developing stages until flowering. Restriction enzyme digestion‐based and PCR‐based DNA markers provide reliable means for identification because of their polymorphism and independence from environmental

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factors. The phylogenetic relations of the Korean native Cymbidium have been analysed by the use of randomly amplified polymorphic DNA (RAPD) (Choi et al., 1998). RAPD study of genetic diversity and identification of Cymbidium cultivars with use of 15 primers has amplified 132 fragments containing 78% polymorphism (Obara‐Okeyo and Kako, 1998). Amplified fragment length polymorphism (AFLP) markers, which need no prior sequence information, are reliable and have high reproducibility. AFLP analysis of Vandaceous revealed that diVerent siblings from the same cross contain 10% polymorphic bands, and the somaclonal variations from the stem tip of the same species contain 0.3%–0.7% polymorphic bands (Chen et al., 1999). Three sequence‐tag‐site (STS) molecular markers converted from AFLP fingerprinting were successful in diVerentiating three Oncidium hybrids (O. Gower Ramsey, O. Sweet Sugar and O. Sharry Baby) (Chen et al., 2006). Chloroplast inheritance based on molecular markers has been tested with a few intergenic hybrid individuals of Phalaenopsis and Doritis orchids, and results suggest that maternal inheritance predominates (Chang et al., 2000). A single‐seed PCR protocol was used to examine the chloroplast DNA inheritance in a Mediterranean orchid, Anacamptis palustris, for the presence of a highly variable minisatellite repeat located in the tRNALEU intron in the chloroplast genome (Cafasso et al., 2005). Approximately 100 viable seeds from each capsule were investigated individually. All examined seeds displayed the maternal cpDNA haplotypes. The internal transcribed spacer regions (ITS1, 5.8S rDNA and ITS2) of the nuclear ribosomal DNA were sequenced from 53 Phalaenopsis species, and a phylogeny was developed on analysis of the molecular data (Tsai et al., 2005). As well, a phylogeny was constructed by use of multiple chloroplast markers, matK, atpH‐atpF and trnD‐trnE (Padolina et al., 2005). Recently, Kao et al. (2007) identified (GA)n microsatellites and mapped them physically to seven Phalaenopsis species and Doritis pulcherrima. In addition, one family of tandem repeated sequences of Pvr I rich in AT at the 50 and 30 ends and consisting of 7‐bp repeat units organized in 13–22 copies in tandem was isolated from P. violacea (Kao et al., 2007). Further attempts to identify microsatellite markers for species identification in Phalaenopsis were achieved by Wu et al. (unpublished data). With the available above‐ mentioned molecular markers, one could construct a genetic map for Phalaenopis orchids. However, obstacles may reside in the cost, diYculties in experimental breeding and long life cycle of Phalaenopis orchids. Another in‐borne problem for an orchid genome project is that the P. equestris used for the construction of both EST and BAC libraries has high hetrozygosity rather than homozygosity.


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G. MICROARRAY ANALYSIS FOR ORCHID FLORAL BIOLOGY

DNA microarrays have become indispensable for functional genomics because they can be used to systematically investigate transcriptional profiles. Various DNA microarray and chip techniques have been developed for the analysis of important organisms (Mockler and Ecker, 2005; Stoughton, 2005). In plants, DNA microarrays and chips have been applied for studies of stress tolerance, growth and metabolic pathways. These are very powerful systems for the study of model systems such as Arabidopsis and rice because of the availability of the complete genome sequences (Arabidopsis Genome Initiative, 2000; International Rice Genome Sequencing Project, 2005), and a large number of full‐length cDNAs have been collected (Kikuchi et al., 2003; Seki et al., 2002). In addition, bioinformatics tools and public databases [e.g., The Arabidopsis Information Resource (Rhee et al., 2003) and RicePipeline (Yazaki et al., 2004)] have facilitated analysis of the DNA microarray or chip data. So far, plentiful ESTs from Phalaenopsis orchid flower buds have been deposited in public databases (Hsiao et al., 2006; Tsai et al., 2006), and thus, the EST clones or sequence data are currently being used to construct cDNA microarrays or oligo‐DNA chips. These microarrays allow for global gene profiling of the orchid flower and can reveal the regulatory mechanism of orchid flower molecular biology.

H. VIRUS‐INDUCED GENE SILENCING FOR FUNCTIONAL GENOMICS STUDY OF ORCHID FLORAL GENES

In RNA interference (RNAi), double‐stranded RNA (dsRNA) specifically suppresses the expression of a gene with its complementary sequence. Short interfering dsRNAs (siRNAs) mediate post‐transcriptional gene silencing and can be used to induce RNAi in many organisms. Experimentally, these molecules can be produced through transcription of inverted‐repeat transgenes or delivered directly into cells. The use of siRNAs has emerged as a technique to knock out expression of specific genes in various organisms and provides a powerful method for studying gene function. An alternative approach to plant loss‐of‐function assay is virus‐induced gene silencing (VIGS). Compared with transforming plants with sense and/or antisense genes, VIGS is much faster. Because of the host range limitation of the currently constructed virus vectors, application of VIGS is restricted to plants such as Arabidopsis, tomato, tobacco, pea, soybean, opium poppy and wheat (Benedito et al., 2004; Constantin et al., 2004; Hileman et al., 2005; Scofield et al., 2005; Zhang and Ghabrial, 2006).

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For functional validation of orchid genes, the most intriguing and challenging studies are of genes involved in reproductive stages because of the diversity of orchid flowers and because the transition from vegetative to reproductive stages in orchids usually takes about 2–3 years. In addition, orchids usually bloom only once a year, which limits the time for conducting experiments. To test whether the CymMV vector could induce gene silencing in all floral organs, a B‐class MADS‐box family gene, PeMADS6, transcribed in all flower organs was selected with a stretch of 150 nt in the 30 region. Lu et al. directly inoculated the constructed CymMV vector into emerging stalks with six nodes (about 8 cm) of Phalaenopsis amabilis var. formosa. Approximately 6 weeks later, the flowers blossomed. Compared with mock‐inoculated plants, plants inoculated with the constructed CymMV vector showed the PeMADS6 RNA level reduced in sepals, petals, lips and columns, to 63 2%, 33 3%, 23 5% and 33 2%, respectively, on real‐time PCR (Lu et al., 2007). Furthermore, small‐molecular weight RNA from inoculated plants was extracted and analysed by northern blot hybridization. PeMADS6 21‐nt siRNA was detected only in plants inoculated with the constructed CymMV vector containing the PeMADS6 region (Lu et al., 2007). Thus, the generation of PeMADS6 siRNA is specific, and the reduction of PeMADS6 RNA level is through the gene‐silencing mechanism. However, PeMADS6‐silenced plants showed no visible morphological changes, perhaps because the knockdown level induced by VIGS was not enough to induce flower morphological change. Knockdown of MADS‐box family genes by inserting a 500‐bp DNA fragment of a PeMADS6 conserved region of the MADS‐box family of genes into CymMV vector resulted in blossoming flowers in inoculated plants, but streaks or patches of greenish tissue appeared in sepals, petals and lips (Lu et al., 2007). With loss‐of‐ function assays such as T‐DNA insertion or transposon tagging, simultaneously targeting all genes with functional redundancy is diYcult. In contrast, VIGS can knock down the RNA level after RNA transcription, regardless of the RNAs transcribed from genes in diVerent genome locations, and thus can silence all genes simultaneously. I. GENETIC TRANSFORMATION SYSTEM FOR VARIOUS ORCHIDS

A crucial challenge in molecular breeding of orchids is the establishment of eYcient and reproducible gene transformation systems. So far, genetic transformation by biolistic bombardment has been conducted in various orchids, including Vanda, Dendrobium (Chai et al., 2007; Chang et al., 2005; Kuehnle and Sugii, 1992; Men et al., 2003), Cymbidium (Yang et al., 1999),


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Phalaenopsis (Belarmino and Mii, 2000) and Oncidium (Li et al., 2005). Although particle bombardment was successfully applied to produce transgenic plants with reporter genes, both the transformation and regeneration rates were relatively low. Agrobacterium tumefaciens‐mediated transformation was used to introduce foreign genes into Dendrobium (Yu et al., 2001), Phalaenopsis (Chan et al., 2006; Mishiba et al., 2005; Sanjaya and Chan, 2007) and Oncidium (Chan et al., 2006; Li et al., 2005; Liau et al., 2003; You et al., 2003). The target tissues are cultured seeds that swell into structures called protocorms and protocorm‐like bodies (PLB) derived from lateral buds, immature flower buds, protocorms or somatic tissues such as shoot tip and leaf. Use of sweet pepper ferredoxin‐like protein (pflp) cDNA, with an HPT II and GUS coding sequence, inserted into PLBs of Oncidium demonstrated the applicability of pflp gene as an antibiotic‐free selection marker in Oncidium (You et al., 2003). Transgenic plants showed enhanced resistance to Erwinia carotovora, which causes soft rot disease, even when the entire plant was challenged with the pathogen (Liau et al., 2003). Chan et al. (2005) used ‘gene stacking’ in Phalaenopsis by double transformation events. First, Phalaenopsis PLBs were transformed with CymMV coat protein cDNA (CP), then transformed with pflp cDNA by A. tumefaciens infection to enable expression of dual (viral and bacterial) disease‐resistant traits. More recently, Chai et al. (2007) reported on a new transformation method with L‐methionine sulfoximine used as a novel agent for selecting transgenic Dendrobium hybrids D. Madame Thong‐In and D. Chao Paya Smile, with the bialaphos resistance (bar) gene used as a selectable marker. The substantial time and economic savings with the new transformation systems that involve pflp and L‐methionine sulfoximine as selection agents may facilitate functional studies of orchid genes.

III. MOLECULAR BIOLOGY OF ORCHID FLORAL DEVELOPMENT For plants, the MADS‐box‐containing transcriptional regulators have been the focus of floral organ specification, development and evolutionary studies (Mu¨nster et al., 1997; Purugganan et al., 1995; Weigel and Meyerowitz, 1994). In the concise ‘ABCDE model’, the organ identity in each whorl is determined by a unique combination of activities, called A, B, C, D and E, of organ identity genes (Theissen and Saedler, 2001; Weigel and Meyerowitz, 1994; Zahn et al., 2005; Fig. 3A). In any one of the four flower whorls, sepals are specified by A and E activity, petals by A, B and E activity, stamens by B, C and E activity, carpels by C and E activity and ovules by C, D and E

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activity. Protein–protein interaction studies have revealed that the MADS‐ box transcription factors are active at the molecular level in a combinatorial manner (Egea‐Cortines et al., 1999; Ferrario et al., 2003; Honma and Goto, 2001; Tsai et al., 2008). The diversification of MADS‐box genes during evolution has been proposed to be a major driving force for floral diversity in land plant architecture (Theissen et al., 2000; Zahn et al., 2005). According to the classical view, the orchid flower is composed of five whorls of three segments, each including two perianth whorls, two staminal whorls and one carpel whorl (Brown, 1810; Figs. 1 and 3B). This structure also conforms to the general flower structure of many other monocotyledonous families. Within the monocots, only well‐known crop species such as rice and maize have been studied thoroughly. However, their highly reduced flowers make them unsuitable for general floral development studies. All expected whorls in the flowers are present in orchids, and their highly sophisticated flower organization oVers an opportunity to discover new variant genes and diVerent levels of complexity within morphogenetic networks. Thus, the Orchidaceae provides a rich subject for investigating evolutionary relationships and developmental biology to verify the validity of the ‘ABCDE model’ in monocots and study how MADS‐box genes are involved in defining the diVerent highly specialized structures in orchid flowers. A. A‐CLASS GENES IN ORCHIDS

To date, almost cloned orchid MADS‐box genes involved in floral development were from Epidendroideae, the largest orchid subfamily containing many more genera and species than all of the other four subfamilies together.

A

B

Fig. 1. Flowers from wild‐type (A) and peloric mutant (B) Phalaenopsis spp. Scale bar ¼ 1 cm.


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So far, four A‐class MADS‐box genes have been identified from Dendrobium. One of them was isolated from D. Madame Thong‐In and named DOMADS2 (Yu and Goh, 2000). The other three genes were cloned from Dendrobium thyrsiflorum and named DthyrFL1, DthyrFL2 and DthyrFL3 (Skipper et al., 2005). The APETALA1/FRUITFULL (AP1/FUL) MADS‐ box gene lineage within the eudicots is recognized as two clades (the euAP1 and euFUL clade), whereas the non‐core eudicots and monocots have sequences similar to only euFUL genes (Litt and Irish, 2003). Sequence analysis showed that these genes contain the C‐terminal FUL‐like motif LPPWML of monocot FUL‐like proteins, but this motif is not present in the sequence of DthyrFL3 (Skipper et al., 2005). Phylogenetic analysis showed that DthyrFL1 and DOMADS2 are orthologous genes; the existence of DthyrFL2 and DthyrFL3 represents a recent duplication event in D. thyrsiflorum (Skipper et al., 2005). DOMADS2 is expressed in both the shoot apical meristem (SAM) and the emerging floral primordium throughout the floral transition and later in the column of mature flowers (Yu and Goh, 2000). The expression pattern of DOMADS2, from the SAM and increase in later stages of floral development, suggests that DOMADS2 is one of the earliest regulatory genes during the transition of flowering. The DthyrFL genes are expressed not only during inflorescence development but also in developing ovules (Skipper et al., 2005). These A‐class genes in orchid may be involved in floral meristem identity and in column and ovule development. Unlike its homologue AP1 in Arabidopsis, DOMADS2 may not be associated with the development of the first two whorls. However, whether the DthyrFL genes are associated with perianth formation is unclear. In addition, a MADS‐box gene OMADS1 in Oncidium orchid involved in floral initiation and formation belongs to the AGL6 subfamily rather than the A‐class genes (Hsu et al., 2003). Transgenic Arabidopsis and tobacco‐overexpressing OMADS1 showed significantly reduced plant size, flowered extremely early and lost inflorescence indeterminacy (Hsu et al., 2003). In Arabidopsis, another A‐class gene without MADS‐box region is APETALA2 (AP2). AP2 encodes a transcription factor with two continuous AP2 domains. AP2 enables A‐class function in Arabidopsis and represses AGAMOUS (AG) in the first and second floral whorls (Jofuku et al., 1994). Only one AP2‐like gene, named DcOAP2, from Dendrobium, has been reported (Xu et al., 2006). Transcripts of DcOAP2 were detected in all the floral organs as was AP2 in Arabidopsis. However, the concurrence of the expression of DcOAP2 and DcOAG1, a putative AG‐like gene in all floral organs, implies that the mechanism underlying the regulation of AG orthologues may be diVerent from that in Arabidopsis (Xu et al., 2006).

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Both the developmental and biochemical aspects of B‐class genes required to specify the identity of petals in whorl 2 and stamens in whorl 3 appear to be conserved in many core eudicots (Irish and Kramer, 1998; Theissen et al., 2000). The B‐class genes in the monocots rice and maize are similar in function to the core eudicots B‐class genes (Ambrose et al., 2000; Lee et al., 2003; Nagasawa et al., 2003; Whipple et al., 2004). The orchid flowers have a petaloid perianth arrangement that could be explained by a ‘modified ABC model’ in that the expression of the B‐class genes has expanded to whorl 1 (van Tunen et al., 1993). In addition, the orchid flowers display an elaborated labellum, which is a highly modified petal. Because the function of A‐class genes is poorly defined in angiosperms, studies of petal development and evolution have generally focussed on B‐class genes (Baum and Whitlock, 1999). The extraordinary floral diversity in orchids may be related to the evolution of B‐class genes. Molecular functions have been studied in more detail in B‐class genes than in any other floral homeotic genes in orchids. So far, various numbers of APETALA3 (AP3)‐like and PISTILLATA (PI)‐like genes have been isolated from several orchids. These genes include one AP3‐like OMADS3 isolated from Oncidium Gower Ramsey, four AP3‐ and one PI‐like genes identified from P. equestris and two AP3‐ and one PI‐like genes cloned from Dendrobium crumenatum (Hsu and Yang, 2002; Tsai et al., 2004, 2005; Xu et al., 2006). All these AP3‐like genes in orchids are members of the paleoAP3 lineage (Fig. 2A). The paleoAP3 genes identified from orchids were subdivided into two subclades. One subclade contains OMADS3, PeMADS5, DcOAP3A and PeMADS2, and the other contains OcOAP3B, PeMADS3 and PeMADS4 (Fig. 2A). This result suggests that the ancestor of Orchidaceae might have had two paleoAP3‐like genes and further gene duplication took place at least in the AP3 clade in the monocots. Interestingly, both OMADS3 and PeMADS5 do not show an obvious paleoAP3 motif, which suggests that they are orthologous genes. Although they share similar expression patterns in orchid floral organs, PeMADS5 is not expressed in vegetative tissues, but OMADS3 is expressed in leaves (Hsu and Yang, 2002; Tsai et al., 2004). Phylogenetic analysis showed that DcOAP3A and PeMADS2 are also orthologous genes (Fig. 2A), but they possess diVerent expression patterns. Similar to OMADS3, DcOAP3A is ubiquitously expressed in all floral organs and in leaves, whereas PeMADS2 is predominantly expressed in sepals and petals (Hsu and Yang, 2002; Tsai et al., 2004; Xu et al., 2006). Recently, we discovered at least three paleoAP3 genes with distinct expression patterns in Oncidium floral organs (unpublished data).

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A

DcOAP3B (Dendrobium crumenatum)

98 86

PeMADS3 (Phalaenopsis equestris) PeMADS4 (Phalaenopsis equestris)

74

LMADS1 (Lilium longiflorum) 71

96

TGDEFA (Tulipa gesneriana) OsMADS16 (Oryza sativa)

66

100

Silky1 (Zea mays) OMADS3 (Oncidium)

76

AP3

PeMADS5 (Phalaenopsis equestris) DcOAP3A (Dendrobium crumenatum)

86

100

PeMADS2 (Phalaenopsis equestris)

100

AP3 (Arabidopsis thaliana)

100

B class

BOBAP3 (Brassica oleracea) 96

DEF (Antirrhinum majus) pMADS 1 (Petunia hybrida)

100

PeMADS6 (Phalaenopsis equestris)

98 78

DcOPI (Dendrobium crumenatum) ORCPI (Orchis italica) LRGLOA (Lilium regale)

99 100

LRGLOB (Lilium regale) OsMADS2 (Oryza sativa)

80

PI

ZMM16 (Zea mays) OsMADS4 (Oryza sativa)

99

ZMM18 (Zea mays)

68 99

ZMM29 (Zea mays)

DOMADS2 (Dendrobium grex Madame Thong-In)

99 91

DthyrFL1 (Dendrobium thyrsiflorum) AIFL (Allium)

50

DthyrFL2 (Dendrobium thyrsiflorum) 99

A class

DthyrFL3 (Dendrobium thyrsiflorum)

87

OsMADS18 (Oryza sativa) LpMADS3 (Lolium perenne)

99

OsMADS1 (Oryza sativa)

100

99

53

OsMADS5 (Oryza sativa) DOMADS3 (Dendrobium grex madame thong-in) SEP4 (Arabidopsis thaliana)

99 100

SEP1 (Arabidopsis thaliana) SEP2 (Arabidopsis thaliana)

90 60

DEFH200 (Antirrhinum majus) FBP2 (Petunia hybrida) SEP3 (Arabidopsis thaliana) DOMADS1 (Dendrobium grex madame thong-In)

55

OM1 (Aranda deborah) 69

DcOSEP1 (Dendrobium crumenatum)

Substitution per 100 residues 0.1

Fig. 2.

(Continued )

E class

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B

PeMADS1 PhalAG1 DthyrAG1 DcOAG1 HAG1 LLAG1 HvAG1 OsMADS3 ZAG1 OsMADS58

C class

HvAG2 WAG NymAG1 PLENA CAG2 SHP1 SHP2 FARINELLI NAG1 ZAG2 ZMM1 OsMADS13 FBP11 FBP7 STK

D class

LMADS2 DcOAG2 ApMADS2 DthyrAG2 Substitution per 100 residues

PhalAG2

0.05

Fig. 2. Phylogenetic relationship of MADS‐box genes of ABCDE class. (A) A‐, B‐ and E‐class genes. (B) C‐ and D‐class genes. Orchid MADS‐box genes are highlighted by open boxes.

Of note, a specialized paleoAP3 gene, PeMADS4, discovered in P. equestris, is specifically expressed in labellum and column that suggests that its function is related to development of orchid labellum and column. Gene duplication is important for generating new genes during evolution (Ohno,


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1970) and may lead to the generation of new organs. Duplication of paleoAP3 like genes in orchids, followed by diversification and specialization of PeMADS4‐like genes, is probably concomitant with the arising of the new floral organ, labellum, in orchids. Overexpression of paleoAP3 genes from Oncidium, Dendrobium and Phalaenopsis under the control of the cauliflower mosaic virus 35S promoter was examined in Arabidopsis (Hsu and Yang, 2002; Xu et al., 2006). Consistently, all results showed the flower morphology of the transgenic Arabidopsis plants overexpressing the orchid paleoAP3 genes indistinguishable from that of the wild‐type plants. Dominant‐negative mutation strategy was further conducted to investigate the functions of OMADS3 and DcOAP3A (Xu et al., 2006); OMADS3 showed a function similar to that of the A‐class genes in regulating flower formation as well as floral initiation, whereas DcOAP3A has a putative B‐class function (Hsu and Yang, 2002; Xu et al., 2006). However, these results may not reflect the real roles these genes play during orchid floral development. Peloric flowers that are actinomorphic mutants with lip‐like petals are widely found in natural populations of species of Veronicaceae, Gesneriaceae, Labiatae and Orchidaceae (Cubas, 2004). From a high frequency of orchid peloric mutants derived from micropropagation, we were able to infer the individual roles played by the diversified paleoAP3 genes in orchids by comparing the expression patterns of the four paleoAP3 genes (PeMADS2, PeMADS3, PeMADS4 and PeMADS5) in wild‐type Phalaenopsis floral organs and in peloric mutants (Tsai et al., 2004). First, we discovered that both PeMADS2 and PeMADS5 were expressed in sepals of wild‐type plants, but only PeMADS2 transcripts were detected in sepals of the peloric mutant, whose morphology was not aVected. This result suggests that PeMADS5 is dispensable, whereas PeMADS2 is crucial for sepal development. Second, the expression of all four PeMADS genes, except PeMADS4, was detected in the wild‐type petals. However, the expression of PeMADS5 was not found in the lip‐like petals of the peloric mutant. This result suggests that PeMADS5 is associated with petal development. Third, the expression of PeMADS4 was concentrated in lips and columns in the wild‐type plant and extended to the lip‐like petals in the peloric mutant. That the PeMADS4 transcript was detected in the lip‐like petal of the peloric mutant suggests that PeMADS4 is required for labellum identity. Fourth, PeMADS3 showed similar expression patterns in the wild‐type plant and the peloric mutant that suggests its important function in inner perianth whorl morphogenesis. So far, only one PI‐like gene have been reported in D. crumenatum, P. equestris and Phalaenopsis spp., DcOPI, PeMADS6 and PhPI10, respectively (Guo et al., 2007; Tsai et al., 2005; Xu et al., 2006). Southern blot hybridization

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results supported that the Phalaenopsis orchid genome contains only one copy of the PI‐like gene (Tsai et al., 2005). DcOPI and PeMADS6 are expressed in all floral organs (Tsai et al., 2005; Xu et al., 2006). However, PhPI10 is predominantly expressed in the lip (Guo et al., 2007), and PeMADS6 is expressed in the undeveloped ovary (Tsai et al., 2005). These results suggest that the expression of PeMADS6 in the ovary has an inhibitory eVect on the development of the ovary, and auxin acts as the candidate signal to regulate repression of PeMADS6 expression there. Furthermore, PeMADS6 is not diVerentially expressed in wild‐type and peloric floral organs, which suggests that PeMADS6 is not responsible for the altered phenotype of the peloric mutant. Ectopic overexpression of DcOPI or PeMADS6 in Arabidopsis demonstrated that both share the angiosperm PI function (Tsai et al., 2005; Xu et al., 2006). Further evidence came from the complementation of the pi‐1 phenotype in Arabidopsis by overexpression of DcOPI and showed that DcOPI is able to substitute PI in Arabidopsis (Tsai et al., 2005). In contrast, PeMADS6 could not complement the pi‐4 mutant (unpublished data). In conclusion, the expression patterns of B‐class genes in orchid floral organs nicely fit the ‘modified ABC model’ in that the expression of the B‐class genes is extended to whorl 1 in plants possessing nearly identical morphology of sepals and petals (van Tunen et al., 1993). The paleoAP3 genes are highly duplicated in the Epidendroideae genome. Diversification and fixation of both these gene sequences and expression profiles might be explained by the subfunctionalization and even neofunctionalization. The driving force behind the specialized labellum and diversified orchid flowers may be linked to the fast evolution rate of paleoAP3 genes. For more insight into the evolutionary history of B‐class genes in orchid, we recently identified DEF/AP3‐like and GLO/PI‐like genes from four subfamilies of Orchidaceae, including Vanilloideae, Cypripedioideae, Orchioideae and Epidendroideae. Results from sequence analyses revealed that the orchid DEF/AP3 can be divided into two clades, PeMADS3/4 and PeMADS2/5, and subdivided into four subclades, PeMADS3, PeMADS4, PeMADS2 and PeMADS5. These four orchid AP3 homologues may have been established by gene duplication and subsequent divergence (unpublished data). Phylogenetic analysis revealed that for the monocot GLO/PI orthologues, those of Orchidaceae appeared to be outside both the two GLO/PI members of Poaceae (OSMADS2/ZMM16 and OSMADS4/ZMM18/ZMM29) and Liliaceae (LRGLOA/LRGLOB). The ancestor GLO/PI homology of Orchidaceae may have occurred at the early time of the monocot evolutionary history and after the separation of Poaceae and Liliaceae. The last common ancestor of Orchidaceae may have contained just one GLO/PI‐like gene. The orchid GLO/PI homologues were recognized as a monophyletic group. The


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original orchid GLO/PI had one copy and a duplicated event occurred after the split of Vanilloideae. The Cypripedioideae appeared to group outside the orchid GLO/PI orthologues of Orchidoideae and Epidendroideae. Further investigation of the GLO/PI orthologue in Apostadioideae will undoubtedly give a better age estimation of the duplication event of the GLO/PI‐like gene in Orchidaceae (unpublished data).

C. C‐ AND D‐CLASS GENES IN ORCHIDS

A gynostemium or column, comprising stamen filaments adnate to a syncarpous style, is normally considered a structure peculiar to the orchids (Dressler, 1993). The development of a column, which involves whorls 3 and 4, would be one of the most interesting subjects to elucidate the evolution of C‐class genes. In most orchid flowers, ovary and ovule development is precisely and completely triggered by pollination, and thus orchids oVer a unique opportunity to study D‐class genes involved in ovule development. More recently, one C‐class gene and one D‐class gene were isolated independently from three orchid species, Phalaenopsis Hatsuyuki (Song et al., 2006), D. thyrsiflorum (Skipper et al., 2006) and D. crumenatum (Xu et al., 2006). PhalAG1, DthyrAG1 and DcOAG1 were classified in the C‐lineage of AG‐like genes, whereas PhalAG2, DthyrAG2 and DcOAG2 were in D‐lineage genes (Fig. 2B). PhalAG1, PhalAG2, DthyrAG1 and DthyrAG2 share similar spatial expression patterns in column, ovary and developing ovules, even though these four genes belong to diVerent lineages (Skipper et al., 2006; Song et al., 2006). One possible explanation is that C‐ and D‐class genes in orchids would act redundantly with each other in floral and ovule development. Although PhalAG1 is expressed in all floral organs at their initiation, its expression quickly decreased and can only be detected in the column and ovary when flowers mature, which is also true for DthyrAG1 (Skipper et al., 2006; Song et al., 2006). We also identified a C‐class gene, PeMADS1, from P. equestris, whose expression pattern was consistent with that of PhalAG1 and DthyrAG1 (unpublished data). In contrast, DcOAG1 is expressed in all mature floral organs, whereas DcOAG2 is expressed in the anther cap and column of D. crumenatum (Xu et al., 2006). The unusual expression pattern of DcOAG1 in monocots suggests that the regulatory mechanism of DcOAG1 is independently evolved in D. crumenatum as in some basal angiosperms such as Illicium and Persea (Kim et al., 2005), but the function of DcOAG1 and Arabidopsis AG is conserved, as supported by the phenotypic similarity between transgenic Arabidopsis expressing either 35S::DcOAG1 or 35S::AG (Xu et al., 2006). The molecular mechanism of orchid gynostemium

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morphogenesis is still a mystery. Mutation of C‐class genes in orchids could possibly shed light on gynostemium morphogenesis. D. E‐CLASS GENES IN ORCHIDS

E‐class genes are required for floral organ identity in all four floral organs and for floral determinacy (Ditta et al., 2004; Kaufmann et al., 2005). They have been shown to form ternary complexes with A‐class and B‐class proteins in a yeast three‐hybrid system and can mediate the interactions between B‐ and C‐class proteins in higher‐order complexes (Honma and Goto, 2001). The first E‐class gene, OM1, was isolated from the supposed bigeneric hybrid Aranda Deborah (Lu et al., 1993). The other three E‐class genes (DOMADS1, DOMADS3 and DcOSEP1) were identified from Dendrobium; two were cloned from D. Madame Thong‐In, and the other was isolated from D. crumenatum (Xu et al., 2006; Yu and Goh, 2000). Phylogenetic analysis showed that OM1 was clustered with DOMADS1 and DcOSEP1, with DOMADS3 separated at a distance from the other three orchid E‐class genes. DOMADS1 RNA is uniformly expressed in both the inflorescence meristem and floral primordium and later exists in all floral organs (Yu and Goh, 2000). The expression pattern of DOMADS1 in mature flowers coincides with that of its counterpart DcOSEP1 in D. crumenatum and with their orthologues in Arabidopsis (Pelaz et al., 2000; Xu et al., 2006; Yu and Goh, 2000). However, OM1 is expressed in mature flowers and not in young developing inflorescence or young floral buds. In mature flowers, it is expressed only in petals and weakly in sepals but not in the column (Lu et al., 1993). Spatiotemporal expression diVerences imply a functional diversification among these genes that is closely related to phylogeny. DOMADS3 transcription begins in the early SAM at the stage before the diVerentiation of the first flower primordium and later can be detected only in the pedicels (Yu and Goh, 2000). The DOMADS3 may function as a regulatory factor in early floral transition and in the development of the pedicel. The expression of E‐class genes overlaps with that of ABC genes in orchids that suggests that the higher‐order MADS‐box complexes are involved in orchid floral development. Recently, one line of evidence that MADS‐box proteins form higher‐order complexes comes from the formation of DcOAP3A‐DcOPI‐ DcOSEP1 and DcOAP3B‐DcOPI‐DcOSEP1 detected by yeast three‐hybrid experiments (Xu et al., 2006). According to these results, we propose a model to explain how MADS‐box genes are involved in defining the highly specialized tepal formation in Phalaenopsis (Fig. 3B). In this ‘orchid tepal model’, we proposed that E‐class DcOSEP1‐like proteins combined with B‐class PeMADS2‐like and

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A

LFY

AP3 PI B class AG

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D class E class Floral organ

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Pe2 SEP

Pe3 Pe5

Pe3 Pe4

Pe2 SEP

Pe3 Pe4

Pe3 Pe4

SEP Pe6

SEP Pe6

SEP Pe6

SEP Pe6

SEP Pe6

SEP Pe6

Fig. 3. Arabidopsis genes regulating flower development. (A) Arrowheads indicate activation and bars indicate repression. Genes involved, at least in part, in the same functions are boxed. In many cases, the eVect may be indirect and the line connecting two genes may represent a chain of events. (B) The ‘Orchid Tepal Model’. In this model, E‐class DcOSEP1‐like proteins combine with B‐class PeMADS2‐like and PeMADS6‐like proteins could specify sepal formation, DcOSEP1‐like proteins combine with B‐class PeMADS3‐like, PeMADS5‐like and PeMADS6‐like proteins could determine petal development and DcOSEP1‐like proteins combine with B‐class PeMADS3‐like, PeMADS4‐like and PeMADS6‐like could specify lip formation in the wild‐type orchid flower (Panel B). The proposed model also illustrates that the formation of ectopic lips at the original petal position could be due to the ectopically expressed PeMADS4‐like interacting with PeMADS5‐like, PeMADS6‐like and DcOSEP1‐like E‐class protein in the peloric orchid flower.

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PeMADS6‐like proteins could specify sepal formation, DcOSEP1‐like proteins combined with B‐class PeMADS3‐like, PeMADS5‐like and PeMADS6‐ like proteins could determine petal development and DcOSEP1‐like proteins combined with B‐class PeMADS3‐like, PeMADS4‐like and PeMADS6‐like could specify lip formation in the wild‐type orchid flower (Fig. 3B). The proposed model also illustrates that the formation of ectopic lips at the original petal position could be due to the ectopically expressed PeMADS4‐ like interacting with PeMADS5‐like, PeMADS6‐like and an DcOSEP1‐like E‐class protein in the peloric orchid flower (Fig. 3B). The model also implies that PeMADS4 is a lip identity gene. These findings are in line with current thoughts on how major evolutionary changes in the genetic basis of organ identity were established by gene duplication and the separation of functions. One note also should be cautioned: we could not be sure that all the genes within each family have been isolated in orchids.

IV. REGULATION OF ORCHID FLOWERING A. EXTERNAL AND INTERNAL REGULATION OF FLOWERING

The genetic analyses of ‘flowering‐time’ mutants illustrated four genetic pathways controlling the floral transition from vegetative phase to reproductive phase. The photoperiod pathway and the vernalization pathway mediate signals from the environment. The autonomous pathway probably monitors endogenous cues from the developmental state. Genes involved in hormone biosynthesis and hormone signal transduction have been suggested to form a distinct hormone promotion pathway (Simpson et al., 1999). The vernalization and autonomous pathways promote the floral transition by reducing the level of the floral repressor FLOWERING LOCUS C (FLC) (Reeves and Coupland, 2000; Sheldon et al., 2000) that negatively regulates two ‘flowering‐time’ genes: FLOWERING LOCUS T (FT) (Kardailsky et al., 1999; Kobayashi et al., 1999; Samach et al., 2000) and SUPPRESSOR OF OVEREXPRESSION OF CO1/AG‐LIKE20 (SOC1/ AGL20) (Lee et al., 2000; Samach et al., 2000). The photoperiod pathway mediates signals from long‐day photoperiods and acts via a transcription factor, CONSTANS (CO) (Araki, 2001; Reeves and Coupland, 2000), which directly activates FT and SOC1/AGL20 (Samach et al., 2000). The gibberellin (GA) pathway has an essential role in SD conditions (Simpson et al.,


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1999; Wilson et al., 1992). The balance between CO and FLC activity may determine the levels of FT and SOC1/AGL20 expression. The photoperiod and GA pathways control the floral meristem identity gene LFY by mediating diVerent cis elements on the LFY promoter (Blazquez and Weigel, 2000). The comparison of flowering regulation between Arabidopsis and monocots has been reviewed by Dennis and Peacock (2007) and not further be discussed here. Several researches have been reported that a number of factors such as heredity, chemical factors, growth rate, nutritional state of the plant, thermoperiodism and photoperiodism determine the flowering time of orchids (Arditti, 1992). However, most of these studies focussed on the physiological level instead of molecular level (Vaz et al., 2004). Although an AGL‐like gene OMADS1 isolated from Oncidium has ability to upregulate FT, SOC, LFY and AP1 expression in transgenic Arabidopsis (Hsu et al., 2003), the actual role of the gene in Oncidium still need to be investigated. However, the long life cycle of orchid and ineYcient transformation systems obstruct the proceeding of functional analyses. Combining the eYcient transformation systems with in vitro flowering system of Psygmorchis pusilla or Dendrobium Madame Thong‐In will advance our understanding of orchid flowering regulation (Vaz et al., 2004; Yu and Goh, 2000). The identification of functional promoters of MADS‐box genes not only will provide important information about the environmental and developmental factors aVecting the regulation of MADS‐box gene expression but also will help to further elucidate the role of this group of genes in plant evolution. B. GENE REGULATION OF FLORAL DEVELOPMENT IN ORCHIDS

Except for the homologue of LFY gene that has been isolated from Orchis italica, so far, no other genes upstream of the ABC model genes have been isolated (Montieri et al., 2004). OrcLFY was isolated by Montieri et al., and the coding regions of 14 orchid species were compared. Their study showed that purifying selection acts on this gene in these orchids. However, no functional analysis results exist for these genes in orchid or Arabidopsis. In addition, ectopic expression of the AGL6‐like OMADS1 in Arabidopsis shows the upregulated expression of the flowering‐time genes FT and SOC1 and flower meristem identity genes LFY and AP1 (Hsu et al., 2003). Transgenic plants ectopically expressing 35S::OMADS1 compensate the late‐flowering defect in gi‐1 or co‐3 mutant plants but not in ft‐1 and fwa‐3

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plants, so FT may be the target for OMADS1 in transgenic Arabidopsis plants. In D. Madame Thong‐In, Yu et al. (2002) isolated and sequenced a 3.5‐kb E‐class DOMADS1 promoter fragment upstream of the transcription start site and predicted the CArG‐box motif and DNA‐binding core sequences (TGAC) of the class 1 knox genes (Yu et al., 2002). Deletion analysis of the DOMADS1 promoter fusion with use of ‐glucuronidase (Gustafson‐Brown et al., 1994) involved the stable orchid transformation systems and assay for the spatial and temporal expression by GUS histochemical assay and RNA gel blot analysis. GUS staining of the full‐length 3.5‐kb promoter sequence well matched previous results of DOMADS1 expression in wild‐type orchid plants, whereby DOMADS1 RNA is uniformly expressed in both the inflorescence meristem and floral primordium and later exists in all floral organs (Yu and Goh, 2000). GUS staining was gradually reduced in flowers and was at a high level in vegetative tissues during the progressive deletion of 50 upstream regions, which suggests that the promoter region between nucleotides 3483 and 110 should contain enhancer(s) in promoting the gene expression in flowers and repressor(s) in suppressing the gene expression in vegetative tissues. The authors also suggest the minimal promoter regions required for normal expression in wild‐type plants and the promoter regions containing cis‐acting elements for spatial and temporal regulation of DOMADS1 expression in individual tissues (Yu et al., 2002). The region between nucleotides 214 and 110 shows temporal regulation at the SAM; that between nucleotides 322 and 214 shows a negative eVect in leaves; that between nucleotides 519 and 323 shows temporal regulation at the SAM and positive regulation at the flower; that between nucleotides 930 and 519 shows spatial regulation at the SAM, inflorescence and flower; that between nucleotides 1189 and 930 shows positive regulation at the inflorescence apex; that between nucleotides 1663 and 1189 shows a negative eVect at the stem and root; that between nucleotides 2442 and 1663 and between nucleotides 3483 and 2442 shows positive eVects at the SAM and flower. In P. equestris, upstream promoter sequences of 1.3‐ to 3.3‐kb fragments for the five B‐class MADS‐box genes PeMADS2, PeMADS3, PeMADS4, PeMADS5 and PeMADS6 were isolated and sequenced. Serial deletion fragments of the promoter regions were cloned and fused to the GUS reporter gene and assayed for their transient expression in various orchid floral organs of flower buds by particle bombardment. Detailed analysis of the promoter regions is currently under way to clarify the regulation of the B‐class genes involved in orchid floral morphogenesis (unpublished data).


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V. MOLECULAR BIOLOGY OF ORCHID SCENT PRODUCTION A. SIGNIFICANCE OF THE ORCHID FLORAL SCENT

The sense of smell is the most basic and comprehensive sense. Humans have noticed the scents that emanate from flowers since 3000 BC. When the Egyptians were learning to write and make bricks, they were already making primitive perfumes and using them for religious rituals. For plants, floral scent volatiles and pigments have evolved to attract insect pollinators and enhance fertilization rates (Kaiser, 1993; Pichersky and Gang, 2000). About 2500 years ago, in ancient China, Confucius glorified the beauty and scent of the orchid flowers that he referred to as ‘lan’. He compared these flowers to the perfect human being and their scent to the joys of friendship. In orchids, large quantities of pollen in masses are spread by bees, moths, flies and birds and the floral scents serve as attractants for species‐specific pollinators (Van der Pijl and Dodson, 1969). These pollinators play an important role in orchid floral diversification, which is advantageous to the evolution of an obviously successful family. Floral signals from distinct modalities elicited both attraction and copulation behaviour, as seen by continuous volatiles attractive to patrolling males and pollination by mimicking the pheromone and posture of female thynnine wasps in Chiloglottis orchids (Australian orchids) (Mant et al., 2005; Schiestl et al., 2003). An enormous variety of orchids pollinated by bees, wasps, flies and bumblebee species cover a whole range of scents, from rosy‐floral and ionone‐floral to aromatic‐floral and spicy‐floral (Arditti, 1992; Van der Pijl and Dodson, 1969). However, our knowledge of the biochemistry of fragrance production and the mechanisms regulating its emission in orchids remains sketchy. B. INVESTIGATION OF ORCHID SCENT COMPONENTS

Floral scent is a composite characteristic determined by a complex mixture of low‐molecular‐mass volatile molecules and dominated by monoterpenoid, sesquiterpenoid, phenylpropanoid and benzenoid compounds and fatty acid derivatives (Knudsen et al., 1993). In ancient China and Japan, orchids, and particularly the attractively scented Cymbium species, have been highly prized for a long time and are all characterized by intense, very pleasant scents. On the American continent, Vanilla was used as a flavouring agent for the traditional Aztec cocoa drink and as a perfume in the preparation of lotion. Many volatile components have been identified in Orchidaceae flowers from the American, African and Australian tropics, as

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well as Asia and parts of Europe (Kaiser, 1993; Hsiao et al., 2006, 2008a). These major compounds include terpenoid, phenylpropanoid and benzenoid in diVerent ratios. Of interest, the linalool and geraniol volatiles exist in the entire scented orchid. With their colourful perianth, Phalaenopsis flowers are pollinated by bees (Christenson, 2001). According to Pijl and Dodson, the genus contains rosy‐ floral, aromatic‐floral, sweet‐floral and spicy‐floral scents; some examples are P. fasciata, P. hieroglyphica and P. schilleriana (Hsiao et al., 2008a; Van der Pijl and Dodson, 1969). In general, numerous species of Phalaenopsis orchids have fragrance, but few strongly scented species exist in the genus (Table II).

TABLE II Scented Species of Phalaenopsis Subgenera Proboscidioides Aphyllae P. wilsonii Parishianae Polychilos P. mannii P. amaboinesis P. bellina P. corningianna P. fasciata P. gigantea P. hieroglyphica P. javanica P. luddemanniana P. mariae P. modesta P. pulchra P. reichenbachiana P. venosa P. violacea P. speiosa P. sumatrana P. tetraspis Phalaenopsis P. schilleriana P. stuartiana P. amabilis P. equestris

Scent strength Scentless Moderate Scentless Faint Strong Strong (linalool and geraniol) Strong Medium Moderate Faint Moderate Medium Faint Strong Moderate Moderate Strong (benzenoid) Strong (linalool and geraniol) Medium Medium Medium Medium Faint Scentless Scentless

Arrangement from personal communication with research members at Taiwan Sugar Research Institute.


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C. DIFFICULTIES IN ORCHID SCENT RESEARCH

The global flower industry thrives on novelty. Domestication of wild species in conjunction with traditional breeding has long been the principle path for generation of novel flowers in the industry. Traits such as flower colour, shape and fragrance are primary novelty markers because they are key determinants of consumer choice. Floral scent is important not only in attracting pollinators but also in consumer choice of flowers because of its sensual associations. However, many modern floricultural varieties have lost their scent with traditional breeding programmes. Breeders in cut‐flower and ornamental markets have focussed on producing plants with improved vase life, shipping characteristics and visual aesthetic values (i.e., colour and shape). Owing to the lack of direct selection or perhaps because of a negative association with many of these traits, many cultivated flowers have lost their scent (Vainstein et al., 2001). Phalaenopsis Alliance is undoubtedly the most widely grown orchid in the world. To date, no simple, eYcient and reliable culture methods for scented orchids have been developed. Although the sympatric speciation of orchids is linked to diVerences in their floral odours, the large genome size, long life cycle and regeneration time and ineYcient transformation system render the orchid scent biology diYcult to explore. Furthermore, several scented and scentless species are cross‐incompatible, which leads to the diYculty of producing scented oVspring via traditional breeding. In some successful cases of cross‐breeding, the oVspring have diluted scent or even totally expel the scent‐producing ability. Thus, to increase crop quality, the molecular breeding of scented species by introducing key enzymes regulating scent production to species/cultivars already with good characters is of interest (Hsiao et al., 2008a). D. MOLECULAR ASPECTS OF FLORAL SCENT

In the last decade, investigations of floral scent in diVerent plants have resulted in the characterization of a large number of genes encoding enzymes responsible for the synthesis of scent compounds. The initial discovery of scent genes was in 1996, when the (S)‐linalool synthase gene encoding an enzyme responsible for the formation of the linalool (monoterpene) was isolated from Clarkia breweri flowers. Thereafter, other genes revealed were iso‐eugenol O‐methyltransferase, benzyl alcohol acetyltransferase and salicylic acid carboxyl methyltransferase, all from C. breweri, and benzoic acid carboxyl methyltransferase from snapdrogen that encode the enzymes responsible for the formation of methyl isoeugenol, benzyl acetate, methyl salicylate and methyl benzoate, respectively (Dudareva et al., 2004;

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Guterman et al., 2006). Development of functional genomic technology in recent years helped in identifying new fragrance genes in vegetative tissues and flowers, including three new genes from the deoxyxylulose‐5‐phosphate (DXP) pathway of isoprenoid biosynthesis in the peppermint Mentha Piperita (Lange et al., 1998; 1999), phenylpropene metabolism in the sweet basil Ocimum basilicum (Gang et al., 2001), diterpene synthesis in Stevia rivaudiana of Asteraceae (Brandle et al., 2002), terpene synthase (TPS) in Arabidopsis (Aubourg et al., 2002; Chen et al., 2003), and rose germacrene D synthase (Guterman et al., 2002). Although many of volatile synthesis enzymes have been identified, the amount of a given compound can be regulated by the pathway on multiple levels, and most genes and enzymes involved in the biosynthesis of scent compounds are still unknown. E. MOLECULAR RESEARCH IN SCENT METABOLISM OF P. BELLINA

P. bellina, classified in the subgenus Polychilos, is native to Malaysia, and numerous commercial varieties have been bred because of its pleasant fragrance. Qualitative and quantitative analyses by GC‐MS of volatile compounds from P. bellina flowers revealed monoterpenoids, phenylpropanoids, benzenoids and fatty acid derivatives (Table III). Monoterpenoids, including geraniol, linalool and their derivatives (Table III), accounted for more than 80% of the total volatiles collected from the P. bellina flowers. Others included nerol; 2,6‐dimethyl‐octa‐3,7‐diene‐2,6‐diol; 2,6‐dimethyl‐octa‐1,7‐diene‐3,6‐diol; 3,7‐ dimethyl‐2,6‐octadienal; geranic acid and 2,6‐dimethyl‐octa‐2,6‐diene‐1,8‐diol (Hsiao et al., 2006). TABLE III Major Classes of Volatiles Emitted by P. bellina and P. equestrisa Amount (ng/flower/h) Class of volatiles Monoterpenes Linalool Linalool derivatives trans‐Geraniol Geraniol derivatives Sequesterpene Phenylpropanoid Benzenoid Fatty acid derivatives Adapted from Hsiao et al. (2006). ND: not detected.

a b

P. bellina (scent)

P. equestris (scentless)a

382.8 16.1 105.4 15.3 39.6 20 163.4 1.6 34.5 5.4 ND 38.6 17.1 40.2 20.8 3.3 1.8

NDb ND ND ND 5.3 2.1 109.0 14 33.2 7 330.5 35.0


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The combination of chemical analysis, genomics and bioinformatics uncovered the scent biosynthesis pathway and the relevant genes (Fig. 4). A total of 2359 individual 50 ESTs were sequenced from the cDNA library of P. bellina flower bud. We developed software for the pathway and literature (http://www.taiwanorchid.net/PaL/), and candidate genes involved in the DXP‐ geraniol‐linalool pathway of Phalaenopsis identified included DXP synthase, DXP reductase, 4‐diphosphocytidyl‐2‐C‐methyl‐D‐erythritol 2‐phosphate cyclase and geranyl diphosphate synthase (GDPS). Scent‐related genes were further identified by comparing diVerentially expressed transcripts for the floral dbESTs of the scented P. bellina and the scentless P. equestris because P. bellina lacks lines of scentless varieties. P. equestris is a native species of Taiwan and has a colourful perianth. No monoterpenoid derivatives were emitted in the scentless P. equestris flowers; instead, fatty acid derivatives, phenylpropanoids and benzenoids were the major volatiles (Table III). Comparisons of the most abundant ESTs by calculating the enrichment factor (defined below) for diVerent transcripts in the floral dbESTs of P. bellina and P. equestris facilitate the provisional identification of scent metabolism genes. The enrichment factor is obtained by dividing the proportion of a certain transcript in the scented species by that in the scentless species. GDPS, epimerase (EPI), lipoxygenase and diacylglycerol kinase

Construction of cDNA library of P. bellina flower bud (remove column) Assembly and editing

1187 unigene database (499 contigs and 688 singletons) P. bellina PaL finder: Data mining/literature mapping with scent metabolism keywords

Scent metabolism pathway in P. bellina

P. equestris Comparison of floral unigene database in P. bellina and P. equestris EST filtering: P. equestris floral unigene database (E-value < 0.1) Arabidopsis and Oryza floral unigene (E-value < 10-7 )

Scent metabolism related genes in P. bellina

Fig. 4. Identification of Phalaenopsis scent metabolism pathway and scent candidate genes involved in the DXP‐geraniol‐linalool pathway [Adapted from Hsiao et al. (2006)].

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show 15‐, 10‐, 10‐ and 8.5‐fold enrichment, respectively (Table IV). GDPS is significantly diVerentially expressed in the scented species (Table IV). All monoterpenes are formed from geranyl diphosphate, which is synthesized from dimethylallyl diphosphate and isopentenyl diphosphate (Tholl et al., 2004). In addition, scent‐related genes were further identified by use of two‐step EST filtering. First, the scent‐related genes in the scent floral bud unigene database were filtered by use of in silico hybridization, with the unigene database of the P. equestris flower bud used as a probe. Second, EST filtering was performed against the collected flower unigenes of rice and Arabidopsis (15350 unigenes) obtained from NCBI public databases. With the EST filtering, we identified transcripts encoding genes for NADPH dehydrogenase (NADPHDH), EPI, molybdopterin biosynthesis cofactors and GDPS. Concomitantly, EPI and GDPS were among the highly expressed transcripts. In addition, our results suggest that NADPHDH and cytochrome P450 are required for the formation of linalool and geraniol derivatives (Fig. 5). Furthermore, transcripts encoding for signal transduction, such as sensor proteins, membrane proteins and mitogen‐activated protein kinase were identified that suggests that scent emission is probably related to stimuli that elicit a series of signal transductions for gene expression and scent production. We also detected ferrochelatase and a Myb family protein, which has been shown to be the regulator of petunia flower fragrance biosynthesis (Verdonk et al., 2005). TABLE IV DiVerentially Expressed Transcripts for Phalaenopsis. bellina (Scent) and P. equestris (Scentless) in the Floral cDNA Librariesa Abundance in floral dbESTs Putative functions

P. bellina (%)b

P. equestris (%)c

Enrichment factord

Geranyl diphosphate synthase Epimerase/dehydratase Lipoxygenase Diacylglycerol kinase Expansin Peroxidase Elongation factor

0.3

0.02

15

1.61 2.12 0.34 0.68 1.36 1.19

0.16 0.21 0.04 0.16 0.39 0.3

10 10 8 4.3 3.5 3.2

Adapted from Hsiao et al. (2006). Number of ESTs divided by 2359 ESTs of P. bellina floral dbESTs. c Number of ESTs divided by 5593 ESTs of P. equestris floral dbESTs. d Proportion (%) of ESTs in P. bellina and P. equestris floral bud libraries and enrichment factor in the scent floral bud library (proportion of P. bellina flower). a b

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O OH

+

OP O OH Pyruvate Glyceraldehyde

2 3 4

3-phosphate

EPI

DXPS

5 O

OP P Isopentenyl diphosphate

OP P Dimethylallyl diphosphate

OH

GDPS OP

OH Deoxyxylulose 5-phosphate

OP P

TPS

OH

DXPR

Cytochrome P450 OH

OH OP

Geranyl diphosphate

O

OH OH 2-C-methyl-D-erythritol 4-phosphate

Linalool oxide

Linalool TPS

OH OPP cytidine OH OH 4-(cytidine 5'-diphospho)2-C-methyl-D-erythritol

Cytochrome P450 monoxygenase

OH

Oxidation

OH

Trans-geraniol

OP

CHO

Oxidation

OP P OH

OH

HO

2-Phospho-4-(cytidine 5⬘-diphospho)-2-C-methylD-erythritol

NADPHDH Citral

2,6-dimethyl-octa-2,6-diene-1,8-diol

COOH Geranic acid

DMEC

O

PP

CH2OH

O OH OH 2-C-methyl-D-erythritol2,4-cyclodiphosphate

Citronellol

Fig. 5. Putative metabolic pathway from pyruvate and glyceraldehyde‐3‐phosphate to scent synthesis and related enzymes in Phalaenopsis bellina. DXPS, deoxyxylulose‐5‐phosphate synthase; DXPR, deoxyxylulose‐5‐phosphate reductoisomerase; DMEC, 4‐diphosphocytidyl‐2‐C‐methyl‐D‐erythritol 2‐phosphate cyclase; EPI, epimerase; GDPS, geranyl diphosphate synthase; NADPHDH, NADPH dehydrogenase [Adapted from Hsiao et al. (2006)].

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Floral scent research has mainly concentrated on the isolation and characterization of enzymes and genes involved in the final steps (TPS) of scent biosynthesis (Pichersky et al., 2006). However, the level of scent biosynthetic enzymes is not the only limiting factor, and the direct precursor (GDP) could also contribute to the regulation of scent production (Aharoni et al., 2004; Dudareva et al., 2004; Guterman et al., 2006; Nogues et al., 2006). We identified a functional homodimeric GDPS that lacks an Asp‐rich motif for scent production in the flower of P. bellina (Orchidaceae, monocot). Spatial and temporal expression analyses of PbGDPS revealed it to be closely related to monoterpene emission, which indicates that it may play a key role in the regulation of scent production in P. bellina flowers (Hsiao et al., 2008b). Meanwhile, the TPS of Phalaenopsis has a dual function to catalyse monoterpene and sesquiterpene products (unpublished data). According to our data, the two steps of enzymes activation may be diVerent regulatory mechanisms in the terpenoid biosynthesis pathway of orchid. The molecular basis and biological importance of these observations need further study. Phalaenopsis spp. are among the most important ornamental orchid flowers for export worldwide. Although most of the Phalaenopsis spp. are scentless, some do have a pleasant scent. However, many modern Phalaenopsis cultivars have lost the ability of scent emission with traditional breeding programmes. The prospective applications for restoring scent in Phalaneopsis follow consumer wishes. Further study of key enzymes, such as GDPS and TPS, involved in the scent biosynthesis pathway are necessary for use of metabolic engineering to improve the production of scent precursors to emit high level scents.

VI. MOLECULAR BIOLOGY OF ORCHID FLOWER COLOUR PRESENTATION A. ORCHID FLOWER COLOUR

Anthocyanin pigments impart colour to the flowers of many plant species. In most plants, these pigments are normally found dissolved in the vacuolar solution of epidermal cells. The major floral diversity of the Orchidacea characterizes the family as one of the most evolved among flowering plants. The pigments within the flower of the bi‐colour Oncidium Gower Ramsey show a mixture of carotenoids and anthocyanins (Hieber et al., 2006). The yellow pigment is an equal mixture of all‐trans and 9‐cis isomers of violaxanthin, with esterification specific to the 9‐cis isomer. The red pigment is composed of the anthocyanins cyanidin and its methylated derivative peonidin (Hieber et al., 2006). The Cymbidium genus contains species with white,


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yellow, green, pink, purple and red flowers but not orange‐coloured flowers. The anthocyanins cyanindin‐ and peonidin‐based were identified in a survey of two species and seven cultivars of Cymbidium (Tatsuzawa et al., 1996). The Dendrobium genus includes members with pink, red, bronze and brown‐ coloured flowers. In Dendrobium and Catlleya Alliance orchids, the yellow colouration has been ascribed to carotenoids (Matsui, 1994; Thammasiri et al., 1986). The oranges and reds are ascribed to the co‐existence of both carotenoids and anthocyanins (Griesbach, 1984; Matsui, 1994; Matsui and Nakamura, 1988; Thammasiri et al., 1986). The anthocyanins are based on cyanidin, with peonidin occurring as a minor pigment. The anthocyanin in blue‐coloured flowers is mainly cyanidin. Peach‐coloured flowers have anthocyanins of pelogonidin glycosides (Kuehnle et al., 1997). The Phalaenopsis genus contains flowers that are white, yellow, orange, red, red‐purple and purple. A sinapyl cyanidin 3,7,30 ‐triglucoside was reported for P. schilleriana (Griesbach, 1990). In the red‐purple flowers of five species and cultivars, including P. equestris, P. intermediates, P. leucorrhoda, P. sanderiana and P. schilleriana ‘Pink Butterfly’, four acylated anthocyanins were isolated and found to be based on 3,7,30 ‐triglucoside (Tatsuzawa et al., 1997). Recently, Kuo et al. identified the anthocyanins of sinapyl cyanidin 3,7,30 ‐ triglucoside for a Phalaenopsis hybrid, Doritaenopsis (Dtps.) Tinny Ribbon Dtps. Plum Rose (personal communication). B. ORCHID FLOWER COLOUR BIOSYNTHESIS GENES

The first and key enzyme in the branch of phenylpropanoid biosynthesis is chalcone synthase (CHS). It catalyses the condensation of the acyl residues from one molecule of 4‐coumaroyl‐CoA and three molecules of malonyl‐ CoA. The CHS gene has been cloned from P. True Lady and Dtps. Queen Scarlet (Hsu and Huang, 1999), and from a Phalaenopsis hybrid (Han et al., 2006). In addition, the orchid CHS gene was cloned from a Bromoheadia finlaysoniana and a Dendrobium hybrid (Mudalige‐Jayawickrama et al., 2005). The Den‐CHS‐4 genes are expressed in all developmental stages of flower buds, with highest expression in the medium‐sized buds of Dendrobium (Mudalige‐Jayawickrama et al., 2005). From phylogenetic analysis, the orchid CHS genes have two subfamilies, orchid CHS1 and CHS2, that diverged before the divergence of the three orchid genera (Han et al., 2006). Production of the three classes of anthocyanins is controlled by the availability of the substrates dihydrokaenpferol (DHK), dihydroquerceetin (DHQ) and dihydromyricetin (DHM) and the enzyme activities of flavonoid 30 ‐hydroxylase (F30 H), flavonoid 30 ,50 ‐hydroxylase (F30 50 H) and dihydroflavonol 4‐reductase (DFR) (Davies, 2000). DFR catalyses the NADPH‐dependent

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conversion of dihroflavonols such as DHK, DHQ and DHM into unstable corresponding leucoanthocyanidins, the immediate precursors of the anthocyanins (Martin et al., 1985; Reddy et al., 1987). Orchid DFR gene has been cloned and identified from B. finlaysoniana (Liew et al., 1998), Cymbidium hybrida (Johnson et al., 1999), a lavender cyanidin‐accumulating Dendrobium Sw. hybrid and a pale orange pelargonidin‐accumulating Dendrobium hybrid (Mudalige‐Jayawickrama et al., 2005), and Oncidium Gower Ramsey (Hieber et al., 2006). The first orchid DFR gene was cloned from B. finlaysoniana. The Cymbidium DFR gene is unable to eYciently reduce DHK, which is an essential step for the production of pelogonidium anthocyanin, which thus explains why Cymbidum flowers lack the orange colour (Johnson et al., 1999). The Dendrobium DFR gene is flower specific and is expressed in all developmental stages of buds, with highest expression in the medium‐sized buds. In addition, the two DFR genes isolated from diVerent colours of Dendrobium hybrids are exactly the same in nucleotide sequence (Mudalige‐Jayawickrama et al., 2005). Three isolated Oncidium DFR genes are expressed throughout flower development (Hieber et al., 2006). In addition to the DFR gene, four other key pigment biosynthesis genes isolated from Oncidium Gower Ramsey include phytoene synthase, phytoene desaturase, carotenoid isomerase and 9‐cis epoxycarotenoid diozygenase (Hieber et al., 2006). Interestingly, the authors of this study found both phytoene desaturase and carotenoid isomerase but not phytoene synthase with high expression in floral tissues than in leaves. In addition, among three DFR genes isolated from Oncidium Gower Ramsey, one contained an insertion at the 30 coding region (Hieber et al., 2006). A putative flavonoid‐30 ,50 ‐ hydroxylase, a member of the P450 family, was cloned from a Phalaenopsis hybrid and transiently bombarded into Phalenopsis petals to show the colour change from pink to magenta (Su and Hsu, 2003). Further study of the orchid biology by characterizing flower pigment biosynthesis and the molecular mechanisms is required.

VII. CONCLUSION Orchid floral molecular biology, although still young, has already oVered new and exciting perspectives on this intriguing plant family. Recent advances in practical genomics and functional genomics methodologies have allowed orchid floral molecular biology to become a standard scientific research topic accessible to many investigators, which has in turn resulted in many exciting new discoveries. Because more and more plant genomics sequence data and functional genomic tools will be available in the future, interesting areas of research will be targeted reverse‐genetics approaches to


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look at the function of putative orthologues or even of floral‐specific expressed genes. Much remains to be learned in terms of loss and gain of function of such genes in the orchid flower. Although functional redundancies will always be a hurdle, the knowledge gained will serve as a basis to better understand the evolution and regulation of orchid floral biology. The next challenging issues and problems to solve include identification of additional genes involved in flower morphogenesis, control of flowering time and symmetry as well as roles of transposable elements. In addition, domains of disease resistance, embryogenesis, interaction with symbiotic fungi and genetic diversity are areas that will benefit from the tools developed for studying flower morphogenesis. On the application side, new genetically engineered varieties with characters of fragrance, new varieties of colours and change of floral morphology (e.g., from zygomorphy to actinomorphy) are promising areas. The current Solexa/Illumina Genome Analysers can produce a sequencing result of 120 Mbp (equivalent to the Arabidopsis whole genome) at a cost of US $1200 in a couple of days. By availing ourselves of the sequencing technology, the orchid genome might be sequenced. If so, Phalaenopsis will be the model plant for Orchidaceae. However, the genome sequencing will first require construction of an orchid physical map by the use of BAC fingerprinting data to obtain the minimum tiling path. In addition, high heterozygoty might be a problem for a future genome project. The resolution for the heterozygoty is to create double haploid populations from tissue culture of pollinia that might be useful for mapping and setting up a genome project. However, this is possible yet very diYcult and will take many years to reach this goal.

ACKNOWLEDGMENTS We thank the National Science Council, National Science and Technology Program for Agriculture Biotechnology and Council of Agriculture of the Taiwan government for financial support. The orchid nurseries in Taiwan including I Hsin Biotechnology, OX Flowers Farm, Taiwan Sugar Corp. and Sunhope Garden Nursery are highly appreciated for their long‐term support of precious plant materials for the orchid research.

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Molecular Physiology of Development and Quality of Citrus

´ S,* FRANCISCO R. TADEO,* MANUEL CERCO JOSE´ M. COLMENERO‐FLORES,* DOMINGO J. IGLESIAS,* MIGUEL A. NARANJO,* GABINO RIÓS,* ESTHER CARRERA,*,1 OMAR RUIZ‐RIVERO,*,1 IGNACIO LLISO,* RAPHAE¨L MORILLON,{ PATRICK OLLITRAULT,{ AND MANUEL TALON*

*Centro de Geno´mica, Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada (Valencia), Spain { Centre de Coope´ration Internationale en Recherche Agronomique, pour le De´velopement (CIRAD), UR 75, Dpt. BIOS, Apdo. Oficial, 46113 Moncada (Valencia), Spain

I. Fruit Quality in Citrus is Mostly a Consumer’s Preference. . . . . . . . . . . . . . . . II. Citrus Flowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. All Fruit Began Being Flowers .............................................. B. Flowering About to Flourish ................................................ III. Citrus Fruiting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Long and Winding Road Towards Ripening ........................ B. Fruitful Ripening .............................................................. C. Degreening and Regreening.................................................. D. The ‘Fit to Be Eaten’ Part.................................................... E. Acidic and Acidless Fruit .................................................... IV. It is Just not Food. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Breaking the Smell ............................................................

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Present address: Instituto de Biologıá Molecular y Celular de Plantas, Universidad Politećnica de Valencia‐Consejo Superior de Investigaciones Cientı´ficas, Universidad Politećnica de Valencia, 46022 Valencia, Spain. Advances in Botanical Research, Vol. 47 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(08)00004-9

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B. Bitterness or Tastelessness ................................................... V. Fruit Quality Only in Quality Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Risk of Freezing.......................................................... B. Poor Quality Water and Water Shortage .................................. VI. Citrus Abscission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. To Fall or not to Fall, that is the Question................................ B. Abscission at a Glance........................................................ VII. Concluding Remarks and Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Citrus is the most economically important fruit crop in the world. Citrus fruits are classified as hesperidiums, berries of very special organization characterized by a juicy pulp made of vesicles within segments. Besides the typical fruit components, citrus fruit contain many organic compounds necessary for human diet and an extraordinary number of metabolites displaying valuable properties for health. In citrus, the concept of fruit quality comprises several other aspects intimately related to human health apart from physical attributes and diet components. Citrus also possess a rare combination of intriguing biological characteristics including an unusual reproductive biology, a non‐climacteric fruit ripening and several specific tree‐traits. The combination of these characteristics suggests that the study of fruit growth regulation in citrus may reveal original mechanisms based on explicit molecular diVerences and on exclusive genes. Citrus is, therefore, an excellent model to study fruit quality because of its peculiar fruiting, singular biochemistry and economical relevance. In this chapter, the progress that has been carried out in the research on the molecular determinants related to development and fruit quality of citrus is reviewed. The review also intends to provide a physiological frame for the implementation of the information generated during the past years. Molecular background is provided on the current status of principal reproductive processes related to fruit quality mainly flowering, fruiting, ripening, and abscission. We also have focused on main characteristic secondary bioactive compounds, as major contributors of aroma and flavour and finally, on the abiotic stresses influencing development and fruit growth.

I. FRUIT QUALITY IN CITRUS IS MOSTLY A CONSUMER’S PREFERENCE In this work, we review our knowledge on the molecular determinants of citrus fruit development. The specific focus of the study is on the molecular physiology of fruit quality under a developmental perspective, an area that complements information presented in two upcoming reviews dealing with the generation of genomic tools and resources (Talon and Gmitter, 2008) and the physiology of citrus fruiting (Iglesias et al., 2008) in the genus Citrus. In here, background information is presented on the genetic and physiological regulation of reproductive growth and development, from flowering to ripening since all these

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processes are rather determinants of the characteristics and attributes of the final product, a ripe fruit. Also revised are major biochemical aspects of the ripening processes that are crucial for fruit quality including colour acquisition, sweetness build‐up and acidity reduction, and secondary metabolism responsible, for example, of fragrance and bitterness. The final part of the review concentrates on main environmental constrains limiting fruit growth and quality, mainly salinity, water deficit and low temperature, and documents progress in the understanding of abscission processes. Citrus refer to all species of three sexually compatible genera, Citrus, Fortunella and Poncirus, within the tribe of Citreae of the subfamily Aurantioideae. Citrus genus contains the majority of the consumed species while Fortunella genus includes some cultivars (kumquats). Poncirus genus is monospecific (P. trifoliata) and plays a central role in rootstock breeding because of its tolerances to many biotic constraints. Citrus genus contains, according to the diVerent taxonomies, between 16 (Swingle and Reece, 1967) and 156 species (Tanaka, 1961). Phenotypical data (Barret and Rhodes, 1976; Fanciullino et al. 2006) as well as molecular analysis (Fanciullino et al., 2007; Herrero et al., 1997; Nicolosi et al., 2000; Ollitrault et al., 2003) clearly demonstrated the existence of three basic taxa (C. maxima—pummelos‐, C. medica—citrons‐ and C. reticulata—mandarins) that have originated all cultivated Citrus. The diVerentiation between these sexually compatible taxa is explained by an initial foundation eVect in three distinct geographic zones and subsequent allopatric evolution. The pummelos originated in the Malay Archipelago and Indonesia, the citrons evolved in northeastern India and the nearby region of Burmaand China and the mandarins were diversified over a region including Vietnam, Southern China and Japan (Scora, 1975; Webber, 1967). The other cultivated species (C. aurantium—sour orange, C. sinensis—sweet orange, C. paradisi—grapefruit, and C. lemon—limon) have likely appeared by recombination among the basic taxa (Nicolosi et al., 2000; Ollitrault et al., 2003). For C. aurantifolia (Mexican lime), a fourth original taxa (C. micrantha) was involved in combination with C. medica (Nicolosi et al., 2000). Citrus is one of the most important fruit crop in the world with a reported production of 105.4 million tons in 2004–2005 (HYPERLINK ‘‘http://www. fao.org’’ www.fao.org). Citrus trees originated in the southeast of Asia and spread and established through all continents. Currently, citrus trees are grown mainly in latitudes between 40 N and 40 S throughout the tropical and subtropical regions of the world. In these regions, the winter temperatures are adequate for tree survival and there is, in general, suYcient water and suitable soils to support tree growth and fruit production. Major citrus production areas are located in South and North America (mainly in Brazil, USA, Mexico and Argentina), along the Mediterranean basin (in Spain,

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Italy, Egypt, Turkey and Morocco), in the south and east of Asia (in China, India and Japan) and in Australia. Beside the importance of citrus fruit as a rich source of ascorbic acid (vitamin C), fibre and phytochemicals (flavonoids, flavonones or limonoids) with contrasted benefit to human health, citrus culture has achieved a considerable social and economical importance due to the development of a large juice processing industry and to the net profit derived from the export and marketing of fresh fruit. The phenotypical variability of citrus is considerable (Table I). It is related to pomological and organoleptic characters as well as to tolerance to biotic and abiotic constraints. Fruit diameter varies from a few centimetres for mandarins, Poncirus and kumquats to more than 30 cm for some pummelos. Albedo, the inner portion of fruit peel, is absent in kumquats and very little developed in mandarins while it constitutes the major part of the citron fruit. Fruit pulp is green, orange, yellow or red according to carotenoid and anthocyanin composition. Acidity can be null for certain tropical oranges but is very high in lemon and lime. The flavours and essential oils are qualitatively and quantitatively much diversified. For instance, the bitterness taste of pummelos and grapefruits is mainly associated with naringin (flavonoid) and limonin (limonoid). The vegetative and reproductive physiology of citrus has been widely studied and a considerable number of cultural practices have been developed in order to obtain adequate tree development and high fruit quality yields. Fruit quality can be defined in a broad sense as the grouping of inherent attributes and characteristics of a fruit. In general, those attributes perceived by the sense of taste and smell are mostly related to internal quality while external standards are based on characteristics perceived by the sight and touch, such as colour or the presence of blemishes, for example. This definition, however, is only a wide generalization since there may be other additional attributes with higher commercial, toxicological and nutritional implications. The objectives of citrus fruit production and the methods applied to achieve it are diVerent depending on the final destination of fruit, that is, the processing industry or the fresh market (Davies and Albrigo, 1994). The most appreciated features of citrus fruit for processing are related to internal fruit quality which is a function of flavour and palatability. Citrus fruit for processing must have no decay symptoms, elevated juice content, high level of TSS (total soluble sugars, mostly sucrose, glucose and fructose), low TA (titratable acidity, mostly citric acid) and minor amounts of bitter components such as the triterpene limonin and the flavonoid narangin. The external appearance of fruits, however, is not particularly important for processing although attractive internal colour in oranges is usually required. Citrus fresh fruit quality standards, on the

TABLE I Nomenclature and Agronomic Characteristics of Citrus Cultivated Species Common use Scion: Propagation by grafting

Genus Citrus

Pummelo

Latin name

Grapefruit

C. maxima (Burm.) Merr C. paradisi Macfad.

Sweet oranges

C. sinensis (L.) Osb.

Clementine

C. clementina Hort. ex Tan. C. unshiu Markow C. aurantium (L.)

Satsuma mandarin Sour orange

Rootstock: Propagation by seeds (polyembryonic)

Poncirus

Poncirus

C. jambhiri Lush C. reshni (L.) Osb. C. reshni P. trifoliata C. sinensis P. trifoliata P. trifoliata (L.) Raf.

Fortunella

Kumquats

Fortunella spp.

Citrus/Poncirus hybrids

Scion

Common name

Rough lemon Cleopatra mandarin ForAl# 5 Carrizo citrange

Main interest or characteristic and area of production Fruit consumption: mainly grown in Asia, Pacific Fruit consumption, juice: mainly grown in USA Fruit consumption, juice: Americas, Asia, Mediterranean basin Fruit consumption: mainly grown in the Mediterranean basin Fruit consumption: mainly grown in Japan Tolerant to salinity; sensitive to tristeza when grafted Tolerant to drought Tolerant to salinity and tristeza Tolerant to salinity and tristeza; semi‐dwarf Sensitive to salinity and iron chlorosis; tolerant to tristeza and cold Highly sensitive to salinity and iron chlorosis; tolerant to tristeza and cold Ornamental, fruit consumption

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other hand, are largely dependent on consumers’ preferences that in addition may change substantially between countries. In general, considerable more emphasis is put in fresh markets on external than on internal fruit quality standards. Although citrus fruit must reach a minimum ‘maturity index’ (the TSS to TA ratio; in general, 7–9 for oranges and mandarins and 5–7 for grapefruits) to be marketable, the essential quality requirements of citrus fruit for fresh market are mostly cultivar characteristic such as fruit size and shape, brightly coloured and firm rind and absence of surface blemishes. Fruit shape and size that are typical for each major citrus group including oranges, mandarins and grapefruits, are intimately associated with the cell division rate of ovary walls during the initial phases of fruit growth. The range of average fruit size in each cultivar is in addition inversely dependent upon fruit load. In many cultivars, low productions result in higher fruit size and vice versa. This relationship that is very obvious in seeded varieties can also be observed in many seedless species and in general, when fruit number is low, peel texture, shape and colour are not appropriate. Therefore, fruit abscission rates during early stages of development and also thereafter may have certain eVects on fruit size and even fruit shape. Seedlessness and easy‐ peeling fruit are also important and desirable fruit features in some particular markets such as those of the European Union.

II. CITRUS FLOWERING A. ALL FRUIT BEGAN BEING FLOWERS

Citrus species are perennial trees requiring a long juvenile period of several years before the first flowers emerge. Subsequently, flowering becomes an annual process in many relevant citrus varieties, broadly dependent on seasonal and climatic conditions. In subtropical regions, the major bloom occurs during the spring flush, after flower induction on quiescent buds by a low temperature period during winter time (Moss, 1969; Nebauer et al., 2006; Valiente and Albrigo, 2004). However, under tropical conditions, sprouting and flowering may occur over the whole year although the main bloom still takes place during the spring (Monselise, 1985; Reuther and Rios‐Castanõ, 1969; Spiegel‐Roy and Goldschmidt, 1996). It is generally believed that in these areas, water stress caused by drought periods in regions with a dry season substitutes the low temperature requirement of citrus as the major flower induction agent (Cassin et al., 1969; Reuther and Rios‐Castanõ, 1969). Similarly, water deficit in subtropical climates has also been proved to increase the ratio of reproductive shoots and the total number of flowers (Southwick and Davenport, 1986).


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In citrus, bud dormancy is released during spring time and reproductive shoots with a variable leaf to flower ratio are commonly formed. Generally, an increase in flowering intensity of the tree does not result in a concomitant increase in fruit production, since in many varieties there is an inverse relationship between the number of flowers and the probability of setting. Thus, through the manipulation of flower number, it is possible to counteract undesirable agricultural behaviours such as the biennial oscillation of crop production (alternate bearing), a process regulating diVerentially carbohydrate‐related gene expression (Li et al., 2003a). DiVerent common practices and treatments aVecting flower production and commercially used to alleviate alternate bearing include pruning, girdling, defoliation, nitrogen fertilization and gibberellin application (Agustı´, 2003; Guardiola et al., 1982). Interestingly, gibberellins play an inhibitory role on citrus flower bud induction and diVerentiation, as in many other woody trees.

B. FLOWERING ABOUT TO FLOURISH

Physiology of citrus flowering has received broad attention during the past decades and hence has been deeply investigated mostly because of its agronomical relevance (Davenport, 1990). However, during the past years, development of citrus genomics, contributing to the growth of EST database and the improvement of transcriptomic approaches (Talon and Gmitter, 2008), is giving rise, in a similar way to other areas, to the flourishing of molecular and functional studies on citrus flowering. Furthermore, the availability of whole genomic sequences from other dicots is likewise contributing to citrus research, oVering useful molecular models for appropriate comparisons (Arabidopsis Genome Initiative, 2000; Jaillon et al., 2007; Tuskan et al., 2006). Thus, from the three available sequenced dicots, Arabidopsis thaliana and Populus trichocarpa are closer relatives to Citrus genus, both being core components of the rosid clade, while Vitis vinifera is part of Vitaceae, that is considered to be a sister family of rosids. It is anticipated that a significant advance in the knowledge of the molecular basis regulating citrus flowering will come from the appropriate exploitation of existing resources from these organisms. Currently, information in this regards is mostly available from Arabidopsis thaliana and Populus (Jansson and Douglas, 2007; Redei, 1975). Arabidopsis flowering has been extensively studied and a wide range of genes involved in the regulation of the process at diVerent levels has been reported in this species (Komeda, 2004). Despite severe limitations to the application of this model to citrus might arise from

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the weedy architecture and annual life cycle of Arabidopsis, other pivotal features like the positive eVect of cold periods (vernalization) on flowering initiation are favouring its utilization to solve citrus issues. Likewise, in Populus, there are many similarities to citrus such as a woody nature and a juvenile period of several years devoid of flowers that are also accompanied by important diVerences; for instance, poplars are deciduous trees while citrus are perennial. Interestingly, the requirement for a period of chilling temperatures to condition poplar trees to break dormancy before the spring bloom (Rohde et al., 2000) may remind the vernalization eVect of Arabidopsis and the cold induction in citrus, suggesting certain conservation of mechanisms underlying adaptation of these species to seasonal changes in temperate climates. Environmental signals and the specific genetic background are the factors responsible for activating the genes involved in the biochemical and physiological changes that lead to floral induction and development, allowing the transition from vegetative to reproductive development in plants (Bernier et al., 1993; Levy and Dean, 1998; McDaniel et al., 1992). The molecular mechanisms involved in this transition have been widely studied in Arabidopsis. In this species, several genes representing a large family of transcription factors containing a highly conserved DNA‐binding domain (MADS‐box) and a secondary domain (K‐box), also conserved and involved in protein– protein interactions, have been identified (Ma et al., 1991). Several studies indicate the existence of flowering integration genes, such as FLOWERING LOCUS T (FT), LEAFY (LFY) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), which in turn activate diVerent floral identity genes (Tan and Swain, 2006). For instance, LFY regulates at least three homeotic genes encoding transcription factors that control flower organ identity (Coen and Meyerowitz, 1991). These genes are APETALA1 (AP1), a gene mostly expressed in petals and sepals (Wagner et al., 1999); APETALA3 (AP3) that is necessary for petal and stamen development (Lamb et al., 2002) and AGAMOUS (AG) that is required for stamen and carpel development (Busch et al., 1999; Parcy et al., 1998). These precedents have to be considered when citrus flowering is reviewed, since citrus genes studied up to now have been isolated and characterized on the basis of their conservation in model species. This is how the occurrence of AP1, LFY, FT and TERMINAL FLOWER 1 (TFL1) genes in citrus has been identified (Endo et al., 2005; Pillitteri et al., 2004a,b). The first work on citrus blooming involving flowering genes reported the constitutive expression in juvenile seedlings of Carrizo citrange hybrid (Citrus sinensis L. Osbeck Poncirus trifoliata L. Raf.) of LFY and AP1 genes from Arabidopsis (Penã et al., 2001). This study showed the eVect of those genes on the shortening of the juvenile phase in this species, since fertile flowers were produced within a


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few months after transformation. On the other hand, AP1 was as eYcient as LFY expression to induce flowering initiation and no severe developmental abnormalities were observed. Transformation stability and inheritance of early flowering phenotype were also verified in the following spring. Later on, it was shown that a citrus orthologue of the FT gene (CiFT1) isolated from Poncirus trifoliata (L. Raf.) and expressed constitutively in transgenic plants, caused a considerable reduction in flowering time (Endo et al., 2005). Moreover, the crossing of ‘Kiyomi’ tangor plants (C. unshiu C. sinensis) with the pollen of these transgenic lines induced sudden flowering of F1 germinated seedlings. These observations indicate that it is possible to shorten the juvenile period in species with agricultural interest and long juvenile periods, an achievement of major relevance since it would allow quicker breeding process (Tan and Swain, 2006). Recently two additional homologs of FT have also been identified (CiFT2 and CiFT3). These genes show high similarity to CiFT1, but diVerent patterns of organ expression: CiFT3 was mainly expressed in vegetative tissues such as leaves and stems, and consequently it is considered to play a principal role in floral induction (Nishikawa et al., 2007). Furthermore, the expression level of CiFT genes in adult Satsuma mandarin (Citrus unshiu Marc.) was increased by cold treatments, an observation that correlates with cold induction of flowering. In contrast, juvenile Satsuma plants, unable to flower, showed a lower and cold‐insensitive expression of CiFT. In Arabidopsis, it has also been shown that FT transcription depends upon CONSTANS (CO) accumulation and therefore that CO and FT genes, that initiate flowering under long‐days, are coordinated to control the blooming process. Interestingly, both genes show daily regulation. CO mRNA accumulates at the end of the day during long days and during night in short days (Sua´rez‐Lo´pez et al., 2001). A recent study by Bo¨hlenius et al. (2006) in poplar has also confirmed the relationship between CO and FT as controllers of the flowering process in trees showing a seasonal dormancy period. In poplar, expression patterns of CO and FT orthologues are diVerent in specimens located in diVerent geographic areas requiring distinct daylengths for growth cessation and floral induction. They also suggested that the slow accumulation of FT over the juvenile years could be due to a more pronounced role of the EARLY BOLTING IN SHORT DAYS (EBS) gene in trees than in Arabidopsis. This gene is a probable chromatin regulator that could gradually release FT repression after each annual cycle of growth and dormancy, leading to CO‐dependent activation of FT transcription. Thus, it is tempting to suggest that this or a very alike conserved mechanism may also operate in citrus since the similitude among the flowering behaviour of citrus and poplar trees is obvious despite several attractive diVerences. Interestingly, these diVerences are not only founded on very basic biological tree‐specific

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traits, for instance, the perennial versus deciduous nature, but also on more particular aspects of the regulation mechanism of flowering induction such as the ability to response to cold, re‐hydration or even fruit load. Definitively, more experimental work is required to characterize at physiological and molecular levels the role of FT on citrus flowering. Another key gene, TERMINAL FLOWER 1 (TFL1), has also been isolated from Washington navel (C. sinensis L. Osbeck) (Pillitteri et al., 2004a). Expression of this citrus gene in Arabidopsis caused a late flowering phenotype and also complemented the tfl1‐2 mutation, indicating that the Arabidopsis gene and the citrus orthologue are interchangeable in vivo and probably perform a similar function in both species. Furthermore, it was determined that the juvenile stage in citrus correlated positively with the accumulation of TFL1 transcripts and negatively with the RNA levels of the regulatory genes LFY and AP1. Finally, a recent work has reported the cloning and functional characterization of several flowering genes also from C. sinensis (Tan and Swain, 2007), that is, it describes the isolation of one AP3‐like (CitMADS8), one WUSCHEL‐like (CsWUS) and two SOC‐like (CsSL1 and CsSL2) genes. Despite the high similarity of CitMADS8 with AP3, the citrus gene was not able to suppress flower identity defects in the Arabidopsis ap3‐3 mutant. However, citrus SOC‐like genes, mostly CsSL1, rescued the late flowering phenotype of the Arabidopsis soc1 mutant and caused early flowering and additional floral homeotic changes in wild‐type plants. Expression of CsWUS in the Arabidopsis wus‐1 mutant allowed partial restoration of wild‐type phenotype since normal flowers showing the right number of organs in the four whorls were produced although no viable seeds were obtained. In summary, these reports show at the molecular level a high degree of conservation of the essential mechanisms regulating flowering processes among annual and woody species. The genes explored in these studies probably constitute main targets for genetic engineering applications in citrus and in the near future will likely make possible the manipulation and control of the flowering stages, a major dream of citrus growers.

III. CITRUS FRUITING A. THE LONG AND WINDING ROAD TOWARDS RIPENING

In subtropical areas, flowering of most citrus species occurs in spring and subsequent fruiting generally spreads until mid‐winter. However, full ripening in early varieties may be reached as soon as September while in late


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species it can be prolonged until the onset of next summer. Fruit development follows a typical sigmoid curve, divided into three stages (Bain, 1958). The first period, or phase I, is an approximately 2‐month period, between anthesis and June drop, characterized by slow fruit growth rates but high cell division. The following stage, or phase II, takes place during the following 4–6 months and constitutes a fast period of growth, where fruit increases in size mostly by cell enlargement and water accumulation. Through these two stages, developing fruitlets switch basic metabolism from ‘utilization’ to ‘storage’ sinks (Mehouachi et al., 1995). In the final stage, or phase III, growth is mainly arrested and fruits undergo a non‐climacteric ripening process (Fig. 1). Citrus bloom profusely since an adult tree may develop until 250,000 flowers and therefore show an extremely high abscission of buds, flowers, fruitlets and sometimes even fruit. Generally, only a small percentage of fruits overcome the June drop and for a normal crop quite less than 1% of them reach ripening. Abscission of reproductive organs is overall continuous during the first stage of citrus fruiting (phase I) although two major and separate waves of elevated flower and fruitlet abscission can often be distinguished at the onset of the phase and during the transition to the rapid fruit growth period (phase II). These drops characterize and limit the magnitude and scope of fruit‐setting. Abscission of flowers, ovaries and young fruits during the fruit‐set period and of mature fruits during the ripening period is a developmentally regulated process that can lead to significant crop losses and compromise final yield in citrus. In addition, most stressful environmental conditions from several biotic and abiotic origins may also induce abscission of reproductive organs (Das, 2003; Go´mez‐Cadenas et al., 2000; Mehouachi et al., 1995; Timmer and Brown, 2000) and even mature leaves (Das, 2003; Go´mez‐Cadenas et al., 1996, 1998; Tudela and Primo‐Millo, 1992; Young and Meredith, 1971). Abscission of leaves has also a major transcendence on reproductive development since leaves are eventually necessary for flower, ovary and fruitlet setting and subsequent growth (Mehouachi et al., 2000). Most of the work associated with the control of reproductive growth in citrus has been focused on the role of plant hormones and carbohydrates, two pivotal factors controlling essential fundaments of the metabolism and development (Goldschmidt and Koch, 1996; Iglesias et al., 2008). Since this last recent work exposes a detailed description of the physiology of citrus fruiting, only the relevant aspects on this subject are mentioned in here and the reader is kindly redirected to that review for deeper inside. In citrus, plant growth regulators strongly aVect reproductive processes including many aspects related to control of growth, ripening and abscission (Talon et al., 1990b). It is generally accepted that gibberellins (GAs) are

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Phases of fruit growth and development I

II

III

A

Growth

Abscission rate Carotenoids

Chlorophylls

B

C

Acidity

Carbohydrates

Time

Fig. 1. Pivotal physiological and biochemical events occurring during citrus fruit growth and development. Representative stages of the three growth phases of citrus fruit according to Bain (1958). Scale bar ¼ 1 cm. (A) Fruit growth and abscission rate. Dashed line during phase III indicates pre‐harvest fruit abscission occurring in certain varieties. (B) Pigments (chlorophylls and carotenoids) in fruit pulp. (C) Carbohydrate content and acidity in fruit pulp.


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involved in set and development of citrus fruits while their deficiency results in fruitlet abscission (see below). The support for this proposal initially came from several studies reporting that exogenous GA3 considerably improves parthenocarpic fruit set and growth of self‐incompatible genotypes such as Clementine that in the absence of cross‐pollination show negligible parthenocarpic fruit set (Soost and Burnett, 1961). Afterwards, a considerable amount of work has shown that gibberellins were deeply involved in the early promotion of citrus fruitlet growth (Ben‐Cheikh et al., 1997; Goto et al., 1989; Turnbull, 1989). For example, it was found that citrus genotypes with reduced fruit set and higher abscission generally contained lower GA levels than seeded and seedless varieties with high natural parthenocarpy (Talon et al., 1992). In vegetative organs, GAs activate cell division and/or cell enlargement processes and therefore are generally associated with the initiation of growth (Zeevaart et al., 1993). For instance, there is interest in modulating the growth habit of citrus rootstocks since this might eventually aVect the development of the scion and facilitate diverse cultural practices (e.g., pruning, pesticide applications and harvesting). Initial work showed that the ectopic overexpression in tobacco of a citrus GA20‐oxidase, a regulatory step of gibberellin biosynthesis (Talon and Zeevaart, 1992), enhanced gibberellin content and shoot growth (Vidal et al., 2001). Later, Fagoaga et al. (2007) generated transgenic Carrizo citrange rootstocks overexpressing this GA20‐oxidase and confirmed that the gene controls the gibberellin flux through the pathway since taller (sense) and shorter (antisense) phenotypes correlating with higher and lower levels of active GA1 were obtained. In these transgenic lines, however, cell division was more aVected than cell elongation as also observed in poplar, in contrast to the eVects found in herbaceous plants (Talon et al., 1991). Auxins have often been reported either to delay or to induce fruit abscission and hence may operate as growth hormones or as abscising agents (see below). It is well established that auxins promote cell enlargement and in citrus endogenous auxins are high in developing ovaries and during the beginning of phase II, the period of cell elongation. However, exogenous auxins do not improve fruit set during phase I but in contrast are very eVective increasing fruit size when applied at the onset of phase II (Coggins and Hield, 1968; Agustı´ et al., 2002). These observations may suggest that auxins when acting as plant growth stimulators are mostly inductors of cell enlargement, the essential factor controlling fruit size during the phase of rapid growth. In addition to hormones, it is well established that in citrus, nutrients, specially carbohydrates, may have regulatory functions on their own and/or through the maintenance of a proper hormonal homeostasis (Goldschmidt

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and Koch, 1996). Although the specific mechanism involved in the response of fruit growth to carbohydrates has not been studied at the molecular level, many observations suggest that sugars may act not only as essential nutrient factors but also as signals triggering specific hormonal responses (Goldschmidt and Koch 1996; Iglesias et al., 2006) and vice versa (Agustı´ et al., 2002; Mehouachi et al., 1996; Powell and Krezdorn, 1977). The idea that in citrus, fruit abscission is connected to carbohydrate availability was initially anticipated by Goldschmidt and Monselise (1977) who suggested that citrus might possess an internal self‐regulatory mechanism that adjusts fruit load to the ability of the tree to supply metabolites. The link among carbohydrates and fruit growth is currently supported by a wide body of evidence including many studies on source–sink imbalances, defoliation, girdling, shading, sucrose supplementation, defruiting and fruit thinning (Goldschmidt and Koch 1996; Syvertsen et al., 2003; Wallerstein et al., 1978; Yamanishi, 1995). Thus, a tough relationship between carbohydrate amounts available for fruitlets and their probability of abscission has been demonstrated (Go´mez‐Cadenas et al., 2000; Iglesias et al., 2003). Hence, photosynthesis activity has been proved to be crucial since high carbohydrate requirements during fruit set increases photosynthetic rate (Iglesias et al., 2002; Rivas et al., 2007). B. FRUITFUL RIPENING

Citrus ripening is a very attractive subject for studying for several reasons. First, ripening and the growth stages and events preceding ripening are determining fruit quality since many quality attributes are acquired along stages II and III of fruit development. Quality properties are ultimately dependent on the regulation of major physiological and biochemical processes channelling fruit growth. These parameters have also a strong economical relevance since they are related to the consumer perception and eventually constrain the success of the citrus industry. Ripe fruit also show several singular anatomical and physiological particularities. Citrus fruits belong to a special type of berry named hesperidium, composed of two major, morphologically distinct regions: the pericarp (peel or rind) and the endocarp (pulp), which is the edible portion of the ripe fruit. The pericarp is further divided into two parts: the exocarp (flavedo), which is the external coloured portion and the mesocarp (albedo), the white layer of the peel. The pulp consists of segments, the ovarian locules, enclosed in a locular membrane and filled with the juice vesicles that are the ultimate sink organ of the citrus tree. During non‐climacteric ripening, active growth in citrus fruits slows down and metabolism shifts to integrate a number of


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biochemical and physiological changes that eventually render an edible organ. In ripe fruits, ethylene production and sensitivity is low, respiration is considerably attenuated and changes in texture and composition proceed gradually (Aharoni, 1968; Eaks, 1970; Goldschmidt et al., 1993). Furthermore, there is no evidence that the whole process of ripening is controlled specifically by any hormone. Finally, citrus fruit are very useful and convenient models to study regulation of non‐climacteric fruit development. Fleshy fruits can generally be divided in climacteric and non‐climacteric according to the pattern of respiration and ethylene production exhibited during initial ripening. Climacteric fruit ripening is controlled by ethylene through the regulation of gene transcription and, therefore, much information has been generated in the areas of ethylene biosynthesis and response (Giovannoni, 2004). In contrast, the mechanisms of ripening in non‐climacteric fruits, including citrus, are mostly unknown although much eVort has been dedicated to detailed physiological and biochemical descriptive studies (Baldwin, 1993). The following paragraphs revise our knowledge on gene expression associated with citrus fruit ripening. In climacteric fruits, the onset of ripening depends on a transition in ethylene production from a low basal rate, inhibited by exogenous ethylene, to an autocatalytic burst. These patterns of ethylene synthesis have been named as system I and system II, respectively (McMurchie et al., 1972). In mature citrus fruit, very low rates of ethylene production are associated with constitutive expression of the 1‐aminocyclopropane‐1‐carboxylate synthase 2 (CsACS2) and the ethylene receptor CsETR1 genes, indicating that citrus certainly possess a system I machinery, typical of non‐climacteric fruits. However, it has been reported that in detached young fruitlets, there may be an additional system II‐like mechanism since a climacteric‐like rise in ethylene production, preceded by induction of the genes for CsACS1, ACC oxidase 1 and the ethylene receptor CsERS1, was unequivocally present (Katz et al., 2004). In addition, citrus fruits are capable to respond to exogenous ethylene, which stimulates external ripening by accelerating respiration, chlorophyll degradation and carotenoid deposition, in contrast to many other non‐climacteric fruit. These diVerences have led to the suggestion that fruit ripening in citrus is controlled by a singular and diVerent mechanism (Fujii et al., 2007). There are multiple observations that although external and internal ripening in citrus generally coincide, peel and pulp behave in most respects as separate organs and thus can be considered as diVerent physiological processes under distinct regulation machineries. For instance, ultimate genetic evidence has been provided by the study of cDNAs resulting from small‐scale

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EST (expressed sequence tag) sequencing projects from developing fruits at stage II (Hisada et al., 1997) and from mature fruits (Moriguchi et al., 1998). The analysis of the diVerences in gene expression of cell wall‐related genes during rind development revealed that the patterns found in flavedo and albedo could reflect the morphological diVerences between these two tissues (Kita et al., 2000). Similarly, Shimada et al. (2005b) used a citrus cDNA microarray containing 2213 independent genes to examine gene expression and reported in a short communication that the expression profile in the diVerent tissues of the fruit was rather diVerent. C. DEGREENING AND REGREENING

External citrus fruit ripening is mostly dependent upon the conversion of chloro‐ to chromoplast and implies the progressive loss of chlorophylls and the gain of carotenoids, changing peel colour from green to orange (HuV, 1983, 1984). The changes associated with external ripening are essentially comparable to the senescence of vegetative chlorophyllous tissues, that in citrus fruits are mostly influenced by environmental conditions, nutrient availability and hormones (Goldschmidt, 1988; Iglesias et al., 2001). In citrus, chromoplast biogenesis is of particular interest and agronomical relevance, since unlike most ripening processes the chloro‐ to chromoplast conversion is reversible, even from fully diVerentiated chromoplasts (Goldschmidt, 1988). Ripe citrus fruit can therefore undergo a natural regreening process. It is well known that ethylene accelerates colour change in citrus fruits through the activation of chlorophyll degradation (Fujii et al., 2007; Garcia‐ Luis et al., 1986; Jacob‐Wilk et al., 1999; Trebitsh et al., 1993) and carotenoid biosynthesis (Eilati et al., 1969; Fujii et al., 2007; Young and Jahn 1972). Thus, ethylene is being used for the last half century to stimulate colour change in citrus fruits during post‐harvest storage. Ethylene treatment not only accelerates degreening but has multiple eVects on gene expression in flavedo such as repression of transcription of genes involved in sugar metabolism and induction of several genes associated with defence, stress responses, amino acid and protein synthesis and secondary metabolism (Fujii et al., 2007). Despite these several eVects, no clear role for endogenous ethylene during natural citrus ripening has yet been demonstrated. For example, it has been shown that chlorophyllase expression during natural ripening is low and constitutive (Jacob‐Wilk et al., 1999) while a strong increase of chlorophyllase transcripts occurs in detached fruits in response to ethylene treatment (Fujii et al., 2007; Jacob‐Wilk et al., 1999). These observations suggest that naturally occurring ripening control could be independent upon ethylene. This idea is further supported by the occurrence of two diVerential groups of chlorophyllase


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genes: one encoding intrachloroplastic enzymes constitutively expressed at low levels and the other encoding extrachloroplastic chlorophyllases. In citrus, this last group may be induced by ethylene and therefore involved in a hypothetical extraplastidic pathway of chlorophyll breakdown (reviewed in Ho¨rtensteiner, 2006). The existence of this additional pathway suggests that natural and ethylene‐induced degreening may take place by diVerent mechanisms reconciling those apparent contradictory observations. In addition to chlorophyll breakdown, colour change also depends on the rate of carotenoid accumulation in the chromoplasts. Just prior to carotenoid build‐up, there is a transition from the carotenoids of the photosynthetic chloroplast to the intensely coloured carotenoids of the chromoplast (Eilati et al., 1969; Gross, 1987). The expression patterns of carotenoid biosynthetic genes and associated abundance of carotenoids during ripening have recently been reported (Alo´s et al., 2006; Kato et al., 2004; Rodrigo et al., 2004). Cytoplasmic and mitochondrial isoprenoids are thought to be formed from acetyl‐coA via mevalonate, while plastidial isoprenoids such as carotenoids, gibberellins, tocopherol and the phytol chain of chlorophylls are formed from the 2‐methyl‐erythritol‐phosphate (MEP) pathway (Lichtenthaler et al., 1997). In the MEP pathway, four molecules of isopentenyl diphosphate form the C20‐intermediate geranylgeranyl diphosphate (GGPP) that can be used to synthesize gibberellins, phytyl diphosphate via geranylgeranyl reductase (CHL‐P) and carotenoids. In the carotenoid pathway, two molecules of GGPP are condensed to form phytoene in a reaction catalysed by phytoene synthase (PSY), the first committed step in carotenogenesis (Fig. 2). Then, four consecutive desaturations, catalysed by the enzymes phytoene desaturase (PDS) and ‐carotene desaturase (ZDS) render lycopene. Cyclization of lycopene, the first branching point, by ‐lycopene cyclase (‐LCY) leads to ‐carotene and its xanthophyll derivatives, while cyclization by ‐LCY and e‐LCY leads to ‐carotene and lutein (Hirschberg, 2001). Then, sequential oxidizations lead to the diVerent xanthophylls (oxygenated carotenoids) present in the fruit. Although diVerent citrus varieties accumulate diVerent carotenoids (see below), a reported general rule is that the ,e branch is mostly characteristic of green fruits while the , branch is predominant in mature fruits (Alo´s et al., 2006; Kato et al., 2004; Rodrigo et al., 2004). This shift between the two branches is pivotal for colour break since it leads to qualitative changes in carotenoid composition and hence it is accelerated or delayed when fruits are treated with ethylene or ripening retardants, respectively (Alo´s et al., 2006). In tomato, extensive research on the regulation of carotenoid biosynthesis has shown that this pathway is mainly controlled at the transcriptional level (Giovannoni, 2004). In this model, a main key regulatory gene is PSY whose expression level appears to be correlated

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GAP + Pyruvate DXS DXP DXR MEP

MEP pathway

IPP

DMAPP GGPP

CHL-P

Phytyl diphospate

CHL-G

Chlorophyll a

Phytoene

CLH Chlorophyllide a

Phytofluene

MCS Pheophorbide a

PSY PDS PDS z-carotene

RCC

PaO

ZDS Neurosporene

RCCR FCCs

ZDS Lycopene b-LCY

b-LCY e-LCY

b-carotene

a-carotene

b-criptoxanthin

a-criptoxanthin

Zeaxanthin

Lutein

NCCs

b-CHX

b-CHX b-CHX

e-CHX

ZEP Antheraxathin ZEP Violaxanthin NSY Neoxanthin

Fig. 2. Regulation of carotenoid and chlorophyll biosynthesis in flavedo of citrus fruits during ripening. Pigment substitution associated with the conversion of chloroplasts into chromoplasts during external citrus fruit ripening depends on the coordinated transcriptional control of genes involved in several plastidic metabolic pathways. During degreening, carotenoid biosynthesis and chlorophyll degradation are induced while chlorophyll synthesis is repressed. Induced and repressed genes are highlighted by red and green colours, respectively. Expression data are from Kato et al. (2004), Rodrigo et al. (2004) and Alo´s et al. (2006). Several steps are omitted for simplification. Nature of colour of each pigment is highlighted on their own names. Abbreviations: CHL‐P, geranylgeranyl reductase; CHL‐G, chlorophyll synthase; ‐CHX, ‐carotene hydroxylase; e‐CHX, e‐carotene hydroxylase; CLH, chlorophyllase; mcs, metal chelating substance (unknown); DMAPP, dimethylallyl diphosphate;


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with total carotenoid content in the mature fruit (Giuliano et al., 1993). More recent reports also suggest the involvement of other upstream genes such as 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXS) (Lois et al., 2000) that catalyses the first step of the MEP pathway (Fig. 2). In citrus, total carotenoid levels correlated with expression of PSY and PDS, repression of e‐LCY and further increase of ‐LCY expression (Kato et al., 2004; Rodrigo et al., 2004). These changes that lead the shift from the ,e branch to the , branch are concomitant as explained above with chlorophyll catabolism and hence the combination of both processes is responsible for degreening, that is, the change from green to coloured tissues. Thus, Alo´s et al. (2006) showed that DXS expression levels peaked at the mature green stage together with carotenoid and chlorophyll concentrations. Natural degreening was accompanied, however, by a marked decrease in transcript levels of DXS and CHL‐P while, conversely, expression of PSY and pheophorbide a oxygenase (PaO), a gene involved in chlorophyll disappearance, increased. This pattern of change is compatible with the enhancement of the metabolic flux through the carotenoid pathway and the simultaneous reduction of chlorophyll synthesis. Furthermore, gibberellin and nitrate, two degreening retardants, delayed both the decrease of DXS expression and the induction of PaO and PSY transcript accumulation. These results suggest that transcriptional control rules the coordination of the synthesis of both chlorophylls and carotenoids (Fig. 2) and that competition for GGPP between PSY and CHL‐P at the branching step is a pivotal element in this coordination. Although the characteristic colour of typical citrus varieties is mainly provided by the accumulation of 9‐cis‐violaxanthin and ‐cryptoxanthin, there are also citrus‐specific carotenoids such as ‐citraurin and ‐citraurinene that provide an attractive colouration and whose biosynthetic basis remains unknown (Oberholster et al., 2001). Citrus contains the greatest diversity of carotenoids of any fruit studied to date and their specific accumulation patterns are responsible for the broad range of colours exhibited by citrus fruits (Gross, 1987). Several studies indicated that diVerences in fruit carotenoid composition might be used to classify citrus varieties, thus linking genetic DXP, 1‐deoxy‐D‐xylulose 5‐phosphate; DXR, 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase; DXS, 1‐deoxy‐D‐xylulose 5‐phosphate synthase; FCCs, fluorescent chlorophyll catabolites; GAP, glyceraldehyde‐3‐phosphate; GGPP, geranylgeranyl diphosphate; IPP, isopentenyl pyrophosphate; ‐LCY, ‐lycopene cyclase; e‐LCY, e‐lycopene cyclase; MEP, 2‐methyl‐erythritol‐phosphate; NCCs, non‐fluorescent chlorophyll catabolites; PaO, pheophorbide a oxygenase; RCC, red chlorophyll catabolite; RCCR, red chlorophyll catabolite reductase; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ‐carotene desaturase; ZEP, zeaxanthin epoxidase; NSY, neoxanthin synthase.

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diversity with carotenoid content. Goodner et al. (2001) reported that the diVerences in the concentration of ‐cryptoxanthin in the juice of mature fruits can be used to diVerentiate oranges, mandarins and their hybrids. More recently, Fanciullino et al. (2006) classified 25 citrus varieties belonging to eight species into three clusters according to the contents of ‐cryptoxanthin and violaxanthin in mature fruit juice. A more comprehensive study on the classification of citrus varieties on the basis of diVerential carotenoid accumulation has been recently carried out by Matsumoto et al. (2007), that quantified 18 carotenoids in flavedo and pulp of 39 citrus varieties at diVerent stages of fruit development and ripening. The patterns obtained in flavedo allowed the classification of the varieties into five clusters: carotenoid‐poor, phytoene‐ abundant, violaxanthin‐abundant, violaxanthin‐ and ‐cryptoxanthin‐abundant, and phytoene‐, violaxanthin‐ and ‐cryptoxanthin‐abundant. According to the patterns of carotenoids in juice sacs, the same varieties were classified into four clusters, since no phytoene‐abundant cluster was detected. Further work is currently being carried out in order to compare the expression levels of the carotenoid biosynthetic genes among the diVerent citrus varieties (Fanciullino et al., 2008). The carotenoid profiles of flavedo and pulp are, with few exceptions, very similar (Matsumoto et al., 2007). However, the increase in carotenoid levels and the related changes in gene expression are generally initiated before in the pulp than in the flavedo although both processes proceed more slowly in the pulp (Kato et al., 2004). Carotenoid content in pulp is also generally lower than in flavedo. An exception to this general rule is the cara‐cara mutant of Washington navel, characterized by an accumulation of lycopene in the pulp while a normal carotenoid profile is present in the flavedo. This mutated phenotype has been explained on the basis of an alternative splicing of LCY (Tao et al., 2005). Other citrus mutants aVected in fruit colour have been identified and are currently being studied. A very useful mutant for this purpose is the so‐called nan (‘navel negra’, black navel) mutant, a spontaneous mutation of Washington Navel impaired in chlorophyll catabolism and thus exhibiting an abnormal brown colour in the ripe flavedo (Alo´s et al., 2008). Under natural conditions, expression of chlorophyll metabolism genes in the nan mutant is normal during ripening. Similarly to its parental cultivar, the mutant responds to exogenous ethylene inducing expression of phytoene synthase, chlorophyllase and pheophorbide a oxygenase and repressing geranylgeranyl reductase. However, no significant loss in chlorophyll content can be observed in the mutant either during natural degreening or after ethylene treatment. Transcript profiling studies had revealed that a citrus orthologue of several SGR (stay green) genes is significantly repressed in the mutant. The involvement of SGR in chlorophyll degradation has been


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demonstrated in transgenic rice leaves (Jiang et al., 2007; Park et al., 2007). Unlike previously described stay‐green mutants, however, the nan mutation appears to be associated, not with the SGR gene itself, but with an upstream regulatory element. The mutant, therefore, accumulates free chlorophylls, that in turn induce other altered responses such as increased ROS and subsequent anti‐oxidant and photo‐protective responses such as carotenoid and ascorbic acid accumulation.

D. THE ‘FIT TO BE EATEN’ PART

During the past decades, research on citrus fruit flavour that depends upon multiple compounds mostly sugars, acids and flavanones, has received considerable input because of both the uniqueness of the physiological processes sustaining this trait and the potential importance of these components to human health. Mature citrus pulp contains a very high percentage of water (85–90%) and many diVerent constituents, including carbohydrates, organic acids, amino acids, vitamin C, minerals and small quantities of lipids, proteins, and secondary metabolites, such as carotenoids, flavonoids and volatiles (Davies and Albrigo, 1994). Total soluble solids comprise 10–20% of the fresh weight of the fruit, and consist mainly of carbohydrates (70–80%), and relatively minor quantities of organic acids, proteins, lipids and minerals. During ripening, in general, there is a decline in titratable acidity (TA) mostly due to catabolism of citric acid (the principal organic acid of citrus juice; Monselise, 1986) and an increase in sugars, usually expressed as total soluble solids (TSS). The TSS to TA ratio is commonly known as the maturity index (MI). In citrus, one of the intriguing observations of the research on internal ripening processes is that citrus maturation does not appear to be strongly aVected by any particular hormonal treatment. Thus, the identification of regulatory genes expressed in citrus pulp during ripening provides precious information on the nature of these mechanisms. To date, the most comprehensive study on the transcriptome profiling of the citrus fruit flesh was presented by Cerco´s et al. (2006) who examined gene expression with a 7 K cDNA microarray, during development and ripening of self‐incompatible Citrus clementina. They reported that as many as 2243 putative unigenes showed significant expression changes while functional classification revealed that genes encoding for regulatory proteins were very abundant (more than 10% of the expression changes). These proteins were overrepresented approximately within the core of the rapid fruit growth phase suggesting that fruits at this stage were reprogramming developmental commands to face the

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complex cellular modifications associated with ripening. The most abundant family of up‐regulated transcription factors in developing citrus pulp was the NAC family. These genes are grouped in a very large family of plant‐specific transcription factors characterized by a highly conserved N‐terminal domain (Aida et al., 1997; Olsen et al., 2005). The functions of NAC genes remain unknown although it has been reported that this family is related to a wide range of biological processes (reviewed in Ooka et al., 2003 and Olsen et al., 2005). Its involvement in fruit ripening has been specifically suggested for SENU5, a NAC transcription factor that was up‐regulated during tomato fruit ripening (John et al., 1997). In citrus fruits, three of the up‐regulated NAC factors are orthologues of the Arabidopsis NAC genes AtNAC2, NAP and At3g04070, previously involved in vegetative (Takada et al., 2001) and reproductive developmental processes. For example, Sablowski and Meyerowitz (1998) demonstrated that NAP is implicated in the transition from cell division to cell expansion in Arabidopsis floral organs. The citrus orthologue of NAP might also play a role during fruit development since in citrus a more gradual but equivalent transition takes place between phase I and phase II (Bain, 1958). Similarly, two additional up‐regulated citrus NAC factors are the orthologues of Arabidopsis ATAF1 and ATAF2 factors. These have previously been implicated in the response to wounding and pathogens on the basis of the expression of their potato orthologues (Collinge and Boller, 2001). Furthermore, it has been reported that many genes involved in the response to wounding, pathogens and insects are induced during citrus fruit development (Shimada et al., 2005b). Since citrus fruits are grown in an open field and therefore are permanently exposed to wounding and insect attack, it is reasonable to think that the expression of the two ATAF factors could be related to these conditions. The dry‐summer environment, when citrus fruit expands (phase II), might also be the reason for induction of another citrus NAC factor orthologue to Arabidopsis RD26, a gene induced by drought, salt stress and ABA (Fujita et al., 2004). The results of microarray analyses also identified additional transcription factor families that were apparently deeply involved in fruit development and ripening (Cerco´s et al., 2006) such as MYB and MADS genes. Interestingly, these families have been frequently correlated with development and ripening of other fruit species, including apple and strawberry (Giovannoni, 2004). In addition to transcription factors, genes encoding proteins with potential functions in signal transduction mechanisms were also found, such as protein phosphatases, Ras‐related small GTP‐binding proteins and serine/threonine protein kinases. Further detailed analyses of their physiological significance and occurrence will provide information on the roles of these genes in citrus fruit development.


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1. The sweeter the better Transcriptomic analyses also highlighted a large number of genes encoding enzymes involved in metabolic processes. Most pivotal metabolic changes were related to carbohydrate build‐up, acid reduction, modifications in secondary metabolism, carotenoid accumulation and chlorophyll decreases. It is generally accepted that developing citrus fruitlets constitute main utilization sinks during the cell division period and act as carbohydrate storage sinks during the cell enlargement stage and thereafter (Mehouachi et al., 1995). These analyses suggested that in those stages, carbohydrate synthesis and catabolism are mostly down‐regulated while sugar transport appears to be rather operative (Cerco´s et al., 2006). Sink strength in citrus fruits has been associated with the presence of sucrose metabolizing enzymes, mainly sucrose synthase (Etxeberria et al., 2005; Hockema and Etxeberria, 2001; Komatsu et al., 2002). Enzymatic activity assays also indicated that sucrose accumulation during citrus fruit ripening parallels an increase in sucrose synthase activity (Komatsu et al., 2002). Sucrose synthase in citrus is apparently encoded by three non‐allelic genes, CitSUS1, CitSUS2 and CitSUSA. While CitSUS1 was expressed in green fruits and repressed during ripening, CitSUSA was induced during citrus fruit ripening (Komatsu et al., 2002). Moreover, alkaline invertase and sucrose phosphate synthase activities are mainly detected in the sink cells whereas sucrose synthase activity appears to be associated with the vascular bundles (Lowell et al., 1989; Tomlinson et al., 1991), specifically into the companion cells. In citrus fruit, juice sacs containing sink cells are physically separated from vascular bundles and hence, the above observations indicate a role for sucrose synthase activity in phloem loading and unloading processes (Nolte and Koch, 1993). Thus, it has been suggested that thereafter, sucrose phosphate synthase activity may re‐ synthesize sucrose for further transport to the vacuoles (Komatsu et al., 2002). Post‐phloem apoplastic transport of sucrose into the juice vesicles certainly occurs with very little hydrolysis (Koch and Avigne, 1990) indicating that sucrose import into the sink cells may well require sucrose transport through the apoplastic route into the vacuoles of the sink cells. In this regard, Etxeberria et al. (2005) demonstrated the existence of Hþ‐sucrose symporters in the plasma membrane and also provided evidence for a subsequent endocytic transport system from the apoplast to the vacuole, a mechanism that allows direct incorporation of sucrose into the vacuole bypassing membrane transport. E. ACIDIC AND ACIDLESS FRUIT

Developing fruits also accumulate during the first half of phase II a considerable amount of organic acids in the vacuoles of the juice sac cells that are progressively catabolized over the second half of phase II through phase III.

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The physiological roles of organic acids are not clear; however, Hockema and Etxeberria (2001) suggested that low pH could result in enhanced sink strength, thus facilitating sugar accumulation. Organic acids contribute significantly to the overall fruit quality and the regulation of acidity loss during ripening, although practically unknown, is a main constraint of the citrus industry. Citric acid accounts for most of the TA in fruit juice (80–90%) that also contains malate (9–15%) and minor quantities of succinate and isocitrate (Baldwin, 1993). Thus, acidity loss mostly concerns to citrate utilization. There is considerable evidence obtained comparing acidless and acidic varieties that activity and expression of citrate synthase was not responsible of diVerences in acid accumulation (Sadka et al., 2001). A simple model has also been proposed suggesting that changes in mitochondrial aconitase activity could be determinant in acid accumulation (Sadka et al., 2000a). Acid build‐up, however, appears to be a complex process controlled by many coordinated enzymes. Furthermore, since the export of citric acid from the mitochondria is a cataplerotic process, several anaplerotic reactions are expected to occur for the replenishment of the Krebs cycle intermediates during the acid accumulation phase. Acid reduction during ripening implicates citrate release from the vacuole to the cytosol and further cytoplasmatic metabolism. Recently, a citrate transporter gene encoding for a novel transporter that mediates citrate vacuolar eZux through the electroneutral co‐transport of Hþ and citrate ions has been characterized (Shimada et al., 2006). Once in the cytosol, citrate is apparently metabolized to isocitrate by cytosolic aconitase and then into 2‐oxoglutarate by NADP isocitrate dehydrogenase (Cerco´s et al., 2006; Sadka et al., 2000a,b). Further data on 2‐oxoglutarate utilization were also provided by microarray analyses and subsequent quantitation of selected metabolites (Cerco´s et al., 2006). The proposal advanced in this work was also compatible with a posterior proteomic study of the citrus fruit proteome reported in Katz et al. (2007). This suggestion proposes that citrate is sequentially metabolized to glutamate that is either used for thiamine biosynthesis or catabolized through the gamma‐aminobutyrate (GABA) shunt (Fig. 3). This observation is of special relevance since it links an eYcient major proton consuming reaction with the occurrence of high acid levels. It is known that in higher plants, GABA is biosynthetized through a proton consuming reaction catalysed by glutamate decarboxylase (Bown and Shelp, 1997). Furthermore, several reports in diVerent species including carrot cell suspensions (Carroll et al., 1994) and ripe tomato fruits (Rothan et al., 1997) indicated that glutamate decarboxylase was activated and GABA synthesis was stimulated by increases in the cytosolic levels of Hþ. During citrus fruit ripening, cytoplasmatic acidification after citrate release from the vacuole may trigger the induction of glutamate decarboxylase reducing, thus, cytosolic acidosis.

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FRUIT QUALITY IN CITRUS Carbohydrates

Oxaloacetate Cit-MDH Malate

PEPCK

Phosphoenolpyruvate

PEPC

PK

NADP-ME

Citrate

Pyruvate ACN Isocitrate Pyruvate NADP-IDH Acetyl-CoA

Oxaloacetate

2-oxoglutarate GADH AsAT AIAT Glutamate

Citrate GADC

Malate

GABA Fumarate

Succinate Mitochondria

GS

Iso citrate Glutamine

a-ketoglutarate GABA-AT

Succinyl-CoA

Succinate semialdehyde SSADH Succinate

Thiamine

Fig. 3. Proposed model for the acid metabolism during ripening of citrus fruit pulp. Acid reduction during ripening implicates citrate release from the vacuole to the cytosol and further cytoplasmatic metabolism. Once in the cytosol, citrate is metabolized to isocitrate by cytosolic aconitase and then into 2‐oxoglutarate by NADP isocitrate dehydrogenase. Later, oxoglutarate is sequentially metabolized to glutamate that is either used for thiamine biosynthesis or catabolized through the gamma‐ aminobutyrate (GABA) shunt. GABA is metabolized to succinate that is incorporated into the mitochondrion while malate is released to the cytoplasm and converted to oxaloacetate. This intermediate, through phosphoenolpyruvate carboxykinase, is transformed to phosphoenolpyruvate entering sugar metabolism. Induced genes during ripening (Cerco´s et al., 2006) are highlighted in red. Abbreviations: ACN, aconitase; AlAT, alanine aminotransferase; AsAT, aspartate aminotransferase; Cit‐MDH, cytosolic malate dehydrogenase; GABA, gamma‐aminobutyrate; GABA‐AT, gamma‐aminobutyrate aminotransferase; GADC, glutamate decarboxylase; GADH, glutamate dehydrogenase; GS, glutamine synthetase; NADP‐IDH, NADP isocitrate dehydrogenase; NADP‐ME, NADP‐dependent malic enzyme; SSADH, succinate semialdehyde dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PK, pyruvate kinase.

The suggestion provides a convincing explanation for the strong reduction of both citrate and cytoplasmic acidity that takes place in citrus fruit flesh during development and ripening.

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A strong increase in the expression of phosphoenolpyruvate carboxykinase (PEPCK) has been found to occur in parallel to the decrease in acidity (Ibań˜ez et al., 2007) during ripening. The function of this gene has been related to main metabolic processes in plants, including both gluconeogenesis and respiration (Malone et al., 2007). Particularly in soft fruits, several studies show a pivotal role in the catabolism of organic acids (Famiani et al., 2005), amino acid and carbohydrate metabolism (Lea et al., 2001). This observation may also link the acid catabolism with an increase in the carbohydrate content in mature citrus fruits (Fig. 3). Using LC‐MS/MS, Katz et al. (2007) analysed soluble and enriched membrane fractions of mature citrus fruit to identify the proteome of fruit juice cells. In this approach, the authors identified 1400 proteins from these fractions by searching NCBI‐nr (green plants) and citrus ESTs databases, classified these proteins according to their putative function and assigned function according to known biosynthetic pathways. This work constitutes the first proteomic study of the citrus fruit and oVers an exhaustive listing of predominant proteins in juice cells including proteins involved in sugar metabolism, citrate cycle, signalling, transport and processing. In a parallel approach, major proteins in the albedo of the fruit peel of matured on‐tree fruits, that is, manganese superoxide dismutase, actin, ATP synthase subunit, citrus salt‐stress‐associated protein, ascorbate peroxidase, translationally controlled tumour protein and a cysteine proteinase (CP) of the papain family, were identified through MS/MS (Lliso et al., 2007). This proteomic survey indicated that major changes in protein content in the albedo of the peel during the ageing process were apparently related to the activation of programmed cell death. In addition, transcriptomic studies indicated that the fraction of genes induced during pulp development and ripening was significantly enriched in genes involved in proteolysis and similar results have been found associated with external ripening (Alo´s, 2007; Cerco´s et al., 2006), suggesting an important remodelling of the fruit proteome during ripening. A significant fraction of the unigenes of this functional category corresponded to proteins involved in the ubiquitin‐dependent proteolysis pathway, including 12 ubiquitin‐ligases of the F‐box and RING families with diVerential time‐ and tissue‐specific expression patterns indicating a role of these proteins in the regulation of proteome modification.

IV. IT IS JUST NOT FOOD It is well known that citrus fruit besides the usual fruit components contain many organic compounds necessary for human diet that may also display well‐appreciated characteristics for health. Citrus fruits are the main source


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of vitamin C in developing countries and there is also an increasing demand of ‘high quality fresh citrus’ driven by consumers and by World Health Organization recommendations in developed markets such as North America, Europe and Japan. Furthermore, citrus possesses the largest number of carotenoids found in any fruit, together with an extensive array of secondary compounds (vitamins C and E, i.e. tocopherols, pro‐vitamin A, flavonoids, limonoids, polysaccharides, lignin, fibre, polyphenols, essential oils, etc.). It has been known for many years, that several of these substances exhibit well‐known nutraceutical activities such as anti‐oxidant, anti‐inflammatory, cholesterol and allergic activities and many of them are essential to prevent cardiovascular and degenerative diseases, thrombosis, cancer, atherosclerosis and obesity. It is surprising that many new properties and substances are continuously been found in citrus fruit. For example, it has recently been suggested that perillyl alcohol from citrus peel has a skin cancer chemoprotection eVect (Einspahr et al., 2003), while ‐hemulene exhibits in vitro a high cytotoxic activity against human tumour models (Loizzo et al., 2007). Moreover, sesquiterpenes have also demonstrated therapeutic potential as anti‐cancer agents (Wang et al., 2007). Thus, there are many important aspects of citrus fruit quality apart from physical attributes and diet components that are intimately related to human health (Bakkali et al., 2007). Citrus is, therefore, an excellent model to study fruit quality because of its peculiar fruiting, singular biochemistry and economical relevance. Therefore, there is an enormous potential in the study of the regulation of secondary metabolism associated with citrus fruit growth. Below, we focus our attention on three main characteristic secondary bioactive compounds of citrus fruit, essential oils, limonoids and flavonoids that are also major contributors of aroma and flavour. Plant volatile compounds are secondary metabolites that may act as signals to attract pollinators and seed dispersers, or even predators of enemies or repel harmful organisms and may also play important roles in plant‐ to‐plant communication (Pichersky and Gershenzon, 2002). In fruit, while sugars and acids are liable for sweetness and acidity, aroma and flavour are the overall result of a unique assortment of volatile compounds. In citrus, volatile terpenoids, the principal components of the essential oils are responsible for much of the aroma and flavour of the fruit. Many of them are important fragrance and flavour compounds and it is believed that some of them possess beneficial eVects for human health because of their anti‐oxidant eVects and anti‐cytotoxic properties at the cellular level (Bakkali et al., 2007). During the past years, research in the triterpenes limonoids has considerably increased particularly after the discovery of the potential benefit of citrus limonoids as bioactive compounds to human health. Recently it has been demonstrated, for example, that obacunone, nomilin, limonin,

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deacetylnomilin and limonin‐17‐beta‐D‐glucopyranoside all limonoids from the red Mexican grapefruit are bioactive anti‐oxidant compounds that inhibit the growth of cancer in colon, lung, mouth, stomach and breast in animal and cell culture studies (Mandadi et al., 2007). Moreover, limonin and nomilin induce glutathione‐S‐transferase enzyme activity in liver and small intestine mucosa of mice (Lam et al., 1989) and have been found to inhibit replication of HIV‐1 (Battinelli et al., 2003). Citrus fruits, in particular oranges, are also extremely rich sources of flavonoids, and intake of these compounds in the habitual diet is very healthy because of their anti‐oxidant properties (Franke et al., 2005). These compounds are mainly located in the peel; polymethoxyflavones, that are generally more active in the flavedo and flavanones in the albedo (Kanes et al., 1992; Ortunõ et al., 1997). It is known, for example that nobiletin, a polymethoxyflavone from orange peel, possesses anti‐cancer activity and prevents atherosclerosis (Kawabata et al., 2005; Whitman et al., 2005) and that auraptene is a potential chemopreventive agent against chemically induced carcinogenesis in digestive tract and liver (Sakata et al., 2004). The content of nobiletin and naringin correlate with nitric oxide production inhibitory activity of peel extracts from various citrus fruits, and it has been proposed that these compounds may provide protection against disease resulting from excessive nitric oxide production (Choi et al., 2007). It has been also reported that glycosylated citrus flavanones (naringin, hesperidin and limonin) also protect against colon cancer development (Fenton and Hord, 2004; Vanamala et al., 2006). Overall, the action of these bioactive compounds appears to complement other nutraceutical properties provided by pivotal carotenoids and vitamins (Arias and Ramon‐Laca, 2005; Nishino et al., 2004).

A. BREAKING THE SMELL

Several studies have focused on the composition of essential oils, a blend of diVerent volatile compounds, in many Citrus species. Volatile compound terpenoids along with alcohols, aldehydes, ketones and acids are the major determinants of the aroma and flavour of citrus fruits. The emission patterns of essential oils show diVerential spatial and temporal changes and are also strongly influenced by many environmental conditions such as insect attack. In sweet orange, for instance, flowers at pre‐anthesis emitted myrcene and ocimene, followed by felandrene, linalool and (E)‐‐caryophyllene, while in ovaries and petals of flowers at anthesis, linalool was the main volatile released (Carrera et al., 2007). Aroma and flavour quality of commercial


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juices and oils may change depending upon processing conditions used to extract and concentrate citrus juice (Lin et al., 2002). Citrus essence oils are characterized for having a high content of terpenoid hydrocarbons and relative low amount of terpenoids oxygenated compounds (0.5–5%). Despite their low levels, these oxygenated terpenes such as acyclic isoprenoids alcohols nerol, citronellol and farnesol and their corresponding aldehydes (neral, citronellal and sinensal) are the compounds that provide the characteristic flavour and aroma to the diVerent citrus species (Weiss, 1997). It is believed that the combination of diVerent proportions of volatile terpenes as well as the presence and absence of trace terpenes may have a profound eVect on citrus fruit flavour and aroma (Ho¨gnado´ttir and RouseV, 2003). Among terpenes, monoterpenes and sesquiterpenes are the major components (up to 90%) of citrus essential oils, although only a few of them display aroma activity. Monoterpene (þ)‐limonene, for example, is the major component of most citrus oils, but its apparent aroma activity appears to be due to co‐eluting impurity (Ho¨gnado´ttir and RouseV, 2003). As a rule, high intensity fragrances are associated with reduced oil levels. Thus, the most intense orange essence oil aroma are produced by terpene alcohol linalool (0.7%), fatty aldehydes decanal and octanal, sesquiterpene aldehydes citronellal and ‐sinensal, ‐ionone (a carotenoid degradation product), and the cyclic ester wine lactone. The distinctive fresh sweetness from the orange peel is mainly due to sesquiterpene sinensal, specially the isomer (all‐E)‐‐ sinensal, whose odour detection threshold is as low as 0.05 ppb. The sesquiterpene ketone (þ)‐nootkatone, a derivate from valencene, has been suggested to have a dominant role in the flavour and aroma of grapefruit (Macleod and Buigues, 1964; Shaw and Wilson, 1981). Nootkatone has been obtained with high yield by biotransformation of (þ)‐valencene, which can be obtained cheaply from oranges, through the use of micro‐organism such as fungi and green algae (Furusawa et al., 2005). Citrus oils accumulate in the flavedo peel in secretory cavities, called oil glands, that are specialized structures responsible for the synthesis and accumulation of large amounts of terpenes. In the juice vesicles, citrus oils accumulate in oil bodies (Sinclair, 1984; Weiss, 1997). A typical oil gland is a multicellular secretory structure composed of a package of broadly round cells in shape, in the centre of which an essential oil‐accumulating space is formed. The secretory cells lining the cavity are thought to be responsible for the production and collection of terpenes. The secretory material in the form of droplets is produced in plastids, from where it is transported to the parietal cytoplasm of the secretory cells via smooth endoplasmic reticulum (sER). After fusion of the sER membranes with the plasmalemma, the exudate reaches the apoplast, through which it is driven to the central cavity of the gland.

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As mentioned above, isoprenoids are synthesized through two independent biosynthetic pathways. Cytosolic mevalonic acid (MVA) pathway is responsible for the biosynthesis of sesquiterpenes from the intermediate farnesyl diphosphate (FPP), while monoterpenes are synthesized from geranyl diphosphate (GPP) through the methyl erythritol phosphate (MEP) pathway, which is considered to be compartmentalized in the plastids (Fig. 2) (Rohmer, 1999; Rodriguez‐Concepcion and Boronat, 2002). In turn, isoprenoid intermediates, are synthesized through condensation of the five‐carbon isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DAMPP) by a GPP synthase (McGarvey and Croteau, 1995). Although biosynthetic genes involved in the synthesis of the terpene intermediates DAMPP and IPP have not yet been cloned, it has been reported that cDNA sequences for DXS (deoxyxylulose 5‐phosphate synthase), HDS (hydroxymethylbutenyl diphoshate synthase) and HDR (hydroxymethylbutenyl diphoshate reductase) are down‐regulated in a Satsuma mandarin mutant that exhibits a smoother and tighter albedo rind and low fragrance (Ishikawa et al., 2007). In a recent paper, Yamasaki and Akimitsu (2007) have shown through in situ localization that gene transcription for monoterpene synthesis in rough lemon (Citrus jambhiri) was detected in irregular parenchymic cells surrounding the secretory cavities. The study was performed with three transcripts, two terpene synthases and a gene upstream in the MEP pathway of monoterpene biosynthesis, RlemispF, coding for 2‐C‐methyl‐D‐erythritol 2,4‐cyclodiphosphate synthase, the step previous to HDS. This RlemispF gene, that has a plastid transit peptide was eVectively localized in the chloroplast, indicating that this enzyme belongs to the plastid MEP pathway. Expression of RlemispF gene was clearly detected around developing secretory cavities, although just a weak expression was visible around the mature secretory cavities. Deduced amino acid sequence of RlemispF revealed multiple phosphorylation sites for protein kinases, suggesting putative post‐transductional regulation. Terpene synthases (TPS) are key enzymes responsible for the diversity of terpene products in plant kingdom, and their catalytic properties have been the subject of diVerent studies including domain swapping, site‐direct mutagenesis and molecular modelling (Back and Chappell, 1996). Despite their sequence diversity, terpene synthases share several conserved amino acid residues, including a short aspartate‐rich motif (DDxxD) related to the binding of a divalent metal cofactor (Bohlmann et al., 1998). Elucidation of the three‐dimensional structures of plant terpene synthases and molecular modelling have allowed identification of the active site and determination of the amino acids that might be directly involved in the catalytic process (Hyatt et al., 2007). Some plant TPS, including those that have been isolated from


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diVerent Citrus spp., are multi‐catalytic and multi‐product enzymes with a high promiscuity and amino acid plasticity (Little and Croteau, 2002; Yoshikuni et al., 2006). It has been reported, for example, that the OsTPS3 gene coding for a (E)‐‐caryophyllene synthase accounts for the major inducible volatile sesquiterpenes in rice despite its genome contains about 50 genes encoding for putative TPS (Cheng et al., 2007). Moreover, ‐humulene synthase from conifer grand fir (Abis grandis) produces 52 diVerent sesquiterpenes from the unique acyclic substrate farnesyl diphosphate, through a wide variety of cyclization mechanisms (Steele et al., 1998). Although many monoterpenes and sesquiterpenes have been identified in citrus essential oils and a monoterpene synthase activity was partially purified from lemon in the late 1970s (Chayet et al., 1977), the first citrus TPS gene, CJFS from Yuzu (Citrus junos) was cloned 24 years later (Maruyama et al., 2001). The gene encoded for the acyclic sesquiterpene synthase, (E)‐‐ farnesene synthase, and showed high homology to other plant sesquiterpene synthases genes previously isolated (Crock et al., 1997; Facchini and Chappell, 1992). The second sesquiterpene synthase gene isolated from citrus, Cstps1, coding for a valencene synthase was cloned from orange (Sharon‐Asa et al., 2003). Expression of Cstps1 gene increased towards fruit ripening correlating with valencene accumulation in fruit, an observation also reported in grapevine that showed higher valencene synthase gene expression during late ripening of berries (Lu¨cker et al., 2004b). Although it was initially supposed that valencene plays an important role in the overall flavour and aroma of orange fruit (Shaw and Wilson, 1981), it was later shown that valencene does not exhibit aroma activity (Elston et al., 2005; Ho¨gnado´ttir and RouseV, 2003). Therefore, valencene may simply be an indicator of fruit maturity, which correlates positively with increased orange flavour quality (Elston et al., 2005). Two additional sesquiterpene synthases genes isolated from Washington Navel are currently under characterization (Carrera et al., 2007). Many monoterpene synthase genes have been cloned and characterized. In lemon, four genes isolated from flavedo peel of young developing fruits appear to account for the formation of more than 90% of the lemon content of essential oils (Lu¨cker et al., 2002). Genes CILIMS1 and ClLIMS2 coding for limonene synthases, while ClPINS and ClTS genes encode a ‐penine and a ‐terpinene synthases, respectively. Interestingly, all four lemon TPS are able to form multiple products from GPP, as has been showed by functional expression of other monoterpene synthases from angiosperm such as spearmint, sage, lavender, and Nicotiana suaveolens, and the gymnosperm grand fir (see e.g., Bohlmann et al., 1999). Moreover, co‐expression of

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three lemon monoterpene synthases resulted in emission of ‐pinene, limonene and ‐terpinene in leaves and flowers of tobacco transgenic plant, in addition to the terpenoids emitted by wild‐type plants and other several side products (Lu¨cker et al., 2004a). Further swapping domain experiments between ClPINS and ClTS and between ClLIMS2 and ClTS demonstrated that a region of 200 amino acids within the C‐terminal domain of these monoterpene synthases, is responsible for determining product specificity (El Tamer et al., 2003). Two terpene synthases from rough lemon (C. jambhiri), RlemTPS1 and RlemTPS2, homologous to (þ)‐limonene and ()‐‐ pinene synthases genes from lemon, respectively, have been shown to be specifically expressed by in situ hybridization in the epithelial cells with dense cytoplasm that surround secretory cavities, the structures responsible for accumulation of essential oils (Yamasaki and Akimitsu, 2007). RlemTPS1 and RlemTPS2 genes were expressed in both developing and mature secretory cavities (Yamasaki and Akimitsu, 2007). Five novel monoterpene synthases genes, CitMTSE1, CitMTS3, CitMTS61, CitMTS62 and CitMTSL3, were isolated from Satsuma mandarin (Citrus deliciosa) (Shimada et al., 2004; Suzuki et al., 2004). Functional expression of five monoterpene synthases demonstrated that CitMTSE1 gene encode d‐limonene synthase; CitMTS3, CitMTS61 and CitMTSL3 genes encode ‐terpinene synthases; and CitMTS62 encode ‐pinene synthase. With the exception of CitMTSL3, whose expression was not detected, the other TPS genes were expressed in peel flavedo at early stages of fruit development. CitMTSE1 and CitMTS3 genes were also highly expressed in flowers at anthesis. These results suggest that a diVerent transcriptional regulation mechanism might be controlling spatial expression of monoterpene synthases in citrus. In addition, two additional monoterpene synthases genes, CitMTSL1 and CitMTSL4, coding for 1,8‐cineole and (E)‐‐ocimene synthases, respectively, were also isolated from Satsuma mandarin (Shimada et al., 2005a). Gene transcripts for both genes were comparatively abundant in flowers and then decreased towards fruit development. The transcript accumulation patterns of CitMTSL1 and CitMTSL4 were well correlated with the biosynthesis of 1,8‐cineole and (E)‐‐ocimene. Recently several citrus mutants have been found with main alterations in the regulation of terpene biosynthesis. Thus, independent transcriptomic analyses of a Satsuma (Ishikawa et al., 2007) and a Clementine (Carrera et al., 2007) mandarin mutants with a smoother flavedo and low fragrance, has revealed down‐regulation of genes related to the biosynthesis of isoprenoids. In a similar approach, transcriptomic analysis of fruits from the alf (altered fragrance) mutant of sweet orange has shown major changes in expression profile of genes coding for a new putative TPS and an O‐methyltransferase (OMT).


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Both TPS and OMT genes showed a diurnal cycle of expression as reported for 1,8‐cineole synthase (CIN) and (E)‐‐caryophyllene synthase (OsTPS3) genes from N. suaveolens and rice (Oryza sativa), respectively (Cheng et al., 2007; Roeder et al., 2007). Interestingly, down‐regulation of TPS gene expression correlated with a dramatic reduction of volatile terpene profile in fruits and flowers of the alf mutant (Carrera et al., 2007). B. BITTERNESS OR TASTELESSNESS

Limonoids are synthesized via terpenoid biosynthetic pathway, starting with squalene cyclization through the cytoplasmatic acetate‐mevalonate pathway. Limonoids are highly oxygenated triterpenes and contain a furan ring attached to the D‐ring (Hasegawa and Miyake, 1996). Further oxidations and skeletal rearrangement give rise to diVerent groups of limonoids (Endo et al., 2002; Hasegawa, 2000). Limonoids occur as limonoids aglycones that are neutral, insoluble and bitter, and also acidic, soluble and tasteless glycosides (Manners, 2007). Although many plant limonoids have been isolated, their occurrence is limited to species from order Rutales, including Citrus spp. Only in citrus fruits, there are about 40 limonid aglycones and other 17 limonoid glucosides and more predominant limonoids are limonin, the most abundant limonoid aglycone, and limonin glucoside, the most represented glucoside (Hasegawa and Miyake, 1996). Citrus limonoids occur in significant amounts in seeds and fruits, citrus seed being the major source of limonoid aglycones; up to 1% of their fresh weight (Hasegawa et al., 1980). Limonoids exhibit a diverse range of biological activities like insecticidal, insect anti‐feedant and growth regulating activity on insects as well as anti‐ bacterial, anti‐fungal, anti‐malarial, anti‐cancer, anti‐viral and several other pharmacological properties (reviewed by Manners, 2007). Interest in limonoids research started when it was shown that limonin was responsible for producing delayed bitterness in citrus fruits, that is, the formation of bitter limonoids from non‐bitter monolactone precursors (Emerson, 1949). This phenomenon is referred to as delayed bitterness and is a major problem for both fresh fruit and juice producers worldwide. Bitterness in citrus is caused in diVerent ways by two distinct compounds. Bitterness caused by flavonoids is common only in bitter citrus fruits such as grapefruit, bitter orange and pummelo while bitterness caused by limonin occurs in both bitter varieties and non‐bitter species (McIntosh et al., 1982). Bitter limonoids in concentrations above 6 ppm reduces the acceptability of citrus juices to consumers, restricting marketing for commercial citrus varieties. In the juice of citrus fruits, limonin‐based bitterness generally develops after physical or freezing damage. Mechanical disruption of juice sacs

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initiates the biochemical transformation of precursor limonate A‐ring monolactone (LARL) into the bitter limonoid aglycone limonin. The reaction is catalysed by the enzyme limonin D‐ring lactone hydrolase, under acidic conditions and is depending upon LARL availability (Fong et al., 1992). Natural debittering of citrus fruits, that is, the conversion of limonoids aglycones into tasteless limonoid glucosides, occurs during fruit ripening through the activity of limonoid glucosyltransferase (LGTase) (Hasegawa and Hoagland, 1977; Fig. 4). During natural fruit ripening of sweet orange, limonin glucoside concentration generally increases while LARL concentration decreases (Fong et al., 1992). However, higher susceptibility to delayed bitterness of Navel orange than, for example, Valencia orange is apparently due to a less eYcient mechanism to convert LARL to the tasteless limonin glucoside. It has also been shown that CitLGT expression increases during

O

O

Limonoid glucosyltransferase (debittering)

O

O GLU COOH O O

O O

Limonin glucoside Tasteless

O O

O

OH COO− O O

o

Limonate A ring lactone Tasteless

o 3 o 2 A 19 1

o Limonin D-ring lactone hydrolase (bittering)

A 5 4

11

17 o D 16 15 o

12 C

9 B 8 7 6

o o

Limonin bitter

Fig. 4. Limonid‐based bitterness and debittering of limonoids in citrus. Transformation of the precursor limonate A‐ring monolactone (LARL) into the bitter limonoid aglycone, limonin, is catalysed by the enzyme limonin D‐ring lactone hydrolase. Debittering of citrus fruits, that is, the conversion of limonoids aglycones into tasteless limonoid glucosides, occurs thorough the activity of limonoid glucosyltransferase.

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fruit development suggesting that transcriptional regulation of CitLGT gene controls the conversion of limonoid aglycones to glucosides. Metabolic engineering of limonin‐based bitterness in citrus transgenic fruits free from limonin bitterness was recently achieved by expression of a limonoid UDP‐ glucosyltransferase (LGTase) previously isolated from albedo of Satsuma mandarin (Endo et al., 2002; Kita et al., 2000; Moriguchi et al., 2003). Flavonoids (flavonols, flavanals, anthocyanidins, flavones and flavanones) are also important secondary plant metabolites with multiple functions and properties aside from conferring bitterness (Fig. 5). Their wide spectrum is the result of chemical variants and substitutions of the basic structure C15 flavan nucleus (Shirley, 1996). More than 60 diVerent flavonoids have already been identified in citrus (Gattuso et al., 2006). The tasteless flavanone hesperidin is the predominant flavonoid in most citrus fruits although its content depends upon the cultivar, environmental growing conditions and 3x malonyl-CoA and p-coumaryl-CoA CHS OH Naringerin chalcone

OH

OH

Anthocyanins DFR

CHI OH

O

F3H Dhk

Flavanone naringerin 7-O-GlucosyIT

FLS

Naringerin-7-O-glucoside

OH O

O

Flavanols

Cm 1,6 RhaT

Cm 1,2 RhaT

D-glucose 1-2 I-rhamnose

Dhq

I-rhamnose 1-6 D-glucose

7

OH O

O 7

OH O Naringin (bitter)

OH

Flavanones

O

Narirutin (tasteless)

Fig. 5. Flavanone‐based bitterness and debittering of flavanones in citrus fruit. Bitter and tasteless flavanones, naringin and narirutin, respectively, are synthesized from a derived branch of the flavonoid biosynthetic pathway. Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; 7‐O‐glucosylT, 7‐O‐glucosyltransferase; Cm1,2RhaT, 1,2 rhamnosyltransferase; Cm1,6RhaT, 1,6 rhamnosyltransferase; F3H, flavanone 3‐hydroxylase; DFR, dihydroflavonol reductase; FLS, flavonol synthase; Dhk, dihydrokaempferol; Dhq, dihydroquercetin.

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maturity stage (RouseV et al., 1987). In sweet orange, the polymethoxyflavones sinensetin, nobiletin, tangeretin and heptamethoxyflavone, and the flavanones hesperidin, isonaringin and narirutin (Del Rıó et al., 1998a,b) are more abundant, while neoeriocitrin and eriocitrin are typical to sour orange (RouseV et al., 1987). Several biosynthetic genes of the phenylpropanoid metabolic pathway such phenylalanine ammonia lyase (PAL), chalcone synthase (CHS) and chalcone isomerize (CHI), flavanone 3‐hydroxylase (F3H), dihydroflavonol 4‐reductase (DFR), flavonol synthase (FLS), and UDP‐glucose flavonoid glucosyl transferase (UFGT) have been cloned and characterized, and functional proteins expressed in vitro (Cotroneo et al., 2006). Chalcone isomerase (CHI) catalyses the intramolecular cyclization chalcones into flavanones. Flavanones, a flavonoid subgroup, that greatly contribute to the bitter flavour of grapefruit, bitter orange and pummelo, have also been the subject of intensive work. The source of bitterness in bitter citrus is provided by the flavanone naringenin 7‐O‐glucoside. In pummelo (Citrus maxima), it has been shown that the key flavour‐determinant step of the flavanone‐glycoside biosynthesis is catalysed by rhamnosyltransferase enzymes (Frydman et al., 2004). They demonstrated that 1,2‐rhamnosyltransferases (Cm1,2RhaT ) from pummelo catalysed the transformation of flavavones and flavones to the bitter 7‐O‐neohesperidosides, while 1,6‐rhamnosyltransferases catalysed biosynthesis of the tasteless rutinosides. Bitter species, such as grapefruit and pummelo, accumulated bitter flavanone‐7‐ O‐neohesperidosides (naringin, the major flavonoid glycoside in grapefruit) responsible, in part, for their characteristic juice flavour, while non‐bitter species, such as mandarin and orange, accumulated only tasteless flavanone‐ 7‐O‐rutinosides. The Cm1,2RhaT gene was highly expressed in young fruits and leaves but barely detected in mature fruits, correlating with the developmental pattern of accumulation of flavanone‐neohesperidosides previously reported (Bar‐Peled et al., 1993). Moreover, CitFLS transcripts that lead to flavonols, increased in the peel of Satsuma mandarin during fruit maturation. CitFLS has been characterized as a bifunctional enzyme with an alternative non‐specific 3‐‐hydroxylase activity (Lukacin et al., 2003). Anthocyanins are also an important subgroup of flavonoids, responsible for the colour of many plants, flowers and fruits, including the typical dark‐ red colour of the pulp of ‘blood’ oranges (Dugo et al., 2003; Maccarone et al., 1983, 1985; Markakis, 1982). It has been demonstrated that low temperature regulates expression of anthocyanin biosynthetic genes including PAL, CHS, DFR and UFGT and induces simultaneous anthocyanin accumulation in red orange. Upregulation of these genes, except for PAL, can be reverted to normal values after translation of oranges to the original conditions


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(Lo Piero et al., 2005). Furthermore, it has also been suggested that the reduction of DFR gene expression in non‐red orange cultivars such as Navel in comparison with that observed in red orange might be related to absence of anthocyanins in non‐red oranges (Lo Piero et al., 2006).

V. FRUIT QUALITY ONLY IN QUALITY ENVIRONMENTS It has been mentioned above that fruit development and size and hence fruit quality depends to a large extent upon growth rate and fruit load. Fruit quality, however, is also determined by many environmental conditions such as temperature, light intensity, soil humidity and biotic and abiotic conditions, particularly during the cell enlargement and maturation stages of fruiting (Davies and Albrigo, 1994; Iglesias et al., 2008; Spiegel‐Roy and Goldschmidt, 1996). Thus, internal and external attributes of quality can be considerably modified by the environment including the selection of the rootstocks and cultural practices. Temperature has a major impact on the development of standard rind colour in citrus fruit. Colour break in citrus that is dependent on changing pigment proportions in the flavedo, the outer portion of citrus fruit rind, is aVected by environmental temperatures that modulate both chlorophyll degradation and synthesis of carotenoid and other fruit pigments. In citrus fruit, lycopene synthesis is delayed at high temperatures whereas anthocyanin deposition is stimulated with prolonged cool temperatures. Furthermore, in late‐harvested cultivars of citrus, high temperatures and humidity may stimulate the reversion of chromoplasts to chloroplasts causing fruit rind re‐greening. Higher temperatures in general produce higher fruit size although internal ripening may not be profoundly altered. Fruit position on the tree, in contrast, has a minor influence, but still, fruit on the top and peripheral parts of canopy are more exposed to higher solar radiation and may contain higher concentrations of total soluble solids and vitamin C. Indeed, fruit permanently exposed to sunlight may be lighter, smaller and contain less juice and elevated TSS concentrations. On the other hand, insuYcient light availability decreases fruit size and rind colour. Climate is obviously a major determinant of quality; regions with dry and warm summers and humid and cold winters, for example, produce more brilliant fruit and thick peels. Diseases also cause fruit drop reducing yield and usually have a considerable negative eVect on fruit quality because external lesions produce no marketable fruit. The rootstock has a large eVect on scion vigour and size and tolerance to biotic and abiotic stressful environmental conditions and, therefore,

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influences many quality standards including yield, fruit size, rind colour and juice quality. Rootstocks that confer high vigour to the scion generally induce high yields, but produce relatively poor quality fruit with coarse and thicken fruit rind, poor colour and low levels of TSS and acids (Castle, 1987). Less vigorous rootstocks yield smaller fruit with smoother texture and higher TSS and acidity. Cultural practices also have a major influence on development and quality. Yields and fruit size and rind colour decrease in high‐density citrus tree plantings due to shading conditions. In many regions, rejuvenation pruning, hedging and topping are necessary to increase yield and to improve fruit size and rind colour. It is also well known that nutrition and fertilization practices strongly influence fruit quality and that among the mineral elements, macro‐elements such as potassium (K), nitrogen (N) and phosphorous (P) have the greatest influence (Embleton et al., 1963, 1978). There are many studies reporting and describing the eVects of nutritional imbalances on fruit growth and quality. Usually, the reported eVects are circumscribed to specific scion–rootstock combinations and environmental conditions and although there may be some inconsistencies several nutritional eVects appear to be general in many citrus species and varieties. For example, potassium that is a main regulator of ionic balances and hence is implicated in water accumulation and fruit growth, is pivotal to develop adequate fruit size and rind thickness. Thus, low leaf K content results in small fruit size and reduced rind thickness which predisposes the fruit to splitting and creasing problems. In contrast, excess of K produces large, coarse fruit with thick rinds. Similarly, excessive leaf N levels increase fruit size and rind thickness and coarseness and also juice content. Higher N levels appear to decrease ascorbic acid and modify TSS and acidity. Phosphorous levels have less eVect on fruit quality than K or N since increased leaf P levels slightly decrease fruit size, TSS, acidity, ascorbic acid and rind thickness. Several adverse environmental conditions primarily freeze temperatures, salinity and drought reduce yield due to increased fruit abscission and have very detrimental eVects on fruit quality. Citrus fruit is usually damaged after several hours below 2 8C (Yelenosky, 1985). Severely freeze‐damaged fruit drop few days after but moderately damaged fruit may remain on the tree for several weeks. In freeze‐damaged fruit, juice sacs are generally dried out and thus accumulate lower juice content. Other freezing eVects are loss of turgor and firmness, reduction of carotenoid levels and deposition of hesperidin crystals. Moderate salinity produces similar eVects to those produce by water shortage. High salinity, however, may induce fruit abscission, reduce fruit size and yield and hasten internal and external ripening. Excess of water,


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irrigation or raining and flooding conditions decrease juice and quality since TSS and acidity are reduced while juice percentage increases (Hilgeman, 1977). Excessive water may also increase oleocellosis (rupturing of oil glands in the peel) during harvest and enhances the incidence of physiological peel disorders which render unmarketable fruit (Davies and Albrigo, 1994). On the contrary, severe drought results in massive organ abscission while moderate water shortage reduces juice percentage and increases peel thickness and the TSS and acid content. Fruit size can also be reduced with moderate droughts after June drop. Irrigation is generally needed and used to minimize the deleterious eVects of water shortage on growth, yields and quality of citrus fruit. High environmental humidity, in general, delays external ripening. Because of their main relevance, below it is revised the information generated in recent years on the eVects of these three prominent and often environmental constrains on citrus gene expression. A. THE RISK OF FREEZING

Most commercially important varieties of citrus are seriously aVected by the eVect of low temperature, that is, are cold‐sensitive and therefore susceptible to freezing. Studies on cold hardiness in citrus are mostly developed in Poncirus trifoliata (L.), an interfertile Citrus relative that can tolerate temperatures as low as –26 8C after acclimation. Poncirus rootstocks can confer tolerance to scion varieties and hence are also used as a source of cold tolerant genes (Yelenosky, 1985). Using subtractive hybridization (Sahin‐ Cevik and Moore, 2006a) and diVerential display techniques, researches have compared gene expression among sensitive species and Poncirus searching for tolerant genes. For instance, Zhang et al. (2005) reported that cold up‐ regulated several genes such as betaine/proline transporter, water channel protein, aldoketo reductase, early light‐induced protein, nitrate transporter, tetratricopeptide‐repeat protein, F‐box protein and ribosomal protein L15. Acclimation temperature regime also induces expression of several citrus dehydrins (Cai et al., 1995; Hara et al., 2001; Porat et al., 2002b) that appear to play a significant role on cold tolerance. For instance, expression of a C‐repeat‐binding factor (CBF) and one of its targets, cor19, a cold‐induced gene, accumulated both earlier and to higher levels in Poncirus. Moreover, cor19, cor11 (Cai et al. 1995) and cor15 belong to an unusual group 2 LEA gene family responsive to low temperature. These dehydrins diVer from most other plant dehydrins in having an unusual K‐segment similar to that of gymnosperms and a serine cluster (S‐segment) at an unusual position at the carboxy‐terminus (Porat et al., 2002b). Citrus, however, also possess the typical plant angiosperm‐type K‐segment consensus sequence. According

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to Hara et al. (2001), dehydrins may facilitate plant cold acclimation protecting membrane systems by acting as radical‐scavenging proteins. These authors also reported that transgenic tobacco expressing citrus dehydrins contained fewer peroxidized lipids than the wild type when incubated at low temperatures (Hara et al., 2003). Using in vitro assays, Sanchez‐Ballesta et al. (2004) reported the isolation of a new member of the Citrus dehydrin, Crcor15, sharing a high sequence homology with cor15. This gene, however, did not appear to be involved in the chilling response. Sahin‐Cevik and Moore (2006b) have also identified in Poncirus several other proteins induced during cold acclimation with high homology to LEA and heat‐shock proteins (HSPs). Similarly, genes showing homology to transcriptional factors such as bZIP, AP2 domain, RAV2‐like, and WRKY1 DNA binding and zinc finger proteins were also induced. In Arabidopsis, bZIP has been reported to be a cold‐responsive gene that may induce gene expression through cis‐elements including the abscisic acid response element (ABRE) (Jakoby et al. 2002). Nine‐cis‐epoxycaretonoid dioxygenase (NCED) and glutathione S‐transferase (GST ) genes involved in ABA synthesis and response to oxidative stress, respectively, also showed enhanced transcription. During post‐harvest, chilling injury in citrus fruit can be reduced by previous short heat treatments that activate diVerent molecular responses. Storage of grapefruit cultivars at low temperatures (0–8 8C), for example, leads to the development of chilling injuries resulting from the loss of cellular integrity caused by damage to membranes (Porat, 2004). These detrimental eVects can be considerably attenuated if fruit are stocked at temperatures between 11 and 13 8C (Kader and Arpaia, 1992). Similar eVects have been described in many other cultivars (Sanchez‐Ballesta et al., 2003; Porat et al., 2002b). For instance, in chilling‐sensitive Fortune mandarin, the induction of phenylalanine‐ammonia lyase (PAL) gene expression was concomitant with the development of chilling symptoms. However, a pre‐treatment of 3 days at 37 8C, prevented both cold‐induced damage and PAL mRNA accumulation (Sanchez‐Ballesta et al., 2000) and activated the anti‐oxidant system. Moreover, cold up‐regulated an acidic class III ‐1,3‐glucanase (CrGlcQ), that has been proposed to play a role in reducing chilling‐induced peel damage (Sanchez‐Ballesta et al., 2006). Genes diVerentially expressed in the chilling response have mostly been related to lipid membrane and cell wall enzymes, to main regulators of secondary metabolism and hormonal homeostasis, and to oxidative and general stress responses (Sanchez‐Ballesta et al. 2003; Sapitnitskaya et al. 2006). In many instances, the beneficial eVect of heat conditioning reducing chilling injury has been attributed to the induction of HSPs and to its persistence during cold exposure. Thus, it has been reported that this treatment


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followed by storage at 2 8C increased the expression of two dehydrin genes (cor15 and cpDHN), four HSPs (HSP18‐I, HSP18‐II, HSP22 and HSP70) and a sodium proton antiport gene, cNHX1, known to be involved in salt tolerance (Porat, 2004; Porat et al., 2002a,b; Rozenzvieg et al., 2004). Moreover, Sapitnitskaya et al. (2006) showed that among other genes, HSP19‐I and HSP19‐II, dehydrins, several anti‐oxidant genes and desaturases and lipid transfer proteins were specifically regulated by the heat treatment. Transcriptional factors such as TFIIB or the WRKY family have been identified as putative tolerance‐associated genes when a heat pre‐treatment occurs (Sanchez‐Ballesta et al., 2003).

B. POOR QUALITY WATER AND WATER SHORTAGE

The vegetative and reproductive development of the salt sensitive genus Citrus is gravely impaired by salinity (Greenway and Munns, 1980; Storey and Walker, 1999). Since citrus plants adjust osmotically very rapidly and with high eYciency under saline conditions (Ban˜uls and Primo-Millo,1995; Maas, 1993), salt‐induced damages in leaves were primarily associated with excessive accumulation of sodium (Naþ) (Lloyd et al., 1990) and chloride (Cl) ions (Ban˜uls and Primo‐Millo, 1992). At present, however, it is accepted that Cl is the main toxic component of the salt (Ban˜uls and Primo‐Millo 1992, 1995; Moya et al., 2003; Romero‐Aranda et al., 1998).The physiological basis for the tolerance to salt stress thus is mostly related to the plant ability to restrict Cl transport to the shoot, a mechanism particularly dependent upon the rootstock nature (Maas, 1993; Storey and Walker, 1999). Nevertheless, much of the recent studies on plant tolerance to salinity have been focused on Naþ, while the role of its frequently occurring counterion has been relatively ignored. A recent study comparing transcriptomic and ionic profilings on the salt tolerant rootstock Cleopatra mandarin (an eYcient Cl excluder) and the sensitive Carrizo citrange (a chloride includer) proposes that Cl is the most important component of the gene expression response of citrus plants to salinity (Colmenero‐Flores et al., 2006). The study has also shown radical diVerences in the metabolism regulation and hence in the strategies to cope with salinity since the tolerant rootstock in contrast to the sensitive one repressed functional gene categories related to photosynthesis while activated many responses to stresses (Fig. 6). Interestingly, the superior tolerant rootstock ForAl #5 combines highly eYcient Cl exclusion and active photosynthetic system capabilities, suggesting that the fundamental mechanism of tolerance in citrus is certainly related to Cl exclusion, although this trait

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Fig. 6. Proposed model for chloride homeostasis in citrus. (A) Under salt stress conditions, the sensitive Carrizo rootstock shows low chloride exclusion capability, induction of genes involved in photosynthesis and carbon assimilation, moderate reduction of photosynthetic metabolism, and partial induction of responses to stresses. Eventually, high shoot Cl build‐up gives rise to leaf intoxication and collapse of metabolism. Tolerance in Cleopatra is linked to high chloride exclusion, repression of metabolism, and rapid induction of stress responses. Salt damage in Cleopatra is in this


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may also be moderated by a particular combination of physiological characteristics that are species specific (Lo´pez‐Climent et al., 2007). Moreover, it has been suggested that chloride accumulation is a polygenic trait with a strong heritable basis (Sykes, 1992). Indeed, intergeneric crossing between citrus has been used to identify quantitative trait loci (QTLs) involved in general and tissue‐specific Naþ and Cl accumulation (Tozlu et al., 1999). Nevertheless, a solid hypothesis on the molecular basis of salt tolerance in citrus is currently lacking because major components of chloride transport in the plant kingdom are mostly unknown at the molecular level. Electrophysiological studies, on the other hand, are currently providing substantial information on this area (reviewed in Allen and Sanders, 1997; Roberts, 2006; Tyerman and Skerrett, 1999, White and Broadley, 2001) that may allow a first tentative insight on the regulation of chloride accumulation. Since the symplastic pathway dominates Cl transport in higher plants (Pitman, 1982), it is reasonable to suggest that Cl exclusion can be regulated at the following steps: (i) root uptake; (ii) root eZux; (iii) xylem loading and (iv) shoot to root transport. According to this suggestion, there are a number of putative elements that have been described in several plants and may be

way considerably reduced. The high tolerant ForAl #5 combines elevated chloride exclusion ability with high metabolic activity. This suggests that rootstock tolerance depends mostly upon Cl exclusion although physiological performance may modulate the general response. Induction: upward arrow; repression: downward arrow. Data are from Brumo´s et al. (2007) and Lo´pez‐Climent et al. (2007). (B) Chloride exclusion through the symplastic pathway can be regulated at diVerent levels: (i) root uptake; (ii) root eZux; (iii) xylem loading; and (iv) shoot to root retrieval. The figure shows putative location and function of major Cl‐related transporters that may be involved in Cl homeostasis and hence in salt tolerance (cation transporters have been omitted for clarity). It is proposed that under moderate external Cl content, net Cl influx is directly regulated by active uptake transport (low‐aYnity Cl/Hþ symporters) and passive eZux channels (ECH). At high salinity, plasma membrane depolarization activates the outward‐rectifying depolarization‐activated anion channels (OR‐DA) allowing passive Cl influx. Xylem loading of Cl mediated by anion channels (XPCH) may have special relevance in chloride exclusion in citrus. It has been found that the tolerant rootstock restricts chloride loading to the xylem in comparison with the salt‐sensitive rootstock, suggesting that this may be the main cause of the net chloride uptake observed in the roots of the tolerant genotype. These observations are compatible with the suggestion that the xylem parenchyma channel(s) are the main target for chloride exclusion mechanism and hence for salt tolerance in citrus. Lastly, voltage‐dependent chloride (ClC) and vacuolar (VCl) chloride channels might have roles in Cl vacuolar compartmentalization. Abbreviations: Cl/Hþ, high‐ and low‐aYnity Cl/Hþ symporters; ECH, eZux channel; OR‐DA, outward‐rectifying depolarization‐ activated channel; XPCH, xylem parenchyma channels; ClC, chloride channel; VCl, vacuolar chloride channel conductance.

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involved in Cl homeostasis and hence in salt tolerance. In Fig. 6, we propose a first tentative model for chloride homeostasis in citrus rootstocks diVering in salt tolerance, including putative location and function of major Cl‐ related transporters described to date. Such a model includes, for example, high‐ and low‐aYnity Cl/Hþ symporters that govern active root Cl uptake under normal non‐saline condition (Babourina et al., 1998; Epstein, 1972; Felle, 1994; Lee, 1982). Net chloride uptake under moderate external Cl, thus, may result from the combined action of influx activities and passive eZux channels (Britto and Kronzucker, 2006). At high salinity, plasma membrane depolarization activates the outward‐rectifying depolarization‐ activated anion channels (OR‐DAAC), allowing passive Cl influx to maintain electroneutral Naþ uptake (Cerana and Colombo, 1992; Lorenzen et al., 2004; Skerrett and Tyerman, 1994). In these conditions, linkage of Cl absorption to water use (Moya et al., 2003), growth habit and morphological factors (Moya et al., 1999, 2002) further suggests that diVusive mechanisms through the apoplast may also operate in citrus (Syvertsen et al., 1989). Other pivotal regulatory step is the xylem loading of Cl that is mediated by anion channels such as the xylem parenchyma quickly activating anion conductance (X‐QUAC) (Gilliham and Tester, 2005; Kohler et al., 2002). In citrus, this step may have special relevance since leaf Cl concentrations are primarily associated with transport processes in the root (Fernańdez‐Ballester et al, 2003; Storey and Walker, 1999). For example, root Cl uptake and shoot translocation are lower in the tolerant Cleopatra mandarin that appears to principally restrict loading of Cl into the xylem, thus, reducing Cl transport to shoots (Brumo´s et al., 2007). Few genes involved in chloride homeostasis have been described in the plant kingdom. The voltage‐dependent Cl channel (CLC) gene family has been identified and partially characterized in plants (Geelen et al., 2000; Hechenberger et al., 1996; Lurin et al., 1996). It has been reported that one CLC member encodes an endosomal anion transporter with NO3/Hþ antiporter activity involved in nitrate compartmentalization (De Angeli et al., 2006) while another member has been identified as a chloride channel implicated in luminal pH regulation (Fecht‐Bartenbach et al., 2007). This family likely includes putative Cl channels with important roles in Cl compartmentalization since it has been reported that the soybean GmCLC1 gene responds to salinity and water deficit and encodes a protein located in the tonoplast (Wing‐Yen et al., 2006). In addition, other proteins that are potentially involved in Cl homeostasis are currently under characterization. In citrus, for instance, we have found a chloride responsive gene, CcNRT1‐B, encoding a putative active anion transporter homologous to low aYnity NO3/ Hþ symporters that belongs to the proton‐dependent oligopeptide transporter


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(POT) family (Brumo´s et al., 2007). Moreover, in Arabidopsis, a gene encoding a Naþ:Kþ:Cl cotransporter, AtCCC, involved in developmental processes and in long‐distance Cl transport has been characterized (Colmenero‐Flores et al., 2007). A rather reduced number of studies on the molecular genetic response of citrus plants to salt stress have been reported. Genes, in principle, associated with the response of citrus to salinity were at first obtained from a cDNA expression library of citrus salt‐treated cell suspensions. For instance, the salt‐induced csa citrus gene encoding a phospholipid hydroperoxide glutathione peroxidase (PHGPX) was selected in this way (Ben‐Hayyim et al., 1993). However, further studies suggested that this gene was likely not specifically involved in salt tolerance since the gene was strongly induced in salt‐sensitive cells (Avsian‐Kretchmer et al., 1999). The analysis of the early responses of the csa gene at the mRNA transcript and protein levels led to the conclusion that the salt‐induced expression of the csa gene was mainly mediated by oxidative stress. An important source of Citrus stress‐responsive genes can be found in the EST collections deposited in public databases (for instance, Forment et al., 2005). In particular, the analysis of unique EST clusters associated with salinity tolerance reported by Terol et al. (2007) provided evidence on the involvement of a high number of stress‐associated genes in the response of Citrus to salinity. A succinct listing of these genes includes several Naþ/Hþ antiporters that are probably involved in sodium detoxification; stress‐ related protein kinases, a calcineurin B gene homolog and a mechanosensitive ion channel‐domain containing protein that are likely implicated in NaCl‐associated signal transduction mechanisms; several aldehyde dehydrogenase involved in stress‐derived aldehyde detoxification; two genes of the inositol metabolism; genes associated with lipid metabolism such as the phosphoinositide‐specific phospholipase C and two lipoxygenases; a membrane‐associated salt‐inducible protein; two diVerent NCED genes involved in ABA biosynthesis; two genes involved in the metabolism of the osmolyte and regulatory molecule trehalose; HSPs and molecular chaperons; as well as many uncharacterized stress‐responsive genes (see Terol et al., 2007 and references therein). Citrus species vary in their tolerance to drought and flooding (Bhusal et al., 2002). It is widely accepted that water status of plants is hormonally regulated by abscisic acid (ABA) that further plays additional roles in the regulation or adaptation to other several environmental stresses. ABA appears to be the primarily hormonal signal that mediates between the adverse environment condition and the response against the stress (Go´mez‐Cadenas et al., 1996, 1998; Mehouachi et al., 2005). Research on genetic regulation of ABA

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synthesis has been almost exclusively centred on the analysis of the NCED genes. Three diVerent reports have shown gene expression of several NCED genes diVerentially regulated in diVerent tissues (Kato et al., 2006; Rodrigo et al., 2006; Agustı´ et al., 2007b). According to these reports, one of these genes (CsNCED1/CitNCED3/CcNCED3) was up‐regulated in the flavedo during natural fruit maturation, in dehydrated leaves and fruits and after exposure to ethylene. This gene was likely to play a primary role in the biosynthesis of ABA in both leaves and fruits. A second gene (CsNCED2/CitNCED2/ CcNCD5), expression of which was only detected in the flavedo of fruits, appears to play a subsidiary role restricted to chromoplast‐containing tissue. Additional, information on gene expression in response to water deficit in citrus is limited to a few reports on dehydrins. In mandarin leaves, it has been reported that Crcor15 was rapidly induced after water limitation suggesting its implication in the protective system against osmotic stress (Sanchez‐ Ballesta et al., 2004). Interestingly, other two cold‐regulated dehydrin genes isolated in Citrus, cor11 and cor19, were found to be repressed (Cai et al., 1995) or weakly stimulated (Hara et al., 2001). Moreover, Hara et al. (2005) reported a further citrus dehydrin, CuCor15, that may be involved in the reduction of metal toxicity in plant cells under water‐stressed conditions and Sahin‐Cevik and Moore (2006b) also showed that a gene coding a RING‐H2 finger protein was up‐regulated after cold or water stress. High‐throughput analyses of gene expression in citrus challenged with major abiotic stresses are currently underway in several laboratories and will soon produce valuable information that might eventually lead to discovery of novel genes.

VI. CITRUS ABSCISSION A. TO FALL OR NOT TO FALL, THAT IS THE QUESTION

The shedding or abscission of citrus organs is an active and highly coordinated physiological process involving a vast range of changes in cell wall structure, metabolism and gene expression (Burns, 2002; Goren, 1993; Iglesias et al., 2008; Talon and Gmitter, 2008, Talon et al., 1997). Abscission is dependent on the activation of specialized tissues called abscission zones (AZs) located at well‐defined positions in the plant. Bud and flower abscission during the early fruit growth period, or phase I, takes place at AZ‐A located between the twig and the peduncle whereas AZ‐C located in the calyx


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between the pericarp and the nectary or floral disc is mostly responsible of later fruitlet and fruit abscission. Along phase I, AZ‐A becomes progressively inoperative while AZ‐C is gradually activated. At the end of this period, during the ‘June drop’ and also at ripening in certain citrus cultivars, AZ‐C is fully operative. Mature leaves are shed through the activation of the laminar AZ (LAZ) located at the interface between the petiole and the leaf blade. In contrast, senescent leaves fall through the activation of the branch AZ located at the branch to petiole junction. Fruit drop in citrus appears to be regulated by several plant hormones acting as abscission promoters or inhibitors. Gibberellins, for example, may act as inhibitors of the abscission of flowers and ovaries occurring at the onset of the slow period of citrus fruit growth. The endogenous GAs found in citrus fruits are mainly members of the early‐13‐hydroxylation biosynthetic pathway (Goto et al., 1989; Talon et al., 1992; Turnbull, 1989) leading to the active 3‐hydroxylated‐gibberellin GA1. In this pathway, the precursor GA53 is metabolized to GA1 by the action of two enzymes, GA 20‐oxidase (GA20ox) and GA 3‐oxidase (GA3ox) acting consecutively. Transcript levels of CcGA20ox1 are abundant in developing tissues of citrus such as leaves and apical shoot (Vidal et al., 2003) and are associated with the GA changes observed during fruit growth. In developing fruits, the levels of GA53, GA20, GA9 and GA1 are low just before and after anthesis and increase approximately twofold at anthesis. This transitory rise in GA levels can be detected in seeded genotypes as well as in seedless cultivars possessing high or normal ability for setting (Talon et al., 1990a). In seeded cultivars, pollination induces GA increases at anthesis, whereas in parthenocarpic species, the rise is developmentally regulated. It has also been shown that in seeded varieties, exogenous GA arrested ovary drop of non‐pollinated ovaries. Repeated applications of the inhibitor of GA biosynthesis paclobutrazol to pollinated ovaries decreased the amount of GA and increased ovary abscission (Ben‐Cheikh et al., 1997). These observations indicate that the increase in GA detected in ovaries is a preventive signal of citrus fruit abscission. The role of auxins during abscission is rather elusive, since auxins have been reported either to delay or to induce citrus fruit drop. Abscission of mature citrus fruit, for example, can certainly be commercially postponed by treatments with diVerent synthetic auxins (Agustı´ et al., 2006; Anthony and Coggins, 1999). Furthermore, applications of synthetic auxins to the abscission zone or local treatments with the auxin transport inhibitor 2,3,5‐triiodobenzoic acid result in decreases or increases, respectively, in the sensitivity of AZ‐C to abscission promoters (Yuan et al., 2002, 2003). Similarly, abscission of debladed leaf explants and excised pistils can also be delayed by

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auxins (Einset et al., 1981; Lewis and Bakhshi, 1968) and auxin applications also reduced the sensitivity of the leaf LAZ to abscission promoters (Sagee et al., 1980). Generally, it is believed that during the initial stages of fruit growth and development, auxins might act as inhibitors of abscission, but once the process has been initiated auxins apparently stimulate abscission (Agustı´ et al., 1995). The balance between indole‐acetic acid (IAA) and ABA has also been argued to be the signalling factor triggering mature fruit abscission in citrus. Thus, it has been reported that IAA levels in the AZ of mature ‘Valencia’ orange fruit are higher during the ‘less responsive period’ to abscising agents than during the ‘responsive’ stage (Yuan et al., 2001), in contrast to the tendency observed for ABA. Sagee et al. (1990) also suggested that the abscission promoter ethylene modified endogenous IAA levels in citrus leaves increasing auxin conjugation. Interestingly, several genes coding for enzymes involved in auxin metabolism (IAA amino acid hydrolase, IAA glucosyltransferase and GH3‐like) have been isolated in the calyx AZ of citrus fruit (Burns, 2002). It has also been observed that the expression of GH3‐like and IAA glucosyltransferase genes increased in abscission‐ activated flowers of citrus (Lahey et al., 2004; Li et al., 2003b). Ethylene, on the other hand, has an extensive and unequivocal promotive eVect on citrus abscission (Goren, 1993). In fact, ethylene hastens abscission when applied either to organ explants or to the tree canopy. It is known that methyl‐jasmonate (Me‐JA); coronatine, a structural analog of JA; and ABA, all three abscission agents, stimulate abscission in citrus leaf explants or ripening fruit through ethylene as the last hormonal eVector (Burns et al., 2003; Hartmond et al., 2000; Sagee et al., 1980). Furthermore, the abscission chemical 5‐chloro‐3‐methyl‐4‐nitro‐1H‐pyrazole (CMNP) stimulates mature fruit abscission when in contact with the fruit peel, through the activation of ethylene production as well (Yuan et al., 2001). This abscission‐stimulating chemical induced expression of several ethylene biosynthesis genes in mature fruit peel and calyx AZ‐C (ACS1, ACS2 and ACO) and in leaf blade and LAZ (ACS2 and ACO) (Yuan et al., 2005). However, citrus may possess at least in part an ethylene‐independent abscission pathway since inhibition of ethylene perception with 1‐methylcyclopropene had little eVect on ethephon‐ and CMNP‐induced fruit abscission (Pozo et al., 2004). It has also been reported that Me‐JA induced the expression of CsACO1 in fruit AZ‐C and leaf LAZ whereas coronatine and CMNP increased expression of a gene associated to JA biosynthesis, 12‐oxophytodienoic acid reductase (12‐oxo‐PDA) in mature fruit AZs, in leaf blade and LAZ (Burns et al., 2003). Several plant growth regulators have also been related to abscission of citrus organs subjected to stressful environmental conditions. Citrus trees have a peculiar behaviour regarding leaf and fruit abscission induced by


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water stress. These organs usually do not fall during the period of water stress but they suddenly do after re‐hydration (Addicott, 1982). Using this system as a model for the study of water stress‐induced abscission, it was possible to elucidate the spatial and temporal patterns of change in the hormonal homeostasis in response to water deficiency (Go´mez‐Cadenas et al., 1996; Mehouachi et al., 2005; Tudela and Primo‐Millo, 1992). In these works, it was suggested that the sequence of events leading to organ abscission is as follows: water stress ! ABA accumulation in roots ! ACC accumulation in roots ! ACC transport from roots to shoots ! ACC oxidation to ethylene in leaves ! abscission. The accumulation of ABA in roots in response to water stress is mainly due to the enhanced expression of CsNCED3, a gene of the carotenoid cleavage dioxygenases family (Agustı´ et al., 2007b). The ethylene biosynthetic genes associated with increases in ACC synthase activity and ACC amounts in roots (Tudela and Primo‐Millo, 1992) and ethylene production in leaves (ACC oxidases) have not been characterized until now. Similarly, the essential observation linking carbohydrate and abscission during fruitlet growth was the finding that carbon shortage increased ABA and ethylene and that both were involved in the induction of early abscission (Go´mez‐Cadenas et al., 2000). The simplest interpretation of these observations suggested that ABA acts as a sensor of the intensity of the nutrient shortage modulating the levels of ACC and ethylene, the final activator of abscission. Collectively, the above findings identify ABA, ACC and ethylene as major components of the mechanism regulating organ abscission in citrus. The proposed hormonal sequence also oVers a plausible explanation for the naturally occurring abscission (Zacarias et al., 1995) and for the drop induced through a variety of stimuli such as salinity (Go´mez‐Cadenas et al., 1998). Biotic stresses can also have major impact on citrus fruit drop. The infection of citrus petals with the fungus Colletotrichum acutatum, for example, causes a disease known as post‐bloom fruit drop (PFD) characterized by necrotic brown lesions in petals and premature drop of young fruit (Timmer and Brown, 2000). PFD has been associated with increases in several plant growth regulators in infected tissues. Thus, ethylene production increased in flowers and the amounts of IAA and JA were also enhanced in petals (Lahey et al., 2004). The application of both auxin transport and action inhibitors and JA biosynthesis inhibitors after fungus inoculation improved fruit retention suggesting an inductive role of IAA and JA on young fruit abscission (Chen et al., 2006). As mentioned above, increases in ethylene, IAA and JA are generally associated with the accumulation of transcripts of ethylene (ACC synthase and ACC oxidase) and JA biosynthesis (12‐oxo‐PDA) and auxin metabolism (GH3‐like and IAA glycosyltransferase).

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Abscission of leaves, ovaries, fruitlets or fruits from the parent plant occurs through loss of cell adhesion and cell wall breakdown in the separation layer of the AZs (Goren, 1993). Information about the initial regulatory signals acting at the onset of the abscission process is rather scarce. Recent work points to the involvement of MAP kinases and GTP‐binding proteins. Thus, two partial cDNA sequences with high homology to MAP kinases (pk41 and pk42) have been isolated in AZs from ethylene‐treated fruits (Zhong et al., 1996). The expression of both protein kinases increased in fruit AZ 8 h after ethylene treatment. Yuan et al. (2005) have associated the activity of GTP‐ binding proteins with the abscission response produced by ethylene. The authors showed that guanfacine, a G‐protein‐coupled 2A‐adreno‐receptor selective antagonist, blocked ethylene‐induced leaf abscission and to a lesser extent ethylene‐induced fruit abscission through the reduction in the expression of ACC synthase 1 and ACC oxidase genes in leaf LAZ and fruit AZ‐C. The abscission agent CMNP promotes ethylene production and induces uncoupling of the energy pathway in mitochondria and chloroplast (Alferez et al., 2005). Application of CMNP to mature fruits reduced ATP levels and induced an increase in phospholipase A2 (PLA2) and lipooxygenase (LOX) protein activities and in the levels of lipid hydroperoxide (LPO). Arachidonic acid, an inhibitor of PLA2 activity, blocked the increased eVect induced by CMNP in both enzyme activities and also the accumulation of LPO and reduced abscission, suggesting that lipid signalling could be involved in the herbicidal abscission‐stimulating eVect of CMNP. On the contrary, ethephon treatments are able to induce abscission without symptoms of senescence (Alferez et al., 2006). An additional study dealing with water stress‐induced abscission of citrus leaves has suggested that Ca2þ signalling may be very active in leaf AZ at the onset of abscission since expression of several genes encoding Ca2þ eZux carriers, calmodulin (CaM) and CaM‐like proteins, Ca2þ‐dependent protein kinases, endoplasmic reticulum Ca2þ‐binding proteins, Ca2þ‐binding EF‐hand‐containing proteins and CaM‐binding proteins was observed early during re‐hydration after water stress release (Agustı´ et al., 2007a). The eVective separation of cells during abscission is a consequence of increases in the activity of at least three hydrolytic enzymes secreted to the cell walls: endo‐1,4‐‐glucanases (cellulases, Cel), polygalacturonases (PG) and pectin‐methylesterases (PME). It has been shown, for example, that during ethylene‐induced activation of calyx AZ in young and mature fruit explants, increasing activities of both Cel and PG were detected (Burns et al., 1998; Goren and Huberman, 1976; Greenberg et al., 1975; Huberman and


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Goren, 1979; Riov, 1974). In contrast, synthetic auxins (2,4‐dichlorophenoxyacetic acid) delayed both kind of activities and also abscission. Only three Cel isoenzymes out of seven found in AZ‐C were apparently associated with abscission (Goren and Huberman, 1976). In ethylene‐activated calyx AZs, two diVerent Cel genes (acidic cellulose Cel‐a1 and basic cellulose Cel‐b1) have also been isolated (Burns et al., 1998). The expression level of Cel‐b1 showed a moderate and continuous increase between 4 and 40 h after ethylene treatment whereas the expression of Cel‐a1 dramatically increased 24 h after treatment. Likewise, Wu and Burns (2000) identified two polygalacturonases (PGI and PGIII) in the AZ of CMNP‐treated citrus fruit. PGI and PGIII expression was detected 24 h after CMNP application and increased dramatically at 48–72 h after CMNP treatment. Finally, two additional genes with high homology to PMEs (CsPME1 and CsPME3) have been found in ethylene‐activated fruit AZs (Nairn et al., 1998). CsPME1 appears to be specific of young tissues and fruit AZs whereas CsPME3 increased in ethylene‐treated leaf and fruit. Although activity of PMEs has not been clearly related to abscission in citrus (Rasmussen, 1973; Ratner et al., 1969), the data suggest that all these genes encoding hydrolytic enzymes are likely associated with abscission of citrus organs since their temporal expression patterns in the AZs during citrus abscission are rather similar. Another genes mostly associated with cell wall metabolism such as a ‐galactosidase, a ‐1,3‐glucanase and a chitinase (Wu and Burns, 2000, 2004) or a lipid transfer protein possibly involved in cutin transport to the separation layer (Wu and Burns, 2003) have also been isolated from citrus AZs and over‐ expressed in AZs during organ abscission. Recent studies on citrus abscission are focused on the development and analyses of EST collections from activated AZs as powerful tools to scrutinize the molecular mechanisms controlling organ separation. The citrus EST collections currently deposited in public databases include a set of 4500 ESTs, out of 240,000, associated with abscission, from sweet oranges and Clementine mandarins (Burns, 2002; Forment et al., 2005; Terol et al., 2007). The analysis of this huge amount of information is rendering new insights in the abscission process. For instance, Burns (2002) reported the identification via subtractive cDNA library screening, of 144 ESTs from the calyx AZ of CMNP‐treated mature fruit. Additional ethylene‐induced sequences expressed in leaf LAZs and flower AZs were also isolated using diVerential display techniques. This study revealed three major gene functional categories likely associated with the abscission process: hormonal regulation and signal transduction, cell wall metabolism, and secondary metabolism and defense (Table I). The Spanish ESTs collection that provided the bulk of the abscission‐associated EST set deposited in public databases was obtained

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from two standard cDNA libraries of ethylene‐activated fruit AZs (AZ‐A and AZ‐C), a standard cDNA library of calyx AZ during natural mature fruit loosening and a subtractive cDNA library of leaf LAZ activated after a cycle of water stress/re‐hydration (Forment et al., 2005; Terol et al., 2007). The homology search of this EST set using Blast2GO (Conesa et al., 2005) identified 3720 unigenes candidate to contribute to abscission. Subsequent analyses discriminated more than 500 unigenes with higher statistical probability (P>0.95) of preferential expression in activated AZs versus other several citrus tissues (Agustı´, 2007). These analyses identified additional gene functional groupings including protein catabolism (ubiquitination/ 26 S proteasome and SUMOylation pathways) and cell communication and signal transduction (LRR repeats‐containing proteins, receptor‐like proteins, histidine kinases, protein kinases and phosphatases, lipid signalling proteins) as new pivotal categories with critical roles in citrus abscission (Table II). Despite this information, major regulators of the abscission process in citrus are still mostly unknown although new data provided by microarrays, in some instances, coupled to laser‐assisted microdissection (LAM) are currently being generated in Florida and Spain to gain insight in the process. Initial transcriptomic profiling studies suggest that ethylene induces the activation of diVerent basic physiological programmes in both the LAZ and in the cortical cells of the petiole surrounding the AZ. On the basis of this information, a two‐stage model of citrus leaf abscission has recently been proposed (Agustı´, 2007). The suggestion consists of an initial activation phase, mostly characterized by the activation of signalling pathways (hormones, phospholipids, calcium and oxygen reactive species) and a second execution phase including degradation of the cell wall by hydrolytic enzymes and sugar‐nucleotide metabolism induction for cell elongation (Fig. 7). The process would end with the promotion of a double defensive program (deposition of physical barriers such as callose and lignin and induction of pathogen defense proteins) intending to protect the living zone remaining attached to the plant.

VII. CONCLUDING REMARKS AND PERSPECTIVES This manuscript has reviewed major aspects of the molecular physiology of citrus fruit growth and quality. Overall, the information presented above indicates that there is a huge unexplored potential in the study of the regulation and metabolites associated with citrus fruiting. In addition to specific perennial and woody tree‐traits, the uniqueness of the citrus fruit ripening


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Leaf blade

Ethylene LAZ Petiole Leaf blade

Activation phase

LAZ Petiole

Protein metabolism transcription regulation cell signaling: Phospholipids

Ca2+ ROS

Execution phase

Leaf blade LAZ Petiole

Cell wall breakdown: Hydrolases Transferases Lyases Expansins Cell wall expansion: Sugar-nucleotide metabolism Glycosyltransferases Cellulose synthesis Transport

Defense responses: Callose deposition Lignin deposition Defense proteins

Fig. 7. Proposed model for the molecular events leading to ethylene‐induced citrus leaf abscission. Mature citrus leaves are shed through the activation of the laminar abscission zone (LAZ) located at the interface between the petiole and the leaf blade. LAZ cells are highlighted in green in the diagram. A two‐stage model is proposed: (A) the early phase of abscission or activation phase is characterized by

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process oVers many intriguing fruit characteristics that, very likely, cannot be studied in other model plants or climacteric fruit. Hence, it is a pleasant surprise that in recent years, several genetic, genomic, and proteomic tools and technologies have been quickly adapted by the citrus research community to address major challenges of this plant system (Talon and Gmitter, 2008). As described above, critical functional and expression analyses through microarrays with several platforms have been published and analyses of ESTs in public databases have been initiated (Forment et al., 2005; Terol et al., 2007). Moreover, genetic linkage maps have been produced with increasing value and resolution, following the evolution of new marker systems (Deng et al., 2001; Yang et al., 2001) and two communications have reported progress on the construction of physical maps of the citrus genome (Shimizu et al., 2007; Terol et al. 2007). Initial steps for whole genome sequencing of citrus have also been taken; at the beginning of 2007, for example, the Joint Genome Institute reported the production of a low coverage (1.2) whole‐genome shotgun sequence of Citrus sinensis (Roose et al., 2007) and a new collaborative eVort has been initiated to shift focus as the target for sequencing to a haploid genome. Genetic transformation in citrus is also available and although is mostly being used as an alternative to classical genetic breeding, functional and transgenic studies can now be undertaken (Fagoaga et al., 2007). Strategies based on genome‐wide mutagenesis are being explored (Alo´s et al., 2007) since these approaches are non‐transgenic and have particular interest for the industry. Mutants are invaluable plant materials to help understand the relationship between genotype and phenotype. Collections of induced orange and mandarin mutants have been generated through the use of physical and chemical mutagenic agents (Iglesias et al., 2004) and are currently available. These collections have expanded the natural citrus mutant resources and will enable the emergence of new insights into the regulation of citrus fruiting. In these collections, a whole lot of phenotypic characteristics and other deviations from wild‐type standards can be analysed and mutants with altered physiological processes related to fruit shape, size and quality have been identified. Analytical screenings have also identified mutations involving pivotal components of citrus fruit biochemistry. Other innovative resources such as viral‐induced gene silencing the simultaneous activation of several signalling pathways including hormones, phospholipids, calcium and oxygen reactive species in LAZ cells; (B) during the second stage, the execution phase, dissolution of cell walls by hydrolytic enzymes is terminated and sugar‐nucleotide metabolism for cell elongation is induced in LAZ. In addition, a double defensive program including callose and lignin deposition and induction of pathogen resistance is triggered in the petiole that remains attached to the plant.

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TABLE II Listing of Citrus Proteins Encoded by cDNA Sequences Associated with Abscission Processes Cell wall modification and metabolism Acidic cellulasea,b Basic cellulasea Polygalacturonasesa,b Pectin‐methylesterasea Pectin‐acetylesteraseb Pectate‐lyaseb ‐Glucosidaseb ‐Glucosidasesb ‐Mannosidasesb ‐Mannan‐endohydrolaseb ‐Galactosidaseb ‐Galactosidasea Expansina Lipid transfer proteina Secondary metabolism and defense Phenylalanine ammonia‐lyasea Cinnamic acid‐4‐hydroxylasea CaVeic acid O‐methyltransferasea 1,4‐Benzoquinone reductasea 7‐Ethoxycoumarin O‐deethylasea UDP‐galacturonosyl transferasea ‐1,3‐Glucanasea Chitinasea PR proteins, class IVa Disease‐related proteinsa Protein inhibitor pinIIa Catalasea Amine oxidasea Protein catabolism Ubiquitin‐conjugating enzymesa,b RING finger proteinsb F‐box proteinsb SKP1‐like proteinb AAA‐type ATPase family proteinb Upl1 protease family proteinb Hormonal metabolism IAA amino acid hydrolasea IAA glucosyltransferasea GH3 family proteinsa,b Auxin‐responsive proteinb Auxin response factorb ACC oxidasea 12‐Oxophytodienoate reductasea Allene oxide synthasea (Continues)

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TABLE II (continued) Cell communication and signal transduction LRR repeats‐containing proteinsb Phospholipase Db Diacylglycerol kinaseb Histidine kinasesb Receptor‐like kinaseb Protein kinasesa,b Protein phosphatasesb Sequences were isolated from diVerential display and subtractive cDNA library screenings and up‐regulated in citrus abscission zones (aBurns, 2002) or from standard and subtractive cDNA libraries showing a high probability (P > 0.95) of preferential expression in activated AZs (bAgustı´, 2007).

(VIGS) are being developed and work in citrus proteomics is in progress (Katz et al., 2007; Lliso et al., 2007). Thus, current progress in citrus research including the rapid development of genomic and molecular biology resources for this genus may certainly motivate the development of Citrus as a model system for tree and woody perennial reproductive biology. It is hoped that the application of these new resources and tools will help to overcome traditional constrains limiting research in citrus and enable researches to address fundamental tree‐specific traits in order to attain a true understanding of tree function. These studies will likely provide knowledge that may allow optimization of production and enhancement of nutritional value of fruit, the ultimate goal of the Citrus Industry.

ACKNOWLEDGMENTS Work at Centro de Geno´mica was supported by INIA grants RTA04‐013 and 05‐247, INCO contract 015453 and Ministerio de Educacioń y Ciencia grant AGL2007‐65437‐C04‐01/AGR. Help and expertise of A. Almenar, E Bla´zquez, A. Boix, I. Lo´pez, A. Lo´pez Garcıá‐Usach, I. Sanchı´s and M. Sancho are gratefully acknowledged.

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Bamboo Taxonomy and Diversity in the Era of Molecular Markers

MALAY DAS,* SAMIK BHATTACHARYA,{ PARAMJIT SINGH,{ TARCISO S. FILGUEIRAS,} AND AMITA PAL{

*US Environmental Protection Agency, National Health and Environmental EVects Research Laboratory, Western Ecology Division, 200 S.W. 35th Street, Corvallis 97333, Oregon { Plant Molecular & Cellular Genetics, Bose Institute, Kolkata 700054, India { Botanical Survey of India, CGO Complex, Salt Lake, Kolkata 700064, India } Reserva Ecolo´gica do IBGE, C.P. 08770, Brasilia‐DF 70312‐970, Brazil

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Origin, Systematic Position and Habit .................................... B. Geographical Distribution .................................................. C. Use .............................................................................. D. Chromosome Number and Genome Size ................................. II. Implications of Various Morphological Features in Bamboo Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Rhizomes ...................................................................... B. Branching, Bud and Leaf Characters...................................... C. Inflorescence, Flower and Fruit Characters .............................. III. Bamboo Classifications Based on Morphological Features . . . . . . . . . . . . . . IV. Conspectus of Woody Bamboo Genera of the World . . . . . . . . . . . . . . . . . . . . V. Relevance of Molecular Taxonomy in Bamboo . . . . . . . . . . . . . . . . . . . . . . . . . .


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VI. DNA Fingerprinting‐Based Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. RFLP ........................................................................... B. RAPD .......................................................................... C. SCARs ......................................................................... D. AFLP ........................................................................... E. Microsatellites (SSRS)........................................................ F. Expressed Sequence Tag Derived Microsatellites (EST‐SSR) ......... G. Transposon .................................................................... VII. DNA Sequence‐Based Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Organellar Genes ............................................................. B. Nuclear Genes ................................................................ VIII. Summary and Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Morphological Systematics and Identification ........................... B. Molecular Systematics and Identification................................. C. Future Scope of Comparative Genomics ................................. D. Genetic Diversity and Ecology ............................................. . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT A total of 1400 species of bamboos are grouped under the sub‐family Bambusoideae within the family Poaceae. The plant group harbours both herbaceous and woody members while the taxonomy has traditionally been dependent on morphological characters. Classification systems proposed to date need further support, and taxonomic delineation at lower levels often lack suYcient resolution. Infrequent flowering events and extensive genome polyploidization are an additional challenge for the woody group. The tremendous advancement of molecular marker technologies holds the promise to address diVerent needs of bamboo taxonomy (systematics and identification) and diversity studies. One of the most important prerequisites is to apply the appropriate molecular tool at the proper taxonomic level. More studies are required to better understand the population level genetic diversity in bamboo.

I. INTRODUCTION A. ORIGIN, SYSTEMATIC POSITION AND HABIT

Bamboos are members of the sub‐family Bambusoideae within the grass family Poaceae. Grass family is monophyletic and the early diverging lineages recognized within the family are Anomochlooideae, Pharoideae and Puelioideae (Grass Phylogeny Working Group [GPWG], 2001). Anomochlooideae lacks a true spikelet and is sister to the rest of the family members. Pharoideae is the earliest lineage from the true spikelet‐bearing group and was followed by Puelioideae. The earliest fossil evidence for grasses was reported

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sometimes between Paleocene and Eocene ages (Crepet and Feldman, 1991). According to the fossil species of Pharus, the early diversification of the family started between late Eocene and early Oligocene (Poinar and Columbus, 1992) and extensive diversification occurred by Miocene (Thomasson, 1987). Most possibly, the major radiations of the grasses including Bambusoideae happened 40–50 million years ago (Malcomber et al., 2006). Very recently the first petrified bamboo fossil, Guadua zuloagae sp. nov, was reported from the Pliocene age (Brea and Zucol, 2007). Traditionally, the members of the group share some common features that include rhizomatous habit, hollow segmented culms, petiolate blade with tessellate venation, flowers with three or more lodicules, usually with six stamens, and fruit possess small embryo and linear hilum (Soderstrom, 1981). Few synapomorphic features which are unique for Bambusoideae were reported by GPWG (2001). Leaf blade is mainly constituted of mesophyll tissue with asymmetrically invaginated arm cells, while pseudo‐petiole structures are secondary gain for the sub‐family. It is broadly divided into two tribes, that is Bambuseae/woody bamboos and Olyreae/herbaceous bamboos depending on the presence (Bambuseae) or absence (Olyreae) of the abaxial ligule (GPWG, 2001; Zhang and Clark, 2000).

B. GEOGRAPHICAL DISTRIBUTION

Bamboos are distributed all over the world, but major species richness is found in Asia Pacific (China: 626, India: 102, Japan: 84, Myanmar: 75, Malaysia: 50 and few others) and South America (Brazil: 134, Venezuela: 68, Colombia: 56 and few others) while least (5) in Africa (Bystriakova et al., 2003a,b). The herbaceous bamboos with 110 species are mostly concentrated in the Neotropics of Brazil, Paraguay, Mexico, Argentina and West Indies (Judziewicz et al., 1999, Fig. 1). Brazil is the most prominent place representing 89% of the genera and 65% of the species that are reported from the New World (Filgueiras and Goncalves, 2004). The largest natural bamboo forests, known as ‘tabocais’ in Brazil and ‘pacales’ in Peru, cover 600,000 ha across Brazil, Peru and Bolivia (Filgueiras and Goncalves, 2004). The woody bamboos are unique with complex branching patterns, woody culm and gregarious, monocarpic flowering (Fig. 2). There are 1290 species and they are universally distributed except in Europe which has no native species. They are classified into three major groups: the paleotropical woody bamboos (distributed in tropical and sub‐tropical regions of Africa, Madagascar, India,

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80 °W

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180 °E Arctic circle

40 °N Tropic of cancer

0°

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Herbaceous bamboo

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Fig. 1. World distribution of woody (paleotropical, neotropical, temperate) and herbaceous bamboos (modified and compiled from http://www.eeob.iastate.edu/ research/bamboo/maps.html).

Fig. 2. An example of gregarious flowering in woody bamboo (Thamnocalamus spathiflorus subsp. spathiflorus) covering an area of 3.5 km2 at an altitude of 3000 m in Sikkim, India (recorded during August 2006).

Sri Lanka, Southern China, Southern Japan and Oceania, Fig. 1), the neotropical woody bamboos (Southern Mexico, Argentina, Chile, West Indies) and the north temperate woody bamboos (mostly in North temperate zone and few at high elevation habitats in Africa, Madagascar, India and Sri Lanka, http://www.eeob.iastate.edu/bamboo/maps.html).


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C. USE

Bamboos are popularly known as poor man’s timber for their multipurpose use in the rural life of many Asian countries. Thin culms with narrow cavities are popularly used as umbrella handles, fishing rods and flutes, while mature culms are widely used in constructing mud huts, mats, baskets and fences. Highly nutritious leaves (Khatta and Katoch, 1983) as well as young shoots are often used for preparing delicious soups and pickles. Adult culms are useful for the production of high quality charcoal (Park and Kwon, 1998) along with the fibres which are ideal for paper and pulp production. Because of a high growth rate (typically matures within 5–7 years) plus a number of important fuel characteristics such as low ash content, alkali index or heating value, bamboo is a promising energy crop for future (for details, see Scurlock et al., 2000). The fate of a number of endangered as well as wild species is intimately linked with bamboos (http://www.unep.org/cpi/briefs/Bamboo). For instance, leaves of Sasa senanesis, S. kurilensis and S. nipponica constitute a major part of the winter diet for Hokkaido voles (Clethrionomys rufocanus) when most other plants wither (Stenseth et al., 2003). In central Brazil, the wild stands of Actinocladum verticillatum and Filgueirasia spp. constitue a valuable fodder resource for both livestock and wildlife during the dry season when rest of the vegetation sheds their leaves (Filgueiras, 2002). The hollow bamboo culms provide refuge to many invertebrates and the inadvertent link to giant panda is well known. D. CHROMOSOME NUMBER AND GENOME SIZE

The basic chromosome number for most woody members is x ¼ 12, the same as rice, while for herbaceous bamboo it is synapomorphic (x ¼ 11; GPWG, 2001). Occurrence of polyploidization has been reported in the woody bamboos (Pohl and Clark, 1992; Soderstrom, 1981). Cytological studies indicated the existence of two distinct sections, tropical (hexaploids, 2n ¼ 6x ¼ 72) and temperate (tetraploids, 2n ¼ 4x ¼ 48) within the woody group (Clark et al., 1995; Ghorai and Sharma, 1980; Kellogg and Watson, 1993). It was later supported by the flow cytometric estimation of the genomic DNA content (Gielis et al., 1997a). Temperate bamboos had higher DNA content (4.17–5.3 pg) than tropical ones (2.34–3.23 pg). According to the most recent estimate (Gui et al., 2007), the genome size of tetraploid Phyllostachys pubescens is 2034 Mb, which is 5.4‐fold larger than that of the diploid cultivated rice and 1.9‐fold larger than that of tetraploid wild rice, while little less (86.92%) than that of maize genome. Their analysis utilizing 996 genome survey sequences (GSS) covering 0.92 Mb regions revealed that

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23.28% of the genome consisted of repeat elements. Although it indicates that there might be some direct relationship between the higher genome size and the proportion of repeat elements present, more coverage is essential prior to confirming any such presumption. Soderstrom (1981) pointed out that Southeast Asia is a centre of distribution for tetraploid and hexaploid bamboos. Hsu (1967, 1972) reported one diploid species of Phyllostachys and one of Arundinaria from China. Ruiyang (2003) on the basis of an exhaustive chromosome analysis on 185 species from 33 genera and 6 subtribes has shown the variation in chromosome numbers for some species of Bambusa and Dendrocalamus.

II. IMPLICATIONS OF VARIOUS MORPHOLOGICAL FEATURES IN BAMBOO TAXONOMY Bamboo taxonomy like all other plant groups has traditionally been built upon various morphological features and several classification systems have been proposed to date. A. RHIZOMES

Rhizome is the horizontally grown underground plant part that often sends out roots and shoots (culms) from its nodes. The existence of two basic forms of rhizome was first indicated by Riviere and Riviere (1879) on the basis of their observation of two diVerent growth habits in two bamboo genera. The caespitose habit was studied in Gigantochloa while the spreading habit was observed in Phyllostachys. These two diVerent forms of rhizomes were later recognized as monopodial and sympodial type (McClure, 1925) and were further redefined as leptomorph and pachymorph type (McClure, 1966). The terms leptomorph and pachymorph were preferred over monopodial and sympodial as the later terms were more related to the branching patterns and clump forms than the actual morphological forms of the rhizome (McClure, 1966). In sympodial type, the culms usually grow in clumps and the rhizomes are usually short, thickened and spindle shaped (Fig. 3A). Sometimes the neck length (distance between the point of origin of two culms) in sympodial rhizome is relatively longer and thus generates less tufted culms that look like the monopodial rhizome as observed in Melocanna (Fig. 3B). In the monopodial or running type, the rhizome grows horizontally without frequent, upright culm repetition and hence culms always grow in isolation (Fig. 3C). They are usually slender and hollow (Wong, 2004). Sometimes a mixed situation of both monopodial and sympodial types is observed and known as amphipodial type (Fig. 3D). However, the taxonomic importance of

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A

Root

Short neck

B

Long neck

C Bud

D

Monopodial

Sympodial

Fig. 3. Basic rhizome types in bamboo. (A) sympodial (pachymorph) rhizome with short necks, (B) sympodial (pachymorph) rhizome with long necks, (C) monopodial (leptomorph) rhizome with nodal bud and (D) amphipodial (amphimorph) rhizome with mixed sympodial and monopodial types (redrawn from Soderstrom and Young, 1983 with permission from Missouri Botanical Garden Press).

rhizome, at least for the old world bamboo at genera or supra‐generic level, is well recognized (Stapleton, 1997). B. BRANCHING, BUD AND LEAF CHARACTERS

The first extensive study to understand the importance of branch and bud characteristics was undertaken by Usui (1957) and was subsequently carried out by McClure (1966). A fundamental diVerence with respect to the branching pattern was noticed between the tropical and temperate groups. A basic and ancestral branching pattern was observed in most tropical genera, while the temperate group represented both basic (Arundinaria and

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Thamnocalamus) and complex (Fargesia, Yushania and Borinda) branching patterns (Stapleton, 1994b). Branch complements are most informative at the mid culm position as they are not well developed at the lower culm body (Wong, 2004). Chao et al. (1980) have used branching characters to revise few Asian genera. Bud characteristics have been found to be useful for resolving generic level confusions. For instance, McClure (1966) treated Pleioblastus as a synonym of Arundinaria, although they were later diVerentiated by bud closure characteristics. In addition, the taxonomic and evolutionary significance of prophyll gained special attention. Modification of prophylls into protective bud‐scale was a result of fusion events, while sheath reduction can explain insertion of multiple buds as one observed in Chusquea culeou (Stapleton, 1991). Two functionally diVerent forms of leaves are observed in bamboo. The culm leaves (culm‐sheath) play protective roles for younger shoots, while the green foliage leaves are basically for photosynthetic purposes. The basal part of culm leaf surrounds the internode, while the upper part is usually free and known as the sheath blade. The juncture between the two parts is known as the ligule and is an important taxonomic character. Various other characteristic features such as adaxial plus abaxial hairs, auricle (tiny appendages at the base of lamina on both sides) and sheath blade are useful for quick identification of species in the field. Gamble (1896) was the first one to extensively use various culm leaf features at species level and were later employed at generic level too (Nakai, 1925). They are often very informative even for higher taxonomic rank such as sub‐family. For instance, Bambuseae and Olyreae are clearly diVerentiated on the basis of presence or absence of the abaxial ligule. While important culm leaf characteristics were mostly restricted to macro‐ morphology, the anatomical features of foliage leaves gained special attention in bamboo taxonomy. However, conflicts persisted and in many instances the taxonomic delineation based on the anatomical features was not fully supported by the morphological features. In one such study, 11 anatomical features of African and Asian bamboos were investigated (Soderstrom and Ellis, 1982). Arundinaria tessellata shared 10 out of 11 characters with Thamnocalamus spathiflorus and hence the new Thamnocalamus tessellatus was synthesized. However, the closeness between Arundinaria and Thamnocalamus was contradicted by their own datasets that revealed that only 5 out of 11 characters were overlapping between A. tessellata and T. aristatus. Similarly, studies on the leaf anatomy of Sri Lankan bamboo revealed close proximity between Bambusa bamboos and the members of Arundinariinae rather than other species of Bambusinae (Soderstrom and Ellis, 1988). It is possible that leaf anatomical characters


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might not be a good choice for generic delineation but could have potential for lower taxonomic levels. C. INFLORESCENCE, FLOWER AND FRUIT CHARACTERS

On the basis of the flowering cycle, bamboos have been categorized into three major groups (Brandis, 1899): annual flowering (Indocalamus wightianus, Ochlandra sp.), sporadic or irregular flowering (Chimonobambusa sp., Dendrocalamus hamiltonii) and gregarious flowering that occurs at long intervals with synchronized seeds production (Bambusa bambos, B. tulda, Dendrocalamus strictus). A majority of bamboos belong to the third category where the intermast period may range from 3 to 120 years (Janzen, 1976). Like all other grasses, the flowers of Bambusoideae are arranged in bracteate units on the rachilla and are known as spikelets. Each spikelet is subtended by many empty (without flower) bracts called glumes followed by one to several specialized bracts called lemma which provides protection to the flowers (Fig. 4A). In addition to the lemma, each flower is covered by another bract, sometimes membranaceous, called the palea. Both lemma and palea are vegetative in origin (Stapleton, 1997). Flowers are sessile and a proper perianth is substituted by three lodicules. Bracts subtending to bamboo inflorescence followed a gradual reduction process for other grasses in general. It is believed that the fully bracteate inflorescence such as Bambusa had to loose bracts to develop the ebracteate grass panicle (Holttum, 1958). The spikelets of Olyreae are usually unisexual and one flowered. In Bambuseae it is bisexual and both spikelets and pseudo‐spikelets are present. The later is often bracteate and re‐branching (GPWG, 2001; Judziewicz et al., 1999). Pseudo‐spikelets are also characterized by the presence of specialized branch‐bud bearing bracts at the base of the rachilla, which is not observed in a true spikelet (Fig. 4B). Another important fundamental diVerence is that true spikelets are always single, while pseudo‐spikelets are in groups. McClure (1934) first introduced the concept of diVerent types of spikelets in bamboo. In addition to the idea of spikelets and pseudo‐spikelets, he also conceptualized semelauctant or determinate and iterauctant or indeterminate inflorescence types (1966). The inflorescence type that had pseudo‐ spikelets as the basic unit was considered as indeterminate because it was capable of re‐branching and producing new flowers for almost an indefinite period of time. On the other hand, the determinate inflorescence was primarily based on true spikelets that lack the capability of indefinite growth. Keng (1983) proposed two sub‐tribes within the woody bamboos (Bambusoideae) primarily based on the determinate and indeterminate inflorescence types, which was subsequently adopted in the Flora Reipublicae Popularis Sinicae

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A

Terminal bud

Palea Floral bud Lemma Rachis

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ProphyII

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Terminal bud Palea Floral bud Lemma

Rachis Branch bud Glume

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Fig. 4. A comparative account of (A) spikelet and (B) pseudo‐spikelet inflorescences. Left panels represent schematic diagrams and right panels represent (A) spikelet of Chimonobambusa callosa and (B) pseudo‐spikelet of Bambusa tulda.

(FRPS, Keng and Wang, 1996). Recently, a further enhanced version of FRPS has been published which includes several new taxa and provides a more detailed account of the Chinese bamboos (Flora of China, 2007).


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It is generally accepted that typical bamboo flowers are monochlamydeous while the pseudo‐spikelet of Streptochaeta was interpreted as a reduced form with a single terminal achlamydeous flower (Soderstrom, 1981). However, it is not always easy to define the inflorescence types in bamboos and several contradictory interpretations have been noticed in many cases. For instance, the inflorescence of Racemobambos was recognized as iterauctant by Chao and Renvoize (1989), while semelauctant by Dransfield (1992). Similar confusion has also been observed for its allied genus Neomicrocalamus. N. prainii was considered as semelauctant by Keng (1983) and Stapleton (1994c), while iterauctant by Wen (1986) and Dransfield (1992). The use of the term synflorescence (aggregation of spikelet) instead of inflorescence has also been proposed in bamboos (Stapleton, 1997). The various inflorescence types and floral features are of high taxonomic significance at all levels. For instance, presence or absence of lodicule is a key character for generic delineation. The number of stamens is often used to diVerentiate many closely related genera such as Sinobambusa/Indosasa, Indocalamus/Sasa and Arundinaria/Acidosasa. On the basis of the absence of ovary appendage, Holttum (1956) moved the Asiatic species of the genus Oxytenanthera to either Dendrocalamus or Gigantochloa. Soderstrom and Ellis (1988) described a new genus Pseudoxytenanthera, while Majumdar (1989) created a new genus Pseudotenanthera to accommodate some of the Indian and Sri Lankan species. Bamboo fruits are usually one seeded, dry caryopsis structures, while in few cases (Melocanna, Dinochloa, Ochlandra) these are fleshy and pear shaped.

III. BAMBOO CLASSIFICATIONS BASED ON MORPHOLOGICAL FEATURES Based on gross morphological features, many classification systems have been proposed till date. Munro’s description (1868) was one of the earliest attempts that described 170 species under 20 genera. This classification system was primarily based on that of Nees von Esenbeck (1835). Bentham (1881) followed Nees and Munro with slight modifications and recognized four major groups. Gamble used important collections and notes of Wilhelm Sulpiz Kurz (1876), who was contemporary to Munro and had developed a comprehensive treatise on bamboos of British India (1896). Gamble’s classification covered 15 genera and 115 species with elaborate descriptions and basically followed Bentham in placing the genera under four groups. Camus (1913) pooled the information from the work of Munro and Gamble and compiled 490 species

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under 33 genera in his monograph ‘Les Bambuseés’. McClure’s description (1961) of the woody members of Bambusoideae was another landmark that was further improved by Parodi (1961) who included the herbaceous members in his treatment. He further divided the herbaceous members into three tribes: Olyreae, Phareae and Streptochaeteae. Soderstrom and Ellis (1987) considered a total of 11 tribes within Bambusoideae. Five of them were monophyletic and recognized as ‘core’ Bambusoideae with four herbaceous tribes (Olyreae, Anomochloeae, Streptochaeteae and Buergersiochloeae) and one woody tribe (Bambuseae). All the remaining six (Streptogyneae, Puelieae, Guaduelleae, Phareae, Oryzeae and Zizanieae) were considered as ‘peripheral’ tribes. The proposed ‘core’ Bambusoideae of Soderstrom and Ellis (1987) was similar to the circumscription proposed by Roshevits (1946) except for Parianeae. Prat (1960) only considered the woody members within Bambusoideae and moved the herbaceous taxa to Oryzoideae. Clayton and Renvoize (1986) and Renvoize and Clayton (1992) combined the ‘core’ and ‘peripheral’ Bambusoideae together. They also merged Guaduelleae and Puelieae to Bambuseae and Buergersiochloeae to Olyreae. They further subdivided tribe Bambuseae and recognized only 49 genera in three subtribes, that is Arundinariinae (20 genera), Bambusinae (25 genera) and Melocanniae (4 genera). Tzvelev (1989) recognized the members of ‘core’ Bambusoideae in a separate subfamily and all other grasses were placed under Pooideae. Watson and Dallwitz (1992) basically supported Clayton and Renvoize (1986) and Renvoize and Clayton (1992) except Centotheceae, which was included into Bambusoideae. Kellogg and Campbell (1987) considered Bambuseae as monophyletic based on the presence of woody culms and the herbaceous bamboos as either monophyletic or paraphyletic to Bambuseae. In a subsequent study, Kellogg and Watson (1993) also revised that the ‘core’ Bambusoideae as recognized by Soderstrom and Ellis (1987) were not monophyletic, but rather polyphyletic. Dransfield and Widjaja (1995) included 69 woody genera in their description that was further enhanced to 78 in Stapleton’s description (1994a,b,c, 1997). However, one of the most extensive eVorts to study grass phylogeny and sub‐familial classification has been commenced (GPWG, 2001). The study based on 62 grasses recognized Poaceae as a monophyletic family (Fig. 5). The earliest diverging lineages were Anomochlooideae, Pharoideae and Puelioideae. Bambusoideae formed the clade ‘BEP’ along with Pooideae plus Ehrhartoideae and each of them was supported as monophyletic. One of the most significant conclusions is to abandon the long‐standing belief that bamboos are the most primitive grasses as speculated by a large section of bamboo taxonomists

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Anomochlooideae Pharoideae Puelioideae

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Incertae sedis

Fig. 5. The most recent grass classification system proposed by Grass Phylogeny Working Group (2001) (reproduced with permission from Missouri Botanical Garden Press).

(Clayton and Renvoize, 1986; Tateoka, 1957 and many others) based on the reproductive plesiomorphic characters such as bracteates, indeterminate inflorescence or the presence of spikelet like pseudo‐spikelet structures.

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IV. CONSPECTUS OF WOODY BAMBOO GENERA OF THE WORLD Most of the recent classification systems (Dransfield and Widjaja, 1995; Li, 1997; Soderstrom and Ellis, 1987) placed 67 genera of woody bamboos in nine sub‐tribes. These classification systems were largely dependent on various floral characters such as type of inflorescence or ovary appendages. I. SUBTRIBE ARTHROSTYLIDIINAE: 1. Actinocladum, 2. Alvimia, 3. Arthrostylidium, 4. Athroostachys, 5. Atractantha, 6. Aulonemia (Matudacalamus), 7. Colanthelia, 8. Elytrostachys, 9. Glaziophyton, 10. Merostachys, 11. Myriocladus, 12. Rhipidocladum II. SUBTRIBE ARUNDINARIINAE: 13. Acidosasa, 14. Ampelocalamus, 15. Arundinaria, 16. Chimonocalamus, 17. Drepanostachyum (Himalayacalamus), 18. Fargesia (Borinda, Yushania), 19. Ferrocalamus, 20. Gaoligongshania, 21. Gelidocalamus, 22. Indocalamus, 23. Oligostachyum, 24. Pseudosasa, 25. Sasa, 26. Thamnocalamus III. SUBTRIBE BAMBUSINAE: 27. Bambusa (Dendrocalamopsis), 28. Bonia (Monocladus), 29. Dendrocalamus (Klemachloa, Oreobambos, Oxynanthera, Sinocalamus), 30. Gigantochloa, 31. Dinochloa, 32. Holttumochloa, 33. Kinabaluchloa (Maclurochloa, Soejatmia), 34. Melocalamus, 35. Sphaerobambos, 36. Thyrsostachys IV. SUBTRIBE CHUSQUEINAE: 37. Chusquea, 38. Nerolepis V. SUBTRIBE GUADUINAE: 39. Apoclada, 40. Eremocaulon, 41. Filgueirasia, 42. Guadua, 43. Olmeca, 44. Otatea VI. SUBTRIBE MELOCANNINAE: 45. Cephalostachyum, 46. Davidsea, 47. Leptocanna, 48. Melocanna, 49. Neohouzeaua, 50. Ochlandra, 51. Pseudostachyum, 52. Schizostachyum, 53. Teinostachyum VII. SUBTRIBE NASTINAE: 54. Decaryochloa, 55. Greslania, 56. Hickelia, 57. Hitchcockella (?), 58. Nastus, 59. Perrierbambus (?)


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VIII. SUBTRIBE RACEMOBAMBOSINAE: 60. Racemobambos (Neomicrocalamus) IX. SUBTRIBE SHIBATAEINAE: 61. Chimonobambusa, 62. Indosasa, 63. Phyllostachys, 64. Qiongzhuea, 65. Semiarundianria (Brachystachyum), 66. Shibataea, 67. Sinobambusa.

V. RELEVANCE OF MOLECULAR TAXONOMY IN BAMBOO Two major objectives of any taxonomic study are (a) systematic grouping of the taxa of interest through generation of robust, natural classification system based on stable characters that reflect their true evolutionary history and (b) development of reliable identification key(s) for easy taxon determination. Most of the classifications proposed to date for bamboo are primarily dependent on various morphological features and one of the most immediate needs is to test how natural all these systems are. Stapleton (1997) has summarized few important limitations associated with the traditional morphological classifications: (1) Morphology‐based classifications are often superficial as similarities have frequently gained priorities over dissimilarities. (2) Reproductive characters have often earned priority with an assumption of having higher evolutionary significance than the vegetative characters. The importance of many vegetative features such as rhizome or branch patterns was understood later and thus many of the early herbarium specimens were incomplete. (3) In many cases artificiality was enhanced as characters were frequently considered in isolation rather than considered in groups. It is undeniable that vegetative features are quite essential for field identification of the woody members as flowering cycles are often erratic, which severely restricts the opportunity to study fresh reproductive materials. Even if the dried, herbarium samples are available, quite often these lack enough morphological resolution and thus create confusion in the real field condition. Hence, the identification keys are mostly dependent on various vegetative features that need further refinement and re‐investigation. In particular, the taxonomic demarcation of woody bamboos at lower ranks, such as genera and species, are not well resolved to date. There are several species which are known only vegetatively, new species are constantly been described (Clark et al., 2007; Filgueiras and Londonõ, 2006; Triplett et al., 2006) and several undescribed taxa are known to occur in the wild habitat of South and Central Americas.

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Molecular data sets can provide useful information for addressing various aspects of plant taxonomy. Considerable progress has already been achieved in bamboo and this chapter is primarily aimed at reviewing the various molecular tools applied to date and also the potential pitfalls that need to be critically considered. The major challenge associated with any molecular method is to determine the appropriate taxonomic level at which it is most informative and to correlate it with morphologically definable taxonomic groupings.

VI. DNA FINGERPRINTING‐BASED METHODS A. RFLP

In restriction fragment length polymorphism (RFLP), diVerences in the restriction enzyme recognition site sequences between genomes are the basis of polymorphism. These markers are co‐dominant in nature and are useful for marker assisted selection. The technique was introduced to bamboo by Friar and Kochert (1991, 1994) for phylogeny assessment of 61 accessions and 20 species of Phyllostachys. The study supported the earlier observations of the presence of two distinct sections (Phyllostachys and Heteroclada) in Phyllostachys species pool. However, they disagreed to place P. nigra under the section Heteroclada and thus contradicted a previous study (Wang et al., 1980). The regular use of RFLP in plant genotyping as well as bamboo has been limited mainly due to the requirements of large amount of DNA along with the use of radioactive isotopes. B. RAPD

In randomly amplified polymorphic DNA (RAPD, Williams et al., 1990) technology, a single and short arbitrary primer is used. RAPD was utilized to assess phylogenetic relationships among 73 genotypes of Phyllostachys (Gielis et al., 1997b). The resultant phylogeny neither supported the existence of two distinct sections in the Phyllostachys‐species‐complex nor the placement of P. nigra under Phyllostachys, hence deviated from the previous proposal by Friar and Kochert (1994). However, based on a combined application of RAPD and morphometry, it was confirmed that P. nigra belongs to the section Phyllostachys (Ding, 1998) and it was also confirmed by AFLP (Fig. 6A) and ITS sequence data (Fig. 6B) that two distinct sections, Phyllostachys and Heteroclada, do exist in the Phyllostachys species pool. The utility of RAPD was extended to the tropical group as well. B. ventricosa was found close to B. vulgaris var. striata (Nayak and Das, 2003) and was supported by a previous finding that B. ventricosa is a cultivated variety of B. vulgaris (Chua et al., 1996). Similarly, a high level genetic proximity

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Chimonobambusa marmorea Shibataea chinensis Sinobambusa tootsik Neomicrocalamus andropogonifolius

Fig. 6. The existence of two separate sections, Phyllostachys and Heteroclada, was confirmed by both (A) AFLP and (B) ITS sequence‐based phylogeny (Hodkinson et al., 2000; reproduced with permission from The Botanical Society of Japan).

(0.91) was obtained between B. striata and B. vulgaris (Das et al., 2007) that was in compliance with the proposition that B. striata is a somatic mutant of B. vulgaris (Bennet and Gaur, 1990). RAPD‐based neighbour joining tree

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clearly separated the thorny core Bambusa group from the Dendrocalamus group (Sun et al., 2006). However, most of these studies considered limited number of species and hence the phylogenetic relationships need to be further validated by applying wider species and genera range. Studies on population variability are another area that could benefit from RAPD technology (Hsiao and Rieseberg, 1994). It was found more eYcient than micro‐ or minisatellite to assess genetic variations among the clones of P. pubescens in Taiwan (Lai and Hsiao, 1997). Identification of only nine genotypes among a pool of 176 samples clearly suggested the existence of low population genetic variability. Likewise, applying selected primers which were found highly polymorphic at the rank of species (Das et al., 2007) could not detect any polymorphism among 17 geographically isolated populations of B. tulda (Bhattacharya et al., 2006). These two population level studies indicate the possible existence of limited genetic variability that could be attributed to the pre‐dominant vegetative mode of propagation in bamboo. Nevertheless, it is necessary to emphasize that in spite of enormous promise, the reliability and reproducibility of RAPD technique is not beyond doubt. C. SCARS

Sequence characterized amplified regions (SCARs) is an extension of the RAPD procedure (Paran and Michelmore, 1993), but with better reproducibility due to the use of higher annealing temperature. SCARs are co‐dominant and have been proved useful for genotype/varietal identification. Particularly, they are useful at the seedling stage when key morphological features are indistinguishable. We have developed two species‐specific SCAR markers for B. balcooa and B. tulda (Das et al., 2005) to aid the paper and pulp industry for accurate species diagnosis. To authenticate the utility of these markers at the population level, 80 individual plants collected from 16 eco‐geographically diverse populations were screened. D. AFLP

Amplified fragment length polymorphism (AFLP) is a method described as a combination of RFLP‐ and PCR‐based techniques (Vos et al., 1995). It generates dominant markers like RAPD and is highly sensitive to detect polymorphisms among closely related genomes. It has already been demonstrated eYcient in measuring genetic relationships among 15 bamboo species representing four diVerent genera (Loh et al., 2000). Unique banding patterns were obtained in 13 out of 15 species and the cluster pattern helped reveal the polyphyletic nature of the genus Bambusa. However, separation of two Dendrocalamus species in two diVerent clusters emphasized the need to re‐examine


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their status. This technique has also been employed to assess the genetic diversity of the woody American bamboos, namely, Guadua angustifolia, G. amplexifolia, G. macrospiculata, G. superba and G. unicata (Marulanda et al., 2002). Distinct genetic diVerentiations were observed between species. At the accession level, higher genetic diversity was observed for G. amplexifolia, while it was low for G. angustifolia. Study of clonal structure is an integral part of bamboo biodiversity assessment and AFLP proved useful. A study on the population of the dwarf bamboo, Sasa senanensis, revealed high clonal diversity (Suyama et al., 2000). The clonal distribution pattern over a 10 ha study plot indicated a possible relationship between the clone size and the site characteristics where they grow. For example, larger clones were found in the flat areas, while smaller sized clones were found in steep soil that might have interfered with proper rhizome growth. Another population level study re‐confirmed 67 years of flowering interval in P. pubescens and enumerated that a population originating from the seeds of same flowering event may not necessarily have the same flowering interval (Isagi et al., 2004). The temporal variations in flowering cycles among the siblings of P. pubescens reflect heterogeneity among seeds and are not unexpected in the perennial plant group. AFLP has been proved useful in diverse aspects of bamboo systematics, population structure and variability studies (Gielis et al., 2001) due to the high sensitivity of the technique. However, few limitations associated with the technique include high technical skill and diYculty in analysis of the large number of amplified bands in addition to the cost and time involved. E. MICROSATELLITES (SSRS)

Microsatellites or simple sequence repeats (SSRs, Litt and Lutty, 1989) are short tandem repeated sequences of 1–6 nucleotides in length, highly polymorphic, co‐dominant, multi‐allelic, presumed selectively neutral and hence widely used in plant genetic diversity studies. Primers are designed from the conserved genomic regions flanking the repeat sequences and the detected polymorphism reflects variation in the number of repeats among genomes. However, the entire procedures that include construction and screening of genomic library prior to primer designing are cumbersome and cost intensive. This severely limits the wide application of the technique in non‐crop plants like bamboo, as suYcient genomic information is not yet available in the database. In spite of that, they have already been successfully applied to Phyllostachys (Lai and Hsiao, 1997) and Bambusa (Nayak and Rout, 2005). Six microsatellites were isolated from B. arundinacea and their cross‐species amplification was tested in 18 other bamboo species (Nayak and Rout, 2005). This proof of the principle study indicates that informative conserved

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sequences across taxa could be successfully utilized for defining comparative systematic strategies, and thus reduces the eVorts to develop microsatellites for individual bamboo species. F. EXPRESSED SEQUENCE TAG DERIVED MICROSATELLITES (EST‐SSR)

This is another chimeric marker technology, where SSRs are harvested in silico from EST sequences. It has been shown that EST‐SSR markers derived from maize, wheat, sorghum and rice could be successfully utilized to evaluate genetic diversity among 92 temperate bamboo accessions (Barkley et al., 2005). The technique proved sensitive enough to detect contamination in a bamboo plot where Phyllostachys rubromarginata stands were mixed with either P. flexuosa or P. glauca stands. Thus EST‐SSR holds the promise to extrapolate genomic information from crop to non‐crop plants by exploiting genetic collinearity among the members of the grass family. Although EST‐ SSRs are less polymorphic than genomic SSRs, their easy transferability across species border‐line is highly desirable (Yu et al., 2004), particularly in systems like bamboo where much genomic information is not yet available. G. TRANSPOSON

Miniature inverted‐repeat transposable elements (MITEs) are an important member of the transposon family with high abundance in plants (Wessler et al., 1995). MITE‐transposon display (MITE‐TD) is a modification of the AFLP technique, where the conserved sequence stretches of the MITE transposons are targeted. It has been successfully recruited to assess genetic variations among Oryza species (Park et al., 2003). Sensitivity of another transposon family member, Rim2/Hipa‐TD, has been tested positive to clearly diVerentiate japonica and indica ecotypes of rice (Kwon et al., 2005). Retro‐elements like Wis‐2 have been found conserved across grass genomes like wheat, barley, rye, oats and Aegilops and transcriptionally more active in grasses than in dicots (Vicient et al., 2001). The presence of Ac‐like sequences was found in Bambusa multiplex (Huttley et al., 1995), while partial Ac‐like transposon elements were isolated from three bamboo species: Bambusa vulgaris, Sasa veitchii and Phyllostachys edulis (Gielis, 1998). The sequence obtained from B. vulgaris revealed considerable homology to the hAT superfamily of transposons (Keukeleire et al., 2004). A recent study indicates that 23.28% of P. pubescens genome is enriched with repeat elements and majority of them (18.89%) were LTR retro‐transposons, mainly Gypsy/DIRS1 and Ty1/Copia type (Jie et al., 2007). The possible link between transposons and


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flowering event in bamboo has been speculated over the years, although no direct evidence has yet been obtained.

VII. DNA SEQUENCE‐BASED METHODS A. ORGANELLAR GENES

In the early era of plant molecular systematics, chloroplast DNA restriction site polymorphism was extensively utilized to discriminate plant taxa (Olmstead and Palmer, 1994, for detailed account) and grasses were no exception. In one such study, the phylogenetic relationships among 31 grass taxa selected from six diVerent subfamilies were evaluated (Davis and Soreng, 1993). The analysis identified two main clades, one was the Pooideae and the other clade was PACC that included the woody Bambusoideae (Fig. 7A). This PACC clade A Joinvillea *PACC clade Lithachne *Bambuseae Pharus Nardus Brachyelytrum

*Oryzeae *Pooideae, incl. Diarrhena

• Two major groups in grasses are PACC (Panicoideae, Arundinoideae, Chloridoideae and Centothecoideae) and Pooideae; Davis and Soreng (1993) based on chloroplast restriction polymporphism

Fig. 7.

(Continued )

B

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Leersia virginica Ehrharta calycina

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Streptogyna americana

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Lithachne pauciflora Raddia distichophylla Bambusa stenostachya Bambusa aff. bambos Cephalostachyum pergracile

Bambuseae

Chusquea latifolia Chusquea circinata Guadua paniculata Oryzopsis racemosa

Stipeae

Poa pratensis

Poeae

Avena sativa

Aveneae

Hordeum vulgare

Triticeae

Diarrhena obovata

Diarrheneae

Phaenosperma globosum

Phaenospermateae

Brachyelytrum erectum

Brachyelyteae

Danthoniopsis petiolata

Arundinelleae

Thysanolaena maxima

Thysanolaeneae

Zeugites pittieri Chasmanthium laxum

Centotheceae

Setaria viridis Paniceae Panicum virgatum Sorghum bicolor

Andropogoneae

Zea mays Molinia caerulea Phragmites australis

Arundineae

Arundo donax Micraira lazaridisii

Micraireae

Zoysia matrella

Zoysieae

Sporobolus indicus

Eragrostideae

Eustachys petraea

Chlorideae

Eragrostis curvula

Eragrostideae

Aristida longiseta Pharus latifolius

Aristideae Phareae

Pharus lappulaceus Streptochaeta angustifolia

Streptochaeteae

Anomochloa marantoidea

Anomochloeae

Joinvillea ascendens

Joinvilleaceae

Flagellaria indica

Flagellariaceae

• Anomochloeae, Streptochaeteae and Phareae are basal lineages • strong support for PACC and weak support for BOP • core Bambusoideae (Olyreae and Bambuseae) momophyletic; Clark et al., 1995 based on ndhF data

Fig. 7.

(Continued)

C 100 49 100

81

37

3

b

100

3 74 1 91 1

94 6 j

86 2

88 4

95 5

84 3

100 28

98 5

100 13

62 2

1

2 86 4 89 4

1 1

100 10

1

100 10 100 13

95 4

100 14

93 4 88 3

90 2

Panicoideae

58 1

100 15

Centothecoideae

74 1

Chloridoideae

63 2

97 5

Aristida ads Aristida lat Arundo Molinia Phragmites Danthonia Centropodia Bouteloua Orcuttia 98 Chloris Microchloa 7 Astrebla Kengia Aeluropus 1 Monodia Zoysia Sporobolus 100 Eragrostis 13 Pappophorum Uniola Chasmanthium 100 Lophalherum Orthoclada 7 Zeugites 93 Andropogon Hyparrhenia 3 h Sorghum h Tristachya Zea Digitaria 89 Echinochloa Panicum 2 Loudetiopsis

Arundinoideae

100 26

Oryzoideae

100 8 100 20

100 10

1

100 11

Bambusoideae

98 7

Pooideae

100 12 73 1 94 11 100 4 84 12 2 100 13 95 96 5 4 c

78

RESTIO JOINVLLEA Streptochaeta ang Streptochaeta spi Anomochloa Pharus Brachyelytrum Nardus Avena Phalaris Briza Phleum Poa Vulpia Bromus Hordeum Leymus Triticum Brachypodium Melica Nassella Stipa Lithachne Olyra Pariana Chusquea Phyllostachys Sasa Oryza Zizania Ehrharta

• Bambusoideae is close to Pooideae, Hilu et al., 1999 based on matK data

Fig. 7. The gradual progress of our current understanding of bamboo molecular phylogeny based on (A) chloroplast DNA restriction site polymorphism (Davis and Soreng, 1993, with permission to reproduce from American Journal of Botany), (B)

248

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(Panicoideae, Arundinoideae, Chloridoideae and Centothecoideae) was subsequently supported by others (Barker et al., 1995; Cummings et al., 1994). With the inclusion of DNA sequencing technology, employing coding sequences became a regular practice. The transcribed sequences of five 18S and three 26S rRNA were studied from nine grass species those were members of Bambusoideae, Pooideae and Panicoideae (Hamby and Zimmer, 1988). The study revealed Arundinaria as the basal lineage of the grasses. Chloroplast gene sequencing gained acceleration with the introduction of the rbcL gene encoding the large subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase. Barker et al. (1995) on the basis of the rbcL data revealed monophyletic Bambusoideae related to Pooideae and PACC clade, while another study obtained a weakly supported basal position for Bambusoideae (Duvall and Morton, 1996). However, the utility of rbcL sequences is often restricted above family level and not suYcient for sub‐familial resolutions in grasses (Doebly et al., 1990). In particular, the longer generation time of the woody bamboos might cause slower nucleotide substitution rate compared to other grasses (Gaut et al., 1997) and thus less preferable for lower taxonomic groups. Search for additional informative genes continued and new chloroplast genes encoding ribosomal protein S4 (rps4), NADH‐plastoquinone oxidoreductase subunit 5 (ndhF), maturase K (matK) and RNA polymerase subunit (rpoC2) have emerged. The rps4 was targeted to analyze 26 genera of grasses that included three woody bamboos (Nadot et al., 1994). Their analyses showed paraphyly for the bambusoid group that was close to oryzoids and pooids. It was subsequently supported by the rbcL (Barker et al., 1995) and matK (Liang and Hilu, 1996) sequence data. The first extensive eVort utilizing a wide sample of Bambusoid relied on the ndhF gene due to its higher evolution rate than rbcL (Clark et al., 1995). The analyses based on 45 grass sequences resolved the three herbaceous bamboo tribes, Anomochloeae, Streptochaeteae and Phareae, as the basal lineage within the grass family (Fig. 7B). All the other members of Bambusoideae were clearly separated out. One of the two major clades was a weakly supported BOP clade (Bambusoids, Oryzoids and Pooids), while a strong support was obtained for the PACC clade. Monophyly for the core Bambusoid group (Olyreae and Bambuseae) was observed. It was also inferred that many features that are authentic to traditional Bambusoideae are possible synapomorphies for the family. The ndhF sequence data has also been utilized to confirm polyphyly for Apoclada (Guala et al., 2000). ndhF sequence data (Clark et al., 1995, with permission to reproduce from the American Society of Taxonomy) and (C) matK sequence data (Hilu et al., 1999, with permission to reproduce from the Missouri Botanical Garden Press).


249

A consequence of this study was the description of a new genus (Filgueirasia) that was nested inside Apoclada S.L. (Guala, 2003). Nonetheless, the earliest divergence of Streptochaeta and Anomochloa followed by Pharus was subsequently supported by matK sequence analysis from 62 grass species covering 9 sub‐families (Hilu et al., 1999). Bambusoideae was placed in a separate clade with Pooideae (Fig. 7C). By this time Clark and Judziewicz (1996) recognized that monophyly for Bambusoideae could not be retained if the basal lineages, that is Anomochlooideae and Pharoideae, were to be accommodated. Another chloroplast gene rpo C2 was found useful for grass phylogeny assessment for possessing an extra coding sequence that enhances the rate of substitution and insertion/deletion events (Cummings et al., 1994). DNA sequence data from the non‐coding regions of chloroplasts were simultaneously exploited, particularly for the lower taxonomic categories with the assumption that non‐coding regions are under reduced functional constrain than are coding regions and thus exhibit higher level of sequence variations for enhanced phylogenetic resolutions (Gielly and Taberlet, 1994). The rpl 16 intron data was successfully utilized to study relationships among 23 species of Chusquea and 15 taxa from Bambusoideae (Kelchner and Clark, 1997). Monophyly for Chusquea was strongly supported as was also recognized for the herbaceous and woody bamboos within Bambusoideae. The woody bamboo was divided into temperate and tropical bamboos and the tropical group was further subdivided into New World and Old World clades. Zhang (2000) has demonstrated the successful utilization of rpl 16 sequences even for higher taxonomic level. His analysis based on 35 sequences from six major sub‐families supported the existence of two major, monophyletic groups, BOP and PACC, within the grass family. Although Oryzoideae and Pooideae were strongly supported as monophyletic, support for Bambusoideae was weak. The basal lineage of Streptochaeteae, Anomochloeae and Phareae was also supported. An in‐depth study of the clade Arthrostylidiinae and Guaduinae employing about 50 woody species from Brazil based on the rpl 16 intron as well as trnD‐T and trnT‐L sequences is currently under way by Santos‐Gonçalves (personal communication by T.S.F.). Various chloroplast sequences have contributed immensely to our current understanding of grass as well as bamboo systematics. Particularly at deeper levels, the relative ease of the plastid DNA sequencing makes it a powerful tool for phylogenetic reconstructions. However, several events such as recombination, heteroplasmy or haplotype polymorphism can confound these attempts (Wolfe and Randle, 2004, for detailed account) and hence plastid sequence data should always be combined with other sequences to achieve suYcient resolution for a robust phylogeny.

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M. DAS ET AL. B. NUCLEAR GENES

Among the nuclear genes, internal transcribed spacers of 18S‐5.8S‐26S nuclear ribosomal cistron have gained rapid popularity for plant phylogenetic inference. The ITS regions, 500–700 bp long in angiosperms (Baldwin et al., 1995), are flanked by highly conserved sequence stretches and thus amplified by universal primers (White et al., 1990) and sequenced. Taxon‐ specific character‐state changes in the ITS regions are an outcome of concerted evolution and hence insertion‐deletion polymorphisms (indels) are targeted for phylogenetic reexamination (Alvarez and Wendel, 2003). Phylogenetic relationships among the members of Thamnocalamus and allied groups have been extensively studied with ITS sequence data. Monophyly for the Thamnocalamus group was revealed (Guo et al., 2002). It was subsequently supported by combined as well as individual application of the ITS and low copy granule bound starch synthase gene (GBSSI) sequence data (Guo and Li, 2004). However, the tree based on the combined data sets (GBSSI and ITS) had higher resolution than that based on individual data set. ITS sequence was also employed to study genetic variation and phylogeny assessment of 23 alpine bamboo species from three genera, Thamnocalamus, Fargesia and Yushania. It identified T. spathiflorus var. crassinodus and F. spathacea as the basal lineage of alpine bamboos, although the bootstrap support was weak (Guo et al., 2001). However, it did not observe monophyly for Fargesia and Yushania and suggested the need to re‐investigate the delimiting morphological features. Sequence (ITS) and PCR markers (AFLP) were simultaneously applied to re‐examine the phylogenetic relationships of Phyllostachys complex (Hodkinson et al., 2000). Monophyletic origin and existence of two distinct sections within the Phyllostachys species pool were supported (Fig. 6A and B). Heteroclada was further sub‐divided into two groups and another group within the section Phyllostachys was strongly advocated. The application of ITS sequence data was also extended beyond temperate bamboos. In one such study encompassing 21 species of Bambusa, Denrocalamus, Dendrocalamopsis, Guadua, Leleba and Lingnania, the members of Dendrocalamus were shown as close relatives of Bambusa (Sun et al., 2005). In another study, monophyly of Olyreae and Raddia was strongly supported by either single or combined use of ITS and trnD‐T sequence data (Oliveira, 2006). The basal position of Streptochaeta and Pharus, as already established by various chloroplast genes, has also been supported by ITS data, although Anomochloa was not included in this study (Hsiao et al., 1998). A search (as of October, 2007) in the NCBI‐nucleotide database (www.ncbi.nlm.nih.gov) revealed that complete or partial sequence information is already available for 123 bamboo species spanning across 36


251

genera that reflects a wide acceptability of ITS sequence data to a broad section of bamboo taxonomists. Although nuclear markers were mostly dominated by spacer sequences, the use of low‐copy nuclear genes is gaining popularity. For instance, Mathews et al. (2000) on the basis of the analysis of 51 PHYB sequences concluded Anomochloa and Streptochaeta as the first lineage of the grass family, followed by Pharus and Puelia. This is in gross agreements with many early reports based on various chloroplast regions. However, one of the most significant enhancements was to obtain a strong support for the BOP clade that was previously weakly supported by the ndhF data (Clark et al., 1995) and was not supported at all by other plastid sequences (Cummings et al., 1994; Davis and Soreng, 1993; Nadot et al., 1994). A high support for this clade had also been obtained by the combined application of three phytochrome loci (Mathews and Sharrock, 1996). Nonetheless, bi‐parental, nuclear ITS regions are one of the most popular choices for phylogenetic inference at genus level or below due to higher rate of base substitution than most of the organellar genes. In addition their high copy numbers allow easy amplification by targeting the conserved priming sites surrounding 18S and 26S regions. However, there are associated molecular events that could always confound phylogenetic inference (Alvarez and Wendel, 2003) in addition to the limitation due to small number of informative features (Baldwin et al., 1995) and frequent diYculty in alignment due to length variations (Hsiao et al., 1998). One of the most important prerequisites is to target the true orthologous sequences in related taxa that are subjected to phylogenetic re‐investigation. However, in absence of complete homogenization, unintended inclusion of paralogous counterpart is possible and can always delude the eVort. Particularly, such chances are very high in woody bamboos where extensive genome polyploidization is a common occurrence. The other confounding phenomena discussed by Alvarez and Wendel (2003) are existence of large number of rDNA arrays, eVect of secondary structure on base substitution and chances of contamination due to the use of universal primers. Of these, the contamination problem has already been experienced in the woody bamboo where fungal rDNA was inadvertently co‐isolated and hence co‐amplified with the target DNA (Zhang et al., 1997). Epiphyllous fungi are frequently associated with bamboo leaves. Hence fresh leaves should always be suYciently surface sterilized prior to DNA extraction and to avoid any possible contamination. It is also preferable not to rely on a single PCR reaction, but to clone and sequence products amplified under various reaction conditions (Alvarez and Wendel, 2003) to avoid PCR bias or drift (Wagner et al., 1994). However, many of these issues could be equally associated with any rapidly evolving region that is essential for lower taxonomic or recently radiated groups. The use of low

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copy, nuclear genes is gaining quick popularity as it combines the benefit of high substitution rate but lower chances of obtaining the paralogous counterparts (Small et al., 1998). However, their extensive utilization is mainly restricted by experimental diYculties in isolation and characterization due to lack of suYcient sequence information available in the database.

VIII. SUMMARY AND FUTURE DIRECTIONS A. MORPHOLOGICAL SYSTEMATICS AND IDENTIFICATION

Generation of enormous morphological and anatomical data over the years has built up a strong foundation for bamboo taxonomy studies to address both systematic and identification issues. However, one should always keep this in mind that vegetative morphology‐only phylogenetic analyses often lack suYcient resolution and thus should always be compared with the outcome from other data sources. In an attempt to evaluate phylogenetic relationships among 15 tropical woody species, we obtained the dendrogram pattern based on 32 morphological descriptors that was not fully supported by the classification system of Gamble, while the cluster pattern computed from 120 polymorphic RAPD fragments was in gross agreement (Das et al., 2007). Our follow‐up study based on higher number of taxa (25) revealed similar discrepancy between morphological (Fig. 8A) and DNA polymorphism (Fig. 8B)‐based dendrograms, while only the latter was in complete agreement with the classification system. It is surprising since most of the selected culm (Table I) and culm‐sheath characters (Table II) used in this study are widely used for bamboo species characterization. The most probable explanation is that the classification system was developed using vegetative plus reproductive characters, while only vegetative characters were analyzed in the present study due to the unavailability of reproductive organs. This case study clearly shows that chances of potential errors exist for any phylogenetic interpretation in bamboo that is not based on a complete array of morphological features, that is vegetative plus reproductive. Nonetheless, morphology‐based identification keys are very useful for quick identification at the field, yet it needs further precision as morphological features are often influenced by environment due to the event of true parallelism (Kellogg and Watson, 1993). Particularly, the population level understanding of morphological variability needs to be enhanced in bamboo. We have identified a number of morphological variations in diVerent Bambusa species that call for serious attention to reevaluate the identification keys applied in the field. For instance, in few cases striated culms were observed in B. tulda, which resembles that of B. striata (Fig. 9A and B), while bent culms and compressed,

253


A

0.77

A. malig T. spathiflorus subsp.spathiflorus B. burmanica B. oliveriana B. multiplex ‘Rivieriorum’ B. multiplex ‘Variagata’ B. striata B. vulgaris B. wamin D. strictus B. atra M. baccifera B. affinis O. abyssinica D. giganteus G. atroviolacea P. kurzii B. balcooa B. anriculata B. nutans B. tulda B. teres B. polymorpha B. bambos B. bambos var.giganteus 0.54

0.41

0.28

0.16

Distance coefficient

B

A. maling T. spathiflorus subsp. spathiflorus M. baccifera O. abyssinica D. giganteus G. atroviolacea D. strictus P. kurzii B. affinis B. atra B. auriculata B. teres B. tulda B. nutans B. burmanica B. striata B. vulgaris B. wamin B. balcooa B. bambos B. bambos var. giganteus B. oliveriana B. multiplex ‘Rivieriorum’ B. multiplex ‘Variagata’ B. polymorpha

0.00

0.22

0.45 Genetic distance coefficient

0.67

0.90

Fig. 8. Dendogram derived from UPGMA cluster analysis based on (A) 32 key morphological characters and (B) 244 polymorphic RAPD fragments of 25 woody bamboo species.

swollen internodes were recorded for B. balcooa and B. tulda (Fig. 9C and D). Therefore, further investigations covering diverse ecosystems and taxa are essential to confirm a set of morphological features stable across ecotypes.

TABLE I A Comparison of the 15 Key Culm Descriptors Used to Evaluate Phylogenetic Relationships Among 25 Bamboo Species (OTUs): Mean Height (m), Diameter (mm), Length of Fifth Internode (mm), Ratio of Cavity Diameter to Total Culm Diameter, Internode Bending, Colour (Yellow with Striation ¼ 0, Yellow‐Green ¼ 1, Gray‐Green ¼ 2, Pale Green ¼ 3, Bright Green ¼ 4, Glossy Green ¼ 5, Dark Green ¼ 6), Swollen Node, Nodal Ring (Absent ¼ 0, Whitish ¼ 1, Grayish ¼ 2), Nodal Sheath Scar, Hairs at Nodal Ring, Branches Come Out Piercing Culm Sheath, Curved Lower Nodal Branches, DiVerent Culm Leaf and Branch Leaf Size, Modification of Branches (None ¼ 0, Thorns ¼ 1, Spines ¼ 2), Striation on Culm

OTUs

Heighta

Diametera

Internodea

Cavitya

Bendingb

Colour

Swollen nodeb

Nodal ring

Sheath scarb

Hairsb

Branchingb

Curved branchesb

Leaf sizeb

Modifications

Striationb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

4.2 0.8 6.5 0.9 7.0 1.0 18.3 2.7 20.0 2.9 25.0 5.0 15.8 2.5 15.5 2.2 3.0 0.7 2.5 0.05 12.5 3.2 14.0 1.5 20.2 3.2 16.5 0.9 15.5 3.2 18.3 3.2 19.8 2.2 7.0 1.0 30.5 3.2 14.0 2.7 21.0 2.8 15.0 1.2 8.0 1.3 9.14 1.8 5.0 1.5

50 2.0 40.0 6.0 35.0 5.2 45.0 6.7 90.0 13.5 110 20.0 55.0 8.8 70.0 5.5 20.0 4.6 15.0 3.6 60.0 8.7 37.5 4.1 100 8.7 70.0 9.4 70.0 8.7 50.0 8.7 80.0 8.9 120 17.0 130 13.4 50 9.5 60 8.0 55 5.0 80 5.0 35 7.0 15 5.0

110 15 500 75.0 600 90.0 400 60.0 200 30.0 300 10.0 230 36.6 300 30.5 200 45.8 180 45.8 400 65.8 350 38.4 500 65.8 190 18.6 500 65.8 380 65.8 230 25.5 120 18.6 330 34.2 200 71.4 375 26.4 220 15.0 180 15.0 220 43.9 120 20.0

0.33 0.05 0.37 0.06 0.42 0.06 0.22 0.03 0.33 0.05 0.45 0.05 0.36 0.06 0.52 0.05 0.12 0.03 0.12 0.03 0.4 0.07 0.53 0.06 0.45 0.07 0.42 0.06 0.48 0.07 0.4 0.07 0.5 0.06 0.33 0.05 0.69 0.07 0.6 0.11 0.54 0.07 0.32 0.05 0.55 0.06 0.5 0.09 0.33 0.05

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0

1 2 6 3 4 2 2 5 3 3 4 5 1 0 6 6 4 6 2 1 6 4 4 2 1

1 0 0 0 1 1 1 1 1 1 0 1 0 1 0 0 1 1 0 1 0 0 0 0 1

1 1 1 2 1 0 0 1 2 2 1 1 1 2 1 1 2 1 2 0 0 0 1 1 1

1 1 1 1 1 0 0 1 0 0 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1

1 0 0 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 1

0 0 0 1 1 0 0 0 0 0 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0

0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0

0 1 0 1 1 1 1 0 0 0 1 0 1 0 1 1 0 0 1 0 1 0 1 0 0

0 0 0 0 2 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 1 0 0 0 0 0 0 0 0 1 0 1 1 1 1 1 0 0 0 0 0 0 0 0

OTU 1 ¼ Arundinaria maling, 2 ¼ Bambusa aYnis, 3 ¼ B. atra, 4 ¼ B. auriculata, 5 ¼ B. balcooa, 6 ¼ B. bambos var. giganteus, 7 ¼ B. bambos, 8 ¼ B. burmanica, 9 ¼ B. multiplex ‘riviereorum’, 10 ¼ B. multiplex ‘variagata’, 11 ¼ B. nutans, 12 ¼ B. oliveriana, 13 ¼ B. polymorpha, 14 ¼ B. striata, 15 ¼ B. teres, 16 ¼ B. tulda, 17 ¼ B. vulgaris, 18 ¼ B. wamin, 19 ¼ Dendrocalamus giganteus, 20 ¼ D. strictus, 21 ¼ Gigantochloa atroviolacea, 22 ¼ Melocanna baccifera, 23 ¼ Oxytenanthera abyssinica, 24 ¼ Pseudobambusa kurzii, 25 ¼ Thamnocalamus spathiflorus subsp. spathiflorus. a b

Mean SE. Absent ¼ 0, present ¼ 1.

TABLE II A Comparative Account of the 17 Key Culm‐Sheath Descriptors Used to Evaluate Phylogenetic Relationships Among 25 Bamboo Species (OTUs): Ratio of Total Length to Breadth at Base, Ciliate Margin, Pubescent Adaxial Side, Pubescent Abaxial Side, Hair Colour (None ¼ 0, Golden Brown ¼ 1, Brown ¼ 2, Dark Brown ¼ 3, Black ¼ 4), Number of Hairs (Absent ¼ 0, Scanty ¼ 1, Profuse ¼ 2), Ratio of Total Length versus Blade Length, Shape of Blade (Triangular ¼ 0, Acuminate ¼ 1, Lanceolate ¼ 2, Ovate ¼ 3), Blade Reflexed, Hairy Margin of Blade, Ligule Margin (Entire ¼ 0, Dentate ¼ 1, Serrate ¼ 2), Hairs on Ligule, Auricle, Auricle Continuous with Blade, Bristles on Auricle, Auricle Fringed, Variable Culm Sheath Size at DiVerent Culm Height

OTUs

Length/Breadtha

Marginb

Pub. adaxb

Pub. abaxb

Hair colour

Hair number

Total/Bladea

Shape

Blade reflexedb

Hairy marginb

Ligule margin

Hairs on liguleb

Auricleb

Auricle cont.b

Bristlesb

Auricle fringedb

Sheath sizeb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

3.0 0.85 1.55 0.23 3.33 0.49 1.5 0.22 1.59 0.24 2.8 0.37 2.92 0.47 2.60 0.30 2.87 0.66 2.75 0.55 1.15 0.18 2.69 0.30 1.20 0.18 1.78 0.22 1.25 0.18 1.04 0.18 1.65 0.15 0.87 0.13 1.3 0.13 1.39 0.26 1.31 0.17 1.16 0.15 2.1 0.25 3.37 0.67 3.6 0.85

0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1 1 0

0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 1 1 1 1 0 0 1 0 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0

0 4 1 4 4 4 4 4 0 0 4 4 4 3 4 4 3 2 2 1 1 1 3 4 0

0 1 1 2 1 1 1 1 0 0 2 1 2 2 2 2 2 2 2 2 1 1 1 2 0

4.2 0.55 4.43 0.66 2.0 0.29 2.45 0.37 4.15 0.64 3.66 0.62 3.88 0.62 2.43 0.26 2.87 0.56 2.77 0.55 3.42 0.64 2.33 0.26 3.50 0.64 2.5 0.31 3.20 0.64 3.67 0.64 2.75 0.30 2.08 0.31 3.36 0.35 3.85 0.73 3.0 0.39 1.25 0.42 1.55 0.22 1.8 0.36 3.6 0.55

1 3 3 3 0 1 1 1 2 2 3 1 3 3 3 3 1 3 2 0 2 2 2 2 1

1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 1 0 0 1 1

1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 1 1

2 0 0 1 2 1 1 2 0 0 2 2 2 2 2 2 0 2 2 2 0 1 0 1 2

1 0 1 1 1 1 1 0 0 0 0 0 0 1 0 0 1 1 1 1 1 0 0 1 1

1 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 1 0

0 0 1 1 0 1 1 0 0 0 1 0 1 0 1 1 0 0 0 1 0 1 0 1 0

1 0 1 1 0 1 1 1 1 1 1 1 1 0 1 1 1 0 0 0 0 1 0 0 0

0 0 0 1 0 1 1 0 0 0 1 0 1 0 1 1 0 0 0 0 0 1 0 0 0

0 0 0 1 0 0 0 0 0 0 1 0 1 0 1 1 1 0 1 0 1 0 0 1 0

OTU 1 ¼ Arundinaria maling, 2 ¼ Bambusa aYnis, 3 ¼ B. atra, 4 ¼ B. auriculata, 5 ¼ B. balcooa, 6 ¼ B. bambos var. giganteus, 7 ¼ B. bambos, 8 ¼ B. burmanica, 9 ¼ B. multiplex ‘riviereorum’, 10 ¼ B. multiplex ‘variagata’, 11 ¼ B. nutans, 12 ¼ B. oliveriana, 13 ¼ B. polymorpha, 14 ¼ B. striata, 15 ¼ B. teres, 16 ¼ B. tulda, 17 ¼ B. vulgaris, 18 ¼ B. wamin, 19 ¼ Dendrocalamus giganteus, 20 ¼ D. strictus, 21 ¼ Gigantochloa atroviolacea, 22 ¼ Melocanna baccifera, 23 ¼ Oxytenanthera abyssinica, 24 ¼ Pseudobambusa kurzii, 25 ¼ Thamnocalamus spathiflorus subsp. spathiflorus. a b

Mean SE. Absent ¼ 0, present ¼ 1.

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M. DAS ET AL.

Fig. 9. Striations on the culm of (A) Bambsa striata and (B) B. tulda; bent culm with compressed internodes in (C) B. tulda and (D) B. balcooa. B. MOLECULAR SYSTEMATICS AND IDENTIFICATION

Applications of the various molecular tools have enormous potential, but need judicial application based on the objectivity of the study and taxonomic rank under consideration. Utility of the sequence‐based molecular markers over PCR‐based markers for phylogenetic reconstructions is now well established. However, considerable disagreements still exist regarding the number(s) and nature (gene vs. spacer, or organellar gene vs. nuclear gene) of the target sequence(s). It is quite apparent that identifying one universal barcode, like that of mitochondrial CO1 in animals, is a distant hope for plant taxa (Pennisi, 2007). The slow evolution rate of the mitochondrial genes and low copy number of the nuclear genes led the plant taxonomists to focus mainly on various chloroplastic regions in recent years. Five candidate genes (rbcL, matK, rpoC1, rpoB, atpF/H) and two spacers (trnH‐psbA, psbK/I) are in the centre of all interests (Pennisi, 2007). Also a consensus has been developed to use a combination of these target sequences to avoid the inherent problems associated with each one of them. Similar consensus needs


257

720 700 680 660 640 620 600 580 560

au ric B. ula ba ta B. lc B. ba ooa be mb ec oo s h B. ey bl an um a B. e an a c B. h u co ng i n B. t i em ract ei a B. en fle sis x B uo B. . g sa r ha an d in B. an is B. in en m ter sis em m br edia a B. na m cea ul ti B. ple n x B. uta B. old ns po ha B. lym mii si or p n B. o s h a su pin ba os eq a B. ua su lis rre B. cta te xt i B lis B. . t u tu lda ld oi d B. es v B. ali vu da lg ar is

540

B.

Complete nucleotide sequences length (ITS1,5.8S rRNA, ITS2)

to be developed among the bamboo taxonomists to decide suitable target regions that could be universally sequenced, but should reveal enough sequence diversity to diVerentiate closely related taxa and lower ranks. In parallel the high copy, bi‐parental, ITS regions could be exploited since sequence data are publicly available for many species. However, one of the major limitations of bamboo ITS sequences is their considerable length variation that constrain multiple alignment and phylogenetic tree development. We found that in Bambusa the length varied from 599 bp to 707 bp (Fig. 10). Even for the same species (B. bambos, B. chungii, B. intermedia, B. membranacea, B. sinospinosa, B. surrecta), considerable length variation was observed across diVerent groups [represented by bar lines showing standard deviation (SD) in Fig. 6]. It is not clear at this stage whether this is due to diVerential evolution rate that generates this infra‐species heterogeneity. In addition we also propose to set a stringent standard for ITS‐based phylogeny development, similar to MIAME used for gene expression studies, to enhance resolution and reproducibility. Another practical challenge for bamboo molecular taxonomy is to provide tools for rapid and accurate taxon determination. Particularly, species level questions are always critical for the woody group and development of specific DNA tags might be useful for commercial purposes. For instance, bamboo constitute a major non‐wood fibre source for the paper and pulp

Fig. 10. Considerable length variations of spacers and 5.8S rRNA sequences among the members of Bambusa based on the data retrieved from www.ncbi.nlm. nih.gov as of October 2007. Where more than one submission was found for the same species, the mean value was used as the representative and SD represents variations.

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production in India and only 12 species have been identified for their suitability based on certain physical and chemical properties (Ganapathy, 1997). One such candidate is Bambusa balcooa, which was preferred by the pulp and paper industries because of its mechanical strength attributable to the high specific gravity (Bhatt et al., 2003). Our identified strategy of developing species‐specific SCAR‐marker (Das et al., 2005) is quite eVective to identify species, even at the seedling stage when traditional morphological characters often lack enough resolution. C. FUTURE SCOPE OF COMPARATIVE GENOMICS

One of the potentially emerging areas for bamboo biology is the comparative genomic studies, wherein available genomic information of other well‐ characterized cereal crops could be extrapolated to initiate functional genomics in bamboo. In this respect, Arabidopsis would certainly not be a good choice since extensive genome duplications in bamboo, at least for the woody group, might hinder the possibility of obtaining true one‐to‐one orthologs. However, in absence of suYcient genomic information, a reasonable starting point would be to target the collinear regions of the well‐characterized grass genomes and to search for their homologous regions in bamboos. Based on similar principle, EST‐SSR markers derived from maize, wheat, sorghum and rice have already been applied to bamboo (Barkley et al., 2005). The scope of the comparative genomics research in bamboo exists beyond taxonomy. One of the intriguing questions for future is to study the genes and mechanisms that control the unique flowering behaviour in bamboo. In particular floral genes, such as FEA2, BA1/LAX1, FUL, IDS1, KN1 or RCN1/2 that have already been characterized and connected to phenotypes in other grasses (Malcomber et al., 2006, for further details), should be targeted. It is possible to identify their homologous counterparts in bamboo by utilizing the conserved regions and subsequent functional characterization by expressing those genes in rice or maize deletion mutant lines. It would also provide some fundamental knowledge on the level of orthology existing between bamboo and other domesticated grass genomes. Another important area for future research is to characterize the genes regulating unique flowering event and/or long vegetative phase in bamboo. This could be achieved by performing suppression subtractive hybridization analysis where RNA from a flowering clone could be used as a tester and a non‐flowering clone as a driver or vice versa. The diVerentially expressed genes could be partially sequenced to generate ESTs and predicted by sequence alignment with available grass ESTs. A similar approach has recently been undertaken to identify the nuclear‐encoded non‐photosynthesis related genes in an albino mutant of


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Bambusa edulis (Lin et al., 2006). However, such eVorts are very scanty and the absence of genetic map(s) or mapping population is quite apparent. An NCBI search for bamboo revealed only 329 (mostly Bambusa oldhamii) and 998 (mostly Phyllostachys edulis) hits against publicly available EST and GSS collections. In addition databases that provide resources for diVerent bamboo genotypes are also essential since without morphological connections it is always hard to characterize a set of genes. We have summarized a list of web‐sites which are available so far and contain useful information regarding various aspects of bamboo biology (Table III). D. GENETIC DIVERSITY AND ECOLOGY

In contrast to the vast majority of studies done to date on bamboo taxonomy and systematics, investigations on genetic diversity at the population level are in its infancy. Substantial work has been done to develop comprehensive maps that describe the richness and distribution of woody bamboo species in Asia Pacific, Africa, Madagascar and in America (Bystriakova et al., 2003a,b; http://www.ourplanet.com/wcmc, index 14 and 19). The most alarming fact is the finding that 400 species are potentially threatened by destruction of natural forest cover in the Asia Pacific region. Studying distribution patterns is an important component of bamboo biodiversity, while estimation of population genetic diversity is equally essential for designing eVective conservation strategies. Multi‐loci PCR markers such as RAPD or AFLP are useful at the population level due to relatively low cost, fast assay time and their ability to depict polymorphism among closely related genomes. Our studies on B. tulda (Bhattacharya et al., 2006) and Thamnocalamus spathiflorus TABLE III Important Web‐Resources for Bamboo Biologists Web‐resource

Information available

http://www.eeob.iastate.edu/research/ bamboo/index.html http://bamboo-identification.co.uk

Distribution maps, key characters, methods, useful literatures Description of important identifying keys, classification, nomenclature, useful literatures General bamboo information Plantations, tissue culture, bio‐energy Evaluation data on bamboo accessions Sustainable social, economic and environmental benefits of bamboo

http://www.americanbamboo.org/ http://www.oprins.be http://www.ars-grin.gov/cgi-bin/npgs/ html/crop.pl/bamboo http://www.inbar.int/

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(unpublished data from S.B) at diVerent eco‐geographical regions of eastern India indicated a low level of population genetic diversity for these two species. Similar trend was identified in P. pubescens from Taiwan (Lai and Hsiao, 1997) and Guadua angustifolia from Colombia (Marulanda et al., 2002). It is quite possible that only a few clones of individual species acted as the genetic donor within a particular geographic area and thus resulted in low level among population genetic variability. On the other hand, relatively higher clonal variation was found in Sasa senanensis from Japan (Suyama et al., 2000) and G. amplexifolia from Colombia (Marulanda et al., 2002). It indicates that the diVerential reproductive systems might have influence on population genetic diversity in diVerent bamboo species, since it is expected that the allogamous species are usually more diverse than the autogamous ones. However, further studies are required to better understand emphatically the level of population genetic diversity and clonal structure in bamboo.

ACKNOWLEDGMENTS The financial support of the Council of Scientific & Industrial Research, New Delhi, India [Grant No. 38 (1062)/03/EMR‐II] is gratefully acknowledged. MD acknowledges the resident research associateship award from National Research Council, USA, while this review was written. We are thankful to the Director, Botanical Survey of India, Howrah, for allowing us to collect and study the bamboo germplasms, Mr. Pandey, B.S.I, for plant identification, Johan Gielis for providing useful literatures, R. B. Majumder, Jayadri Ghosh, John Fowler, OSU, Jason Londo, EPA, and Gerald F. Guala, USDA, NRCS, National Plant Data Centre for helpful discussions. We also thank the editors of this volume for giving us the opportunity to contribute.

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AUTHOR INDEX

Numbers in bold refer to pages on which full references are listed.

A Abe, H., 69, 78, 91 Aberle, T., 190, 214 Abizanda, L., 191, 197, 198, 200, 208 Abrahamson, J. L. A., 19, 39 Absalon, M. J., 26, 45 Aceto, S., 123, 142 Adams-Phillips, L., 83, 84, 89 Addicott, F. T., 195, 202 Agustı´, J., 191, 196, 197, 198, 200, 202 Agustı´, M., 153, 157, 159, 160, 162, 163, 169, 193, 194, 202 Agustı´, J., 191, 192, 195, 197, 198, 200, 203 Aharoni, Y., 161, 203 Ahmad, N., 26, 27, 28, 39 Ahn, J. H., 67, 74, 93, 122, 140, 141 Aida, M., 168, 203 Akihama, T., 162, 169, 210, 212 Akimitsu, K., 176, 178, 222 Akita, S., 63, 71, 78, 81, 92 Akiyama, K., 109, 143 Alamar, S., 191, 197, 198, 200, 208 Alba, M. M., 24, 38 Albagnac, G., 70, 85, 86, 93 Albert, S., 29, 30, 32, 40 Albert, V. A., 104, 137 Alberts, D. S., 173, 207 Albrigo, L. G., 150, 152, 167, 183, 185, 206, 222 Alferez, F., 194, 196, 203, 217 Alice, L. A., 247, 249, 263 Allen, G. J., 189, 203 Allison, L., 105, 137 Almagro, S., 25, 35 Almela, V., 159, 160, 162, 163, 192, 193, 194, 203, 209 Alos, E., 163, 164, 165, 166, 172, 183, 191, 192, 197, 198, 200, 208 Alquezar, B., 192, 217 Alvarez, I., 250, 251, 260 Amalric, F., 6, 25, 26, 36, 38, 40, 43, 44 Ambrose, B. A., 114, 136, 145 Ammerlaan, A, H, M., 63, 66, 69, 78, 90, 96 Ammerlaan, A., 67, 74, 75, 90, 96 An, G., 67, 74, 96, 114 An, K., 114, 141

Andersen, J., 3, 21, 28, 29, 42 Andersen, J. S., 3, 21, 35, 41 Andre´, C., 16, 43 Andres, F., 191, 197, 198, 200, 208, 221 Angelier, N., 17, 18, 35 Angelov, D., 25, 35 Angenent, G. C., 109, 112, 114, 118, 136, 138, 144 Anthony, M. F., 193, 203 Araki, T., 122, 136, 140, 154, 155, 207 Arana, M. V., 72, 79, 89 Arcas, M. C., 182, 206 Arditti, J., 123, 125, 136 Arellano, M., 17, 35 Arias, B. A., 174, 204 Arias, C. R., 192, 194, 196, 197, 202, 205 Arpaia, M. L., 186, 212 Arribas, R., 191, 197, 198, 200, 208 Asai, T., 71, 85, 94 Asakawa, Y., 175, 209 Asins, M. J., 149, 210 Aspart, L., 24, 35 Aspart-Pascot, L., 24, 35 Aspinall, D., 187, 213 Atkins, D. R., 180, 208 Aubourg, S., 128, 136, 153, 211 Audergon, J.-M., 70, 85, 86, 94 Aury, J. M., 153, 211 Averbeck, D., 173, 204 Averbeck, S., 173, 204 Avigne, W. T., 169, 212 Avila, C., 152, 216 Avsian-Kretchmer, O., 191, 204 Aznar, M., 194, 203 Azum-Ge´lade, M. C., 16, 35, 38 B Babourina, O. K., 190, 204 Bachellerie, J. P., 23, 36 Back, K., 176, 204 Bagnal, D. J., 122, 143 Bahrami, A. R., 172, 214 Bailey-Serres, J., 26, 27, 28, 29, 35 Bain, J. M., 157, 158, 168, 204 Bajon, R., 248, 251, 265

270

AUTHOR INDEX

Bakhshi, J. C., 194, 213 Bakkali, F., 173, 204 Bakker, J., 56, 93, 95 Baldauf, S. L., 51, 54, 55, 56, 57, 58, 59, 89, 93 Baldwin, B. G., 250, 251, 260 Baldwin, E. A., 161, 170, 204 Balestrini, R., 59, 89 Balk, J., 26, 27, 43, 45 Baluska, F., 75, 89 Bandong, S. L., 114, 145 Ban˜uls, J., 187, 195, 204 Bao, S. L., 244, 264 Bar, E., 138, 177, 219 Bar, R., 242, 259, 261 Bar-Peled, M., 182, 204, 208 Barakat, A., 29, 35 Barbezier, N., 19, 24, 32, 37 Barbier-Brygoo, H., 190, 206, 209, 214 Barker, N. P., 248, 250, 251, 260, 263 Barkley, N. A., 244, 258, 260 Barlow, P. W., 75, 89 Barneche, F., 23, 32, 35, 44 Barreiro, M. G., 70, 71, 91 Barret, H. C., 149, 204 Barros, S., 175, 213 Barry, C., 83, 84, 89 Bartels, J., 251, 267 Barthe, G. A., 57, 90 Bartley, G. E., 165, 209 Bartos, J., 103, 138 Basak, J., 241, 242, 252, 261 Baserga, R., 18, 19, 36 Baserga, S. J., 19, 36 Bateman, R. M., 101, 143 Battinelli, L., 174, 204 Battistelli, A., 172, 208 Baum, D. A., 114, 136 Baumann, E., 120, 142 Bauw, G., 28, 32, 33, 45 Baxter, C., 106, 137 Bazett-Jones, D.P., 4, 44 Bean, J. M., 19, 36 Beavis, W., 109, 143 Bechtold, N., 29, 30, 32, 40 Becker, A., 112, 114, 144 Becker, W. M., 62, 72, 79, 89, 92 Bedinger, P., 52, 54, 62, 79, 80, 91 Behrendt, H., 72, 79, 89 Belarmino, M. M., 111, 136 Belfield, E. J., 65, 68, 78, 84, 89 Bellaoui, M., 30, 36 Bellocco, E., 181, 209 Belser, C., 26, 40 Beltran, J. P., 191, 197, 198, 200, 208 Ben-Cheick, W., 159, 192, 193, 223 Ben-Hayyim, G., 187, 191, 204, 217 Ben-Tal, Y., 160, 222 Benavente-Garcıá, O., 182, 206

Bencivenni, C., 65, 78, 94 Bender, C. L., 192, 194, 196, 197, 202, 205 Benedito, V. A., 109, 136 Bennet, A. B., 69, 84, 88, 95 Bennet, R. D., 179, 210 Bennet, S. S. R., 241, 261 Bennett, A. B., 69, 70, 83, 84, 85, 86, 88, 90, 92, 95 Bennett, M. D., 104, 136 Benschop, J. J., 63, 66, 69, 78, 96 Bentham, G., 235, 261 Berardini, T. Z., 109, 143 Berbel, A., 191, 197, 198, 200, 208 Bergstro¨m, G., 102, 136 Bergstro¨m, L. G., 125, 140 Berhow, M., 174, 210, 212 Bernier, G., 154, 204 Bernstein, K. A., 19, 36 Bertrandy, S., 26, 40 Beven, A. F., 10, 14, 44, 45 Bewley, J. D., 79, 89 Beyer, A. L., 25, 29, 37, 38 Bhalla, P. L., 72, 80, 81, 89, 97 Bhat, N. G., 174, 214 Bhatia, A., 57, 95 Bhatt, B. P., 258, 261 Bhattacharya, S., 241, 242, 252, 258, 259, 261 Bhusal, R. C., 191, 205 Bibikova, T. N., 75, 89 Bicknell, K., 27, 36 Bishop-Hurley, S. L., 71, 82, 89 Blaas, J., 178, 214 Blackstone, N., 251, 267 Blanc, G., 29, 35 Blazquez, M. A., 123, 136, 191, 197, 198, 200, 208 Blecker, D., 51, 59, 68, 75, 82, 91 Bleichert, F., 19, 36 Blomqvist, K., 69, 78, 91 Blumenthal, S., 26, 27, 45 Blumwald, E., 170, 172, 202, 212, 219 Bock, R., 105, 138 Boerjan, W., 154, 218 Bogler, D., 248, 263 Bogner, M., 190, 208 Bogre, L., 26, 27, 36 Bo¨hlenius, H., 155, 204 Bohlmann, J., 128, 136, 153, 176, 177, 204, 205, 214, 220, 221 Boisvert, F. M., 3, 36 Boller, T., 168, 206 Bolviken, E., 229, 267 Boman, B., 190, 220 Bomblies, K., 154, 205 Bondarenko, V. A., 25, 35 Bonfante, P., 59, 89 Bonnet, N., 6, 38 Boon, R., 3, 21, 29, 42

AUTHOR INDEX Borba, E. L., 102, 136 Borg-Karlson, A. K., 102, 138 Boronat, A., 165, 176, 213, 217 Borowiec, J. A, 26, 40 Bosch, D., 178, 207 Botıá, J. M., 174, 216 Bouche, G., 26, 27, 36, 44 Bouchez, D., 190, 209 Bouhlal, R., 189, 190, 191, 205 Bourdais, G., 65, 78, 94 Bourdeaut, J., 152, 205 Bousquet-Antonelli, C., 29, 30, 32, 38 Bouteille, M., 13, 16, 18, 39 Bouvet, P., 25, 26, 35, 36, 38, 43, 44 Bouwmeester, H. J., 177, 178, 207, 214 Bowden, G. T., 173, 207 Bowen, P., 177, 214 Bown, A. W., 170, 205 Bradford, K. J., 50, 56, 57, 67, 71, 72, 74, 79, 81, 89, 90, 93 Brandis, D., 233, 261 Brandle, J. E., 128, 136 Brandli, C., 125, 141 Brandt, A. S., 109, 143 Braun, M., 75, 89 Brea, M., 227, 261 Bridts, C., 229, 262 Brilli, F., 132, 142 Britsch, L., 134, 142, 182, 214 Britto, D. T., 190, 205 Broadley, M. R., 189, 222 Brocklehurst, D., 49, 50, 80, 94 Brooks, G., 27, 36 Brown, G. E., 157, 192, 194, 195, 196, 197, 202, 205, 221 Brown, J. W., 23, 36 Brown, J. W. S., 3, 21, 28, 29, 42, 43 Brown, M. E., 27, 43 Brown, R., 112, 136 Brugidou, E., 19, 24, 32, 37 Brummell, D. A., 50, 56, 57, 69, 70, 84, 85, 86, 88, 90, 92, 93 Brumos, J., 187, 189, 190, 191, 197, 198, 200, 206, 221 Brunner, A. M., 155, 204 Bruns, T., 250, 268 Budde, A., 17, 19, 39 Budowski, P, 162, 163, 207 Bugler, B., 27, 36 Buigues, N., 175, 214 Bureau, T., 244, 268 Burgin, M., 72, 79, 94 Burlet, P., 26, 40 Burnett, R. H., 159, 220 Burns, J., 194, 217 Burns, J. K., 192, 193, 194, 195, 196, 197, 202, 203, 205, 206, 210, 212, 213, 216, 217, 222, 223 Burrows, P. A., 105, 136

271

Busch, M. A., 154, 205, 216 Busscher-Lange, J., 112, 138 Bystriakova, N., 227, 259, 261 C Caderas, D., 64, 77, 90, 91 Cafasso, D., 108, 136 Cai, Q., 185, 192, 205 Caizergues-Ferrer, M., 16, 27, 35, 36 Calsa, T. Jr., 105, 138 Camoin, L., 191, 204 Campbell, C. S., 236, 250, 251, 260, 264 Campos, N., 165, 213 Camus, E. G., 235, 261 Caparros-Ruiz, D., 25, 36, 44 Capron, A., 25, 29, 32, 38, 42 Carbonell, E. A., 149, 210 Carbutt, C., 102, 136 Cardemil, E., 177, 205 Carey, R. E., 51, 53, 54, 58, 59, 90, 96 Caristi, C., 181, 209 Carninci, P., 168, 216 Carpenter, R., 134, 141 Carpentier, M., 27, 36 Carpita, N. C., 54, 90 Carrer, H., 105, 138 Carrera, E., 148, 157, 160, 162, 174, 177, 178, 179, 183, 192, 200, 203, 205, 211 Carroll, A. D., 170, 205 Carroll, S. B., 101, 136 Carta, K. M., 26, 40 Cartwright, P., 251, 267 Casadoro, G., 71, 86, 96 Casagrande, A., 153, 211 Casanova, J., 149, 166, 208 Cassin, J., 152, 205 Castle, W. S., 184, 205 Casulli, V., 172, 208 Catala, C., 69, 86, 90 Cavanaugh, A., 18, 22, 39 Ceccardi, T. L., 57, 90 Cerana, R., 190, 205 Cerco´s, M., 148, 157, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 183, 192, 193, 195, 202, 203, 208, 211, 221 Cerda´, A., 190, 208 Cerdido, A., 26, 36, 37, 41 Cermenõ, M. C., 13, 40 Chai, D., 110, 111, 136 Chan, M. T., 111, 136, 137, 141, 145 Chan, Y. L., 111, 136, 137 Chang, C., 110, 137 Chang, C. C., 105, 110, 137, 145 Chang, D. C., 110, 141 Chang, I. F., 29, 35 Chang, S. B., 103, 104, 106, 108, 140

272

AUTHOR INDEX

Chang, W. C., 110, 137 Chao, C. S., 232, 235, 240, 261 Chapkin, R. S., 174, 222 Chapman, B. P., 128, 136 Chappell, J., 176, 177, 204, 207 Charbonnel-Campaa, L., 155, 204 Chase, M. W., 104, 137, 241, 250, 263 Chatterton, N. J., 250, 251, 263 Chaw, S. M., 105, 110, 137 Chayet, L., 177, 205 Chen, C. C., 103, 104, 106, 108, 140, 141 Chen, D., 117, 118, 138 Chen, F., 67, 71, 72, 74, 79, 81, 89, 90 Chen, G., 109, 143 Chen, H., 27, 36 Chen, H. H., 103, 105, 106, 108, 109, 110, 111, 112, 114, 117, 118, 126, 128, 129, 130, 131, 137, 139, 144 Chen, H. I., 106, 108, 114, 117, 118, 144 Chen, H. Q., 192, 195, 206 Chen, J. J. W., 259, 264 Chen, J.C., 66, 94 Chen, L., 88, 97 Chen, T. C., 106, 140 Chen, W. H., 103, 104, 105, 106, 108, 109, 110, 111, 112, 114, 117, 118, 126, 128, 129, 130, 131, 136, 137, 139, 140, 141, 144 Chen, X., 128, 132, 139 Chen, X. L., 108, 137 Chen, X. Y., 177, 179, 206 Chen, Y. C., 110, 137 Chen, Y. H., 103, 104, 106, 108, 111, 137, 140, 141 Chen, Y. Y., 250, 263 Chen, Z. H., 172, 208, 213 Chen, Z. J., 13, 14, 36, 40 Cheng, A. X., 177, 179, 206 Cheng, C. H., 105, 110, 137 Cheng, C. M., 29, 30, 32, 40 Cheng, Y. C., 6, 16, 43 Cheng, Y. J., 166, 221 Cheong, H., 134, 140 Chevalier, C., 170, 218 Cheverud, J. M., 15, 41 Chiang, M. S., 108, 137 Chin, D. P., 111, 142 Chiou, D. W., 104, 141 Cho, E., 122, 141 Cho, H.T., 50, 51, 54, 55, 56, 57, 59, 63, 65, 66, 67, 68, 74, 75, 76, 77, 78, 82, 86, 90, 91, 93 Cho, M., 75, 76, 93 Choi, D., 51, 54, 55, 56, 59, 61, 62, 63, 65, 66, 77, 90, 93 Choi, G., 134, 140 Choi, J. Y., 108, 137 Choi, S. B., 75, 76, 93 Choi, Y. D., 67, 74, 93

Choisne, N., 153, 211 Chonghaile, G. N., 241, 250, 263 Chopra, S., 72, 80, 81, 96 Chory, J., 122, 140 Chou, L. T., 113, 123, 134, 139 Chouinard, L. A., 11, 36 Chourasia, A., 70, 85, 96 Chow, T. Y., 105, 110, 137 Christensen, S. K., 122, 140 Christenson, E. A., 126, 137 Chu, C. D., 232, 240, 261, 267 Chua, K. S., 240, 261 Chua, N. H., 75, 89 Chuang, M. H., 106, 108, 114, 117, 118, 144 Chung, K. R., 194, 195, 206, 212, 213 Chung, Y. L., 106, 140 Chung, Y. Y., 114, 141 Ciceri, P., 114, 136, 145 Ciuni, M., 182, 206 Civello, P. M., 69, 70, 84, 85, 86, 88, 90, 91 Clark, G. P., 3, 21, 27, 28, 29, 43 Clark, L. G., 227, 229, 233, 239, 246, 248, 249, 251, 261, 262, 264, 266, 267, 268 Clayton, W. D., 236, 237, 261, 266 Clegg, M. T., 244, 248, 262, 263 Cleland, R., 49, 66, 95 Clepet, C., 153, 211 Clute, P., 18, 36 Cmarko, D., 6, 36 Coates, R. M., 176, 211 Coddeville, B., 27, 36 Coen, C., 106, 137, 141 Coen, E., 134, 154, 206 Coggins, C. W., 159, 180, 206 Coggins, C. W., Jr., 159, 193, 203, 208 Cogswell, J. P., 19, 39 Cohen, L., 170, 218 Collinge, M., 168, 206 Colmenero-Flores, J. M., 148, 157, 160, 162, 183, 187, 189, 190, 191, 192, 197, 198, 200, 203, 205, 206, 211, 221 Colmer, T. D., 63, 66, 67, 69, 74, 75, 78, 90, 96 Colombo, R., 190, 205 Columbus, J. T., 227, 266 Comai, L., 16, 43 Comella, P., 19, 24, 32, 37 Compagnone-Post, P. A., 25, 29, 37 Conconi, A., 13, 37 Condamine, P., 65, 78, 94 Cone´je´ro, G., 65, 78, 94 Conesa, A., 187, 191, 197, 198, 200, 203, 206, 221 Constantin, G. D., 109, 137 Continella, G., 149, 216 Cooke, R., 24, 29, 35 Cooney, R. V., 174, 208

AUTHOR INDEX Cooper, W., 168, 212 Coppey-Moisan, M., 17, 18, 35 Cori, O., 177, 205 Cosgrove, D. J., 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 72, 74, 75, 76, 78, 80, 81, 82, 86, 89, 90, 91, 92, 93, 94, 95, 96, 97 Costa, M. M., 106, 137, 262 Cotroneo, P. S., 182, 206 Coupland, G., 122, 141, 143, 155, 220 Courtial, B., 190, 209 Cowan, K., 165, 216 Cozzolino, S., 102, 108, 136, 137 Cramer, G. R., 85, 92 Crane, P. R., 101, 137 Cree, C. B., 184, 207 Crepet, W. L., 227, 261 Cresti, M., 72, 79, 80, 96, 97 Crisosto, C. H., 71, 94 Crock, J., 177, 206, 220 Cronn, R. C., 252, 266 Cronquist, 101, 137 Cross, F. R., 19, 36 Croteau, R., 128, 140, 176, 177, 204, 205, 206, 215, 220 Croteau, R. B., 177, 211, 213 Crowell, D. N., 54, 91 Cubas, P., 117, 137 Cultrera, N. G. M., 172, 208 Cummings, M. P., 248, 249, 251, 261 Cushman, J. C., 85, 92 Custer, L. J., 174, 208 D Dagenais, N., 122, 140 Dahal, P., 67, 71, 74, 79, 81, 89, 90 Dahan, E., 170, 218 Dahse, I., 75, 89 Dallwitz, M. J., 236, 267 Damasceno, C. M., 148, 157, 166, 183, 192, 203 Dammann, R., 13, 37 Dane, F., 185, 223 Daniell, H., 105, 137 Daras, M., 26, 40 Darley, C. P., 51, 54, 55, 56, 57, 58, 59, 63, 66, 69, 78, 91, 93, 96 Darwin, C., 101, 137, 138 Das, A. K., 157, 206 Das, M., 241, 242, 252, 258, 259, 261 Das, P., 240, 265 Daugherty, A., 174, 222 Davenport, T. L., 152, 153, 206, 220 Davies, J., 66, 91 Davies, K. M., 133, 138, 140 Davis, J. I., 245, 247, 251, 261 Dayton, D. C., 229, 266 De Angeli, A., 190, 206

De Block, M., 28, 32, 33, 45 De Ca´rcer, G., 6, 10, 22, 26, 37, 41 de Ca´ssia, R., 123, 144 de La Cruz, J., 28, 40 De la Torre, C., 6, 20, 37, 44 de Maagd, R. A., 178, 207 De Mey, J., 17, 18, 44 De Mey, J. R., 17, 18, 35 de Miguel, L. C., 72, 79, 89 de Sousa, I., 86, 87, 91 De Veylder, L., 17, 39 De, L. M., 240, 262 Dean, C., 122, 143, 154, 213 Debergh, P., 243, 262 DeBures, A., 19, 24, 32, 37 Del Rıó, J. A., 174, 182, 206 Delseny, M., 24, 29, 30, 32, 35, 40 Deltour, R., 6, 7, 11, 12, 37 Deluc, L. G., 85, 92 den Boer, B. G., 113, 139 Deng, X. X., 166, 221 Deng, Z. N., 149, 216 Dennis, E. S., 122, 123, 138, 143 dePamphilis, C., 51, 53, 54, 58, 59, 96 DePamphilis, C. W., 111, 112, 145 Depamphilis, M. L., 30, 39 Deragon, J. M., 29, 30, 32, 38 Derenzini, M., 13, 16, 18, 39 Derksen, J., 81, 94 Derrick, K. S., 57, 90 Devic, M., 29, 30, 32, 38, 40 Dez, C., 30, 37 Dhuique-Mayer, C., 149, 166, 208 Di Fazio, S., 153, 221 Di Rosa, A., 111, 112, 114, 142, 144 Diaz, J. J., 25, 26, 36 Diaz-Sala, C., 74, 92 Dick, M., 251, 267 Dıéz, J. L., 4, 37 Dimitrov, S., 25, 35 Dimitrova, D. D., 26, 40 Disch, A., 163, 213 Ditta, G., 120, 138 Ditta, G. S., 120, 122, 142, 143 Dixon, D., 109, 143 Dodson, C. H., 125, 126, 144 Doebley, J., 248, 262 Doi, K., 109, 140, 168, 216 Dolan, L., 66, 91 Dolezel, J., 103, 138 Dong, H., 88, 97 Donoghue, M. J., 250, 251, 260 Doolittle, W. F., 56, 89 Dopico, B., 66, 68, 78, 96 Dotto, M. C., 69, 85, 91 Douet, J., 26, 27, 28, 29, 32, 43 Douglas, C. J., 153, 212 Dove, B. K., 27, 36 Downes, B. P., 54, 91

273

274

AUTHOR INDEX

Doyle, A., 109, 143 Doyle, J. A., 101, 138 Dozier, W. A., 185, 223 Drachko, D. M., 49, 61, 62, 94 Dragon, F., 25, 29, 37 Drake, R., 168, 212 Dransfield, S., 235, 236, 238, 262 Drea, S., 109, 139 Drescher, A., 105, 138 Dressler, R. L., 102, 119, 138 Drews, G. N., 29, 30, 32, 43 Dubois, C., 149, 216 Dudareva, N., 127, 128, 130, 132, 138, 139, 142, 144 Dudzinski, D. M., 54, 59, 61, 62, 63, 97 Dugo, G., 182, 207 Dugo, P., 182, 207 Duke, E. R., 169, 221 Dumbar, T. S., 27, 40 Duncan, S. A., 19, 41 Dundr, M., 6, 17, 30, 37, 39 Dunsmuir, P., 69, 70, 84, 85, 86, 88, 90 Durachko, D. M., 49, 50, 51, 52, 54, 61, 62, 63, 79, 80, 91, 93, 94, 96 Durbin, M., 248, 262 Duret, S., 170, 218 Duvall, M. R., 248, 262 E Eaks, I. L., 161, 207, 215 Earley, K., 13, 14, 40 Ebel, R. C., 185, 223 Ebner von Eschenbach, C., 72, 79, 89 Echeverrıá, M., 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 32, 35, 36, 37, 40, 43, 44 Ecker, J., 109, 142 Egea-Cortines, M., 112, 138 Ehtesham, N. Z., 26, 42 Eikelboom, W., 114, 118, 144 Eilati, S. K, 162, 163, 207 Einset, J. W., 194, 207 Einspahr, J. G., 173, 207 El Tamer, M. K., 177, 178, 207, 214 El-Otmani, M., 194, 203 Elliott, K. A., 84, 95 Ellis, R. P., 232, 235, 236, 238, 266 Elmaci, C., 187, 218 Elston, A., 177, 207 Elzenga, T. M., 63, 66, 69, 78, 96 Embleton, T. W., 184, 207 Emerson, O. H., 179, 207 Endo, T., 154, 155, 161, 162, 168, 176, 178, 179, 181, 207, 208, 210, 211, 216, 219 Endo-Inagaki, T, 162, 181, 212, 215 Enju, A., 109, 143 Enomoto, K.-I., 26, 27, 28, 29, 32, 40

Ephritikhine, G., 190, 206 Epstein, E., 190, 207 Erard, M., 26, 38 Ernst, H. A., 168, 216 Eshdat, Y., 191, 204 Esponda, P., 8, 9, 11, 34, 37 Etxeberria, E., 169, 170, 207, 210 Everaert, I., 240, 263 Eyal, Y., 162, 177, 208, 211, 219 F Facchini, P., 177, 207 Fagoaga, C., 159, 200, 207 Fakan, S., 6, 36, 38 Faleri, C., 72, 79, 97 Faltin, Z., 191, 204 Famiani, F., 172, 208 Fan, L. J., 229, 263 Fanciullino, A. L., 149, 166, 208 Farrell, A., 168, 212 Fasolino, A., 81, 94 Faucher, C., 26, 44 Fecht-Bartenbach, J. V. D., 190, 208 Feldman, G. D., 227, 261 Felle, H. H., 66, 95, 190, 208 Feng, T. Y., 111, 145 Fenton, J. I., 174, 208 Fernańdez-Ballester, G., 190, 208 Fernandes, C. J., 19, 40 Fernańdez-Go´mez, M. E., 6, 7, 8, 9, 10, 11, 13, 16, 17, 19, 20, 23, 41, 42 Feron, R., 72, 81, 94, 95 Ferrante, A., 70, 85, 91 Ferranti, F., 73, 82, 88, 97 Ferrarini, A., 73, 82, 88, 97 Ferrario, S., 112, 138 Ferrin, T. E., 177, 223 Fidlerova, H., 6, 18, 43 Figueiredo-Ribeiro, L., 123, 144 Figueras, M., 22, 43 Filgueiras, T. S., 227, 229, 239, 262 Fils-Lycaon, B., 70, 85, 86, 94 Finnegan, E. J., 122, 143 Fischer, W. N., 190, 210 Flavell, R. B., 10, 14, 38, 45 Fleming, A., 51, 54, 55, 57, 58, 59, 77, 93, 95, 96 Fleming, A. J., 50, 56, 57, 64, 65, 88, 91, 93, 95 Fleurdepine, S., 29, 30, 32, 38 Fluhr, R., 182, 204, 208 Fon, M., 170, 172, 202, 212 Fong, C. H., 180, 208 Fonseca, S., 70, 71, 91 Forment, J., 167, 168, 169, 170, 171, 172, 191, 197, 198, 200, 205, 208 Fougue, A., 152, 205 Fouraux, M. A., 26, 38

AUTHOR INDEX Fox, G. G., 170, 205 Fox, S., 106, 137 Frachisse, J. M., 190, 206, 209 Francisco-Ortega, J., 248, 263 Francke, W., 125, 141, 143 Franke, A. A., 174, 208 Franke, S., 125, 143 Franke-van Dijk, M., 30, 31, 33, 46 Frederiksen, S., 113, 119, 143 Friar, E., 240, 262 Friis, E. M., 101, 137 Frijters, A., 242, 267 Frommer, W. B., 190, 210 Fromont-Racine, M., 28, 38 Fry, S. C., 49, 61, 62, 94 Frydman, A., 177, 208, 219 Fu, Y. M., 103, 104, 106, 108, 137, 141 Fujii, H., 85, 92, 154, 155, 161, 162, 168, 176, 178, 207, 208, 211, 216, 219 Fujinaga, M., 185, 186, 192, 210 Fujioka, T., 72, 81, 97 Fujisawa, H., 168, 203 Fujita, M., 168, 209 Fujita, Y., 168, 209 Fuk-Ling, W., 190, 222 Fukaki, H., 168, 203 Fukaya, T., 185, 186, 192, 210 Fukuda, T., 119, 143 Furon, V., 152, 205 Furusawa, M., 175, 209 Fuster, M. D., 174, 216 G Gadea, J., 167, 168, 169, 170, 171, 172, 191, 197, 198, 200, 205, 208 Gaillard, J. P., 152, 205 Galizia, C. G., 102, 138 Gallagher, J. E., 25, 29, 37, 38 Gallego, F., 165, 213 Gamba, G., 191, 206 Gambale, F., 190, 206 Gamble, J. S., 232, 262 Gan, L. H., 242, 264 Gan, Y. Y., 242, 264 Ganapathy, P. M., 258, 262 Gang, D. R., 125, 128, 138, 142 Gao, T., 88, 97 Garcıá-Sańchez, F., 190, 208 Garcia-Hernandez, M., 109, 143 Garcia-Fernandez, L. F., 6, 9, 11, 26, 37 Garcıá-Go´mez, J. M., 198, 206 Garcia-Herdugo, G., 22, 43 Garcia-Lidoń, A., 174, 216 Garcia-Luis, A, 152, 162, 163, 209 Garcia-Martinez, J. L., 159, 200, 222 Garcia-Puig, D., 174, 216 Gardner, R. C., 71, 82, 89 Gargiulli, C., 181, 209

275

Gas, N., 6, 9, 11, 16, 26, 35, 38, 41 Gaspin, C., 23, 32, 35 Gattuso, G., 181, 209 Gaudio, L., 123, 142 Gaur, R. C., 241, 261 Gaut, B. S., 248, 262 Gautier, T., 13, 16, 18, 38, 39 Gebrane-Younes, J., 17, 18, 44 Geelen, D., 190, 209, 214 Gehring, C., 50, 56, 57, 93, 94 Gehring, C. A., 57, 93 Geiges, B., 30, 32, 45 Gendall, A. R., 122, 143 Gendra, E., 24, 38 Gentile, A., 149, 216 Gepstein, S., 191, 204 Gerats, T., 244, 264 Ge´raud, G., 20, 40 Gershenzon, J., 127, 130, 132, 137, 138, 144, 173, 216 Gewin, V., 106, 138 Ghabrial, S. A., 109, 201 GhaVari, S. H., 27, 40 Ghisolfi, L., 26, 38, 44 Ghorai, A., 229, 262 Gielis, J., 229, 240, 243, 244, 262, 264 Gielly, L., 249, 263 Gilbert, N., 6, 38 Gill, B. S., 109, 138 Gill, D. E., 101, 102, 138 Gilliham, M., 190, 209 Gillis, K., 243, 262 Gilroy, S., 75, 89 Ginisty, H., 25, 26, 38 Giovannoni, J., 83, 84, 85, 89, 91, 161, 163, 168, 203, 209 Gisbert, C., 159, 222 Giuliano, G., 165, 209 Glockner, F., 229, 267 Gmitter, F. G., 148, 153, 192, 200, 207, 218, 220 Goday, A., 22, 43 Godward, M. B. E., 11, 38 Goessens, G., 19, 45 Goh, C. J., 102, 113, 120, 123, 124, 134, 141, 145 Gold, S. E., 122, 143 Goldschmidt, E. E., 152, 153, 157, 159, 160, 161, 162, 183, 209, 211, 212, 213, 220, 221 Golenberg, E. M., 248, 262 Gomez, R.-M., 70, 85, 86, 93 Go´mez-Cadenas, A., 157, 160, 187, 190, 191, 192, 195, 209, 214, 215, 216, 221 Gon˜i, C., 160, 220 Goncalves, A. P. S., 227, 262 Gonzalez, P., 169, 207 Gonza´lez-Camacho, F., 7, 20, 27, 38

276

AUTHOR INDEX

Gonzalez-Carranza, Z. H., 84, 95 Gonza´lez-Melendi, P., 10, 38 Goodner, K. L., 166, 209 Gookin, T. E., 73, 82, 91 Gorab, E., 10, 11, 45 Goren, R., 160, 161, 209, 210, 211, 217, 218, 222, 223 Gosalbes, M. J., 186, 187, 219 Goto, A., 159, 193, 210 Goto, K., 112, 120, 139, 140 Gotz, S., 191, 197, 198, 200, 206, 221 Gou, J. Y., 67, 74, 93 Gouble, B., 70, 85, 86, 93 Goulao, L. F., 86, 87, 91 Goverse, A., 56, 95 Granell, A., 186, 187, 192, 219 Grant, A., 194, 210 Gravendeel, B., 101, 142 Gravina, A., 160, 217 Gray, J. E., 172, 214 Gray-Mitsumune, M., 69, 78, 91 Green, P. J., 23, 36 Greenberg, J. R., 197, 210 Greenway, H., 187, 210 Greenwood, M. S., 74, 92 Gressel, J., 182, 204, 208 Grierson, C., 75, 92 Grierson, D., 168, 212 Griesbach, R. J., 133, 138 GriYth, M. E., 25, 29, 30, 32, 38 Grigoriev, I., 153, 221 Grimplet, J., 85, 92 Grobe, K., 62, 92 Gross, J., 163, 165, 210 Gross, K. C., 71, 92 Gruissem, W., 30, 36 Grummt, I., 17, 18, 19, 22, 23, 38, 39, 40, 44, 46 Guala, G., 248, 263 Guala, G. F., 249, 263 Guardiola, J. L, 152, 153, 162, 163, 209, 210, 216 Guern, J., 190, 214 Gueta-Dahan, Y., 191, 204 Gui, Y. J., 229, 263 Guihua, S., 190, 222 Guilfoyle, T. J., 21, 38, 39 Guilleminot, J., 29, 30, 32, 38, 40 Guiltinan, M. J., 50, 96 Guitton, Y., 24, 35 Gumbert, A., 102, 138 Guo, B., 117, 118, 138 Guo, Z. H., 250, 263 Gurrieri, S., 69, 84, 88, 95 Gustafson-Brown, C., 124, 138 Guterman, I., 128, 132, 138, 139 Guy, C. L., 185, 186, 187, 189, 192, 205, 219, 221 Guyot, R., 23, 29, 32, 35

H Ha, D. T., 26, 27, 36 Hackett, R, 168, 212 Hakola, S., 57, 95 Halaly, V., 66, 94 Halkidou, K., 22, 39 Hamby, R. K., 248, 263 Hames, B., 229, 266 Hamiche, A., 25, 35 Han, F. S., 6, 16, 43 Han, K. H., 110, 145 Han, S. H., 174, 206 Han, Y. Y., 133, 139 Haneji, T., 27, 42 Hanna, A. I., 106, 137 Hannan, R. D., 18, 22, 39 Hans, F., 25, 35 Hansen, T. F., 229, 267 Hanson, L., 104, 137 Haque, J., 19, 41 Hara, A., 174, 219 Hara, M., 178, 185, 186, 192, 210, 219 Hara, R., 133, 144 Haralampidis, K., 19, 24, 32, 46 Haring, M. A., 130, 144 Harley, E. H., 248, 260 Harpster, M. H., 69, 70, 84, 85, 86, 88, 90, 92 Harrison, E. P., 70, 92 Harrison, M. J., 122, 140 Hartmond, U., 193, 194, 196, 210, 223 Hasegawa, J., 174, 213 Hasegawa, S., 179, 180, 181, 207, 210, 215 Hasenstein, K. H., 75, 97 Hashimoto, T., 175, 209 Haughton, P. M., 49, 66, 95 Havelange, A., 154, 204 Hayama, H., 71, 85, 86, 92 Hayashi, I., 243, 260, 267 Hayashizaki, Y., 109, 143, 168, 216 Heazlewood, J. L., 57, 93 Heberle-Bors, E., 26, 27, 36, 72, 81, 92 Hechenberger, M., 190, 210 Heckman, J. W., 123, 145 Hedden, P., 157, 221 Heix, J., 17, 19, 39 Helder, J., 56, 93, 95 Helliwell, C. A., 122, 143 Hellsten, U., 153, 221 Hempel, W. M., 18, 22, 39 Henning, S. M., 174, 208 Heo, Y.-K., 75, 76, 93 Hernandez-Verdun, D., 6, 13, 16, 17, 18, 19, 20, 35, 36, 38, 39, 40, 42, 43, 44, 45 Herrero, R., 149, 210 Hibara, K., 168, 220 Hidaka, T., 179, 181, 207 Hieber, A. D., 132, 134, 139, 142

AUTHOR INDEX Hield, H. Z., 159, 206 Higuchi, S., 72, 81, 97 Hilbers, C. W., 81, 94 Hileman, L. C., 109, 139 Hilgeman, R. H, 185, 210 Hill, T. A., 154, 213 Hilu, K. W., 247, 249, 263 Hilu, W., 248, 264 Hirano, H., 114, 142 Hirose, K., 159, 193, 210 Hirose, Y., 174, 219 Hirschberg, J., 163, 210 Hirschmann, P., 19, 22, 46 Hirt, H., 26, 27, 36 Hisada, S, 162, 181, 210, 212, 215 Hiscox, J. A., 27, 36 Hiwasa, K., 70, 71, 87, 92 Ho¨gnado´ttir, A., 175, 177, 210 Hoagland, J. E., 180, 210 Hockema, B., 192, 194, 196, 197, 202, 203, 205, 210 Hockema, B. R., 169, 170, 210 Hodkinson, T. R., 241, 250, 263 HoVmann-Benning, S., 51, 59, 68, 75, 82, 91 Hofsommer, H. J., 166, 209 Hogers, R., 242, 267 Holland, D., 162, 177, 211, 219, 223 Holttum, R. E., 233, 235, 263 Hon-Ming, L., 190, 222 Honda, G., 177, 214 Honda, T., 133, 144 Honma, T., 112, 120, 139 Hooykaas, P., 30, 31, 33, 46 Hord, N. G., 174, 208 Horikawa, Y., 119, 143 Hornes, M., 242, 267 Ho¨rtensteiner, S., 163, 211 Hotchkiss, M. W., 244, 258, 260 Hotta, I., 109, 140 Houssa, C., 154, 204 Howe, G., 154, 218 Hsiao, C., 250, 251, 263 Hsiao, H. H., 111, 141, 145 Hsiao, J. Y., 242, 243, 260, 263, 264 Hsiao, Y. Y., 106, 108, 109, 112, 114, 117, 118, 126, 127, 128, 129, 130, 131, 132, 139, 144 Hsieh, C. H., 103, 104, 106, 140 Hsieh, H. L., 26, 27, 45 Hsiung, W. Y., 232, 261 Hsu, B. D., 134, 143 Hsu, C. C, 230, 263 Hsu, H. F., 113, 114, 117, 123, 134, 139 Hsu, Y. H., 110, 137 Hu, C. C., 110, 137 Hu, W. L., 177, 179, 206 Hu, Y., 119, 140 Huala, E., 109, 143

277

Huang, C. H., 104, 106, 108, 113, 123, 134, 139, 140 Huang, H. E., 111, 145 Huang, J., 63, 71, 78, 81, 92 Huang, L., 109, 143 Huang, T., 155, 204 Huber, C., 26, 40 Huberman, M., 196, 197, 209, 211 Huddleson, J. P., 26, 27, 28, 39 Huerta, L., 159, 191, 197, 198, 200, 207, 208 Huisman, B. A., 81, 94 Hunt, D. F., 25, 29, 37 Hunt, T., 17, 37 Hunter, D. A., 66, 70, 73, 82, 85, 91, 94 Hutchison, K. W., 74, 92 Huttenhofer, A., 23, 36 Huttley, G. A., 244, 263 Hwang, J. H., 174, 206 Hyatt, D. C., 176, 211 Hyodo, H., 163, 164, 165, 166, 212 I Ibań˜ez, V., 172, 203, 211 Ibarra, F., 125, 143 Ichikawa, H., 119, 143 Idaomar, M., 173, 204 Iglesias, D. J., 148, 157, 159, 160, 162, 166, 167, 172, 183, 191, 192, 195, 197, 198, 200, 203, 205, 206, 207, 211, 215, 221 Ikemoto, K., 174, 219 Ikoma, Y., 155, 161, 162, 163, 164, 165, 166, 192, 208, 212, 215, 216 Im, K.-H., 69, 78, 92 Imin, N., 55, 80, 93 Immink, R. G. H., 112, 138 Inaba, A., 69, 70, 71, 87, 92, 94 Innocente, S. A., 19, 39 Intine, R. V., 30, 39 Inze´, D., 17, 39 Irish, V. F., 109, 113, 114, 139, 141, 143, 154, 213 Irving, H. R., 57, 93 Isagi, Y., 243, 263 Ishida, J., 109, 143 Ishida, T., 168, 203, 220 Ishiguro, S., 26, 27, 28, 29, 32, 40 Ishikawa, M., 109, 140 Ishikawa, S., 176, 178, 211 Ishikawa,T., 243, 263 Ishimaru, M., 71, 92 Ishizawa, K., 78, 94 Ito, A., 71, 85, 86, 92 Ito, M., 177, 214 Ito, T., 28, 33, 39, 119, 143 Iwabuchi, M., 122, 140 Izquierdo, P., 187, 206

278

AUTHOR INDEX

J Jaaskelainen, M. J., 244, 267 Jabalquinto, A. M., 177, 205 Jacob, T., 75, 89 Jacob-Wilk, D, 162, 211 Jacobs, S. W. L., 250, 251, 263 Jacquemond, C., 149, 216 Jahn, O, 162, 223 Jaillon, O., 153, 211 Janson, J. C., 57, 97 Jansson, S., 153, 155, 204, 212, 221 Janzen, D. H., 233, 264 Jasper, F., 75, 89 Jayaprakasha,G.K., 174, 214 Jendrisak, J. J., 21, 38 Jeng, M. F., 112, 139, 144 Jentsch, T. J., 190, 210 Jeon, J. S., 114, 141 Jeong, D. H., 67, 74, 96 Jia, Z. J., 173, 222 Jiang, H., 167, 212 Jiang, L., 29, 33, 39 Jiang, L. F., 244, 264 Jiang, W., 70, 85, 97 Jiang, Y., 70, 85, 97 Jie, Y. G., 244, 264 Jin, L., 229, 263 Jin, Y., 72, 81, 92 Jinnno, K., 174, 216 Jobet, E., 19, 24, 32, 37 Jofuku, K. D., 113, 139 Johansen, B. B., 113, 119, 143 Johansen, I. E., 109, 137 Johansen, L. B., 113, 119, 143 John, I., 168, 212 Johnson, E. T., 134, 140 Johnson, S. D., 102, 136, 140 Jonak, C., 26, 27, 36 Jones, A. M., 69, 78, 92 Jones, J. T., 56, 95 Jones, L., 80, 82, 93 Jones, W. W., 184, 207 Joosen, R., 30, 32, 41 Jordan, E. G., 4, 5, 6, 19, 39, 40, 44 Joseph, G., 26, 38, 44 Joyce, D., 70, 85, 97 Jua´rez, J., 154, 216 Juan, M., 193, 194, 203 Jubin, C., 153, 211 Judziewicz, E. J, 227, 233, 239, 249, 261, 264 Jun, H. Z., 244, 264 June´ra, H. R., 20, 40, 42 Junttila, O., 154, 218 Jurgens, G., 25, 29, 30, 32, 38, 45 K Kader, A. A., 186, 212 Kaiser, P., 27, 36

Kaiser, R., 125, 126, 140 Kako, S., 108, 142 Kalamaki, M. S., 69, 84, 88, 92, 95 Kalendar, R., 244, 267 Kamemoto, H., 133, 144 Kameya, T., 119, 143 Kamiya, A., 109, 143 Kanehisa, M., 51, 52, 94 Kanes, K., 174, 212 Kang, C., 176, 211 Kang, S. H., 174, 206 Kanno, A., 112, 114, 119, 143, 144 Kantety, R. V., 244, 268 Kao, Y. Y., 103, 104, 106, 108, 140, 141 Kapolas, G., 19, 24, 32, 46 Kapos, V., 227, 259, 261 Kardailsky, I., 122, 140 Kasche, A., 72, 79, 89 Kashimura, Y., 71, 85, 86, 92 Kastan, M. B., 26, 45 Kastenmayer, J. P., 23, 36 Katayama, M., 174, 219 Katholnigg, H., 72, 81, 92 Kato, M., 163, 164, 165, 166, 192, 212, 215 Kato, T., 26, 27, 28, 29, 32, 40 Katoch, B. S., 229, 264 Katoh, S., 177, 205 Katz, E., 161, 170, 172, 202, 212 Kaufmann, K., 120, 140 Kawabata, K., 174, 212 Kawagashira, N., 109, 140 Kawai, J., 168, 216 Kaya, H., 122, 140 Keasling, J. D., 177, 223 Kelchner, S. A., 249, 264 Keller, E., 63, 64, 65, 92 Kellog, E. A., 227, 258, 265 Kellogg, E. A., 229, 236, 248, 249, 251, 252, 261, 264, 265 Kende, H., 50, 51, 54, 55, 56, 57, 58, 59, 63, 65, 66, 67, 68, 74, 77, 78, 90, 93 Kender, W. J., 193, 194, 196, 210, 223 Keng, P. C., 233, 234, 235, 264 Kerbauy, G. B., 123, 144 Kerim, T., 55, 80, 93 Keukeleire, P. D., 244, 264 Khan, M. S., 105, 137 Kharrat, A., 26, 38 Khatta, V., 229, 264 Kielak, A., 56, 93 Kiew, R., 242, 264 Kikuchi, S, 109, 140, 145, 162, 168, 219 Kim, D. W., 75, 76, 93 Kim, G. T., 28, 33, 39 Kim, J. H., 58, 93, 244, 264 Kim, J. T., 111, 112, 114, 142, 144 Kim, K., 26, 40 Kim, N. H., 244, 265 Kim, N. S., 244, 264, 265

AUTHOR INDEX Kim, S., 119, 140 Kim, S. G., 122, 141 Kim, S. J., 174, 206 Kim, S. K., 27, 40 Kindbeiter, K., 25, 26, 36 King, L. M., 248, 249, 251, 261 Kish, C. M., 130, 144 Kishimoto, N, 109, 140, 145, 162, 168, 219 Kita, M., 162, 178, 179, 181, 207, 212, 215, 219 Kitsios, G., 19, 24, 32, 46 Kittilsen, M. O., 229, 267 Klein, C., 6, 38 Klein, J., 18, 40 Knowles, A. E., 190, 204 Knox, R. B., 72, 79, 96 Knudsen, J. T., 125, 140 Ko, H. C., 174, 206 Ko, S. Y., 174, 206 Kobayashi, S., 71, 92 Kobayashi, Y., 122, 140, 154, 155, 207 Koberna, K., 6, 18, 43 Koch, K. E., 157, 160, 169, 209, 212, 214, 216, 221 Kochert, G., 240, 262 Koh, J., 119, 140 Kohler, B., 190, 212 Kojima, H., 26, 27, 28, 29, 32, 40 Kojima, K., 109, 140, 145 Koller, T., 13, 37 Komatsu, A., 169, 212 Komeda, Y., 153, 212 Kong, H., 119, 140 Koningsbruggen, S., 3, 36 Koornneef, M., 50, 94 Koshimizu, K., 174, 219 Kossuth, J., 227, 258, 265 Kostenyuk, I., 194, 217 Kostenyuk, I. A., 194, 196, 223 Koyama, K., 169, 212 Koyanagi, S., 72, 81, 97 Kramer, E. M., 113, 114, 139 Krath, B. N., 109, 137 Krebs, M., 190, 208 Krens, F. A., 109, 136 Kressler, D., 28, 40 Krezdorn, A. H., 160, 217 Kriedemann, P. E., 187, 213 Kronzucker, H. J., 190, 205 Kubelik, A. R., 240, 268 Kubo, Y., 69, 70, 71, 87, 92, 94 Kuboi, T., 185, 186, 192, 210 Kudla, U., 56, 93, 95 Kuehnle, A. R., 110, 132, 134, 139, 140, 142 Kuhlemeier, C., 64, 73, 77, 82, 90, 91, 95 Kuhn, A., 19, 22, 46 Kuiper, M., 242, 267 Kumar, P. P., 113, 114, 117, 118, 119, 120, 145

279

Kumazawa, S., 178, 220 Kuniga, T., 166, 176, 178, 211, 215 Kuno, T., 174, 219 Kunze, J., 102, 138 Kuo, H. C., 106, 109, 126, 128, 129, 130, 131, 139 Kuo, J., 108, 137 Kuo, Y. T., 106, 140 Kuoh, C. S., 106, 108, 109, 111, 114, 117, 118, 126, 128, 129, 130, 131, 139, 141, 144 Kurien, P. A., 69, 84, 88, 95 Kurowska, E. M., 174, 222 Kushima, H., 243, 263 Kuttner, F., 30, 32, 45 Kwack, B. H., 108, 137 Kwasniewski, M., 68, 76, 93 Kwiatkowska. M., 18, 40 Kwon, S. D., 229, 265 Kwon, S. J., 244, 264 Kwon, Y. M., 122, 141 L La Malfa, S., 149, 216 Labanauskas, C. K., 184, 207 Labavitch, J. M., 69, 83, 88, 92, 96 Labrador, E., 66, 68, 78, 96 Lacadena, J. R., 13, 40 Lack, A., 102, 142 Lafuente, M. T., 186, 187, 192, 219, 223 Lagunes, P. M., 161, 212 Lahey, K. A., 194, 195, 212 Lahmy, S., 19, 24, 25, 29, 30, 32, 36, 37, 40 Lai, C. C., 242, 243, 260, 264 Lai, Y. H., 259, 264 Lam, L. K. T., 174, 213 Lam, Y. W., 3, 21, 28, 29, 35, 41, 42 Lamb, J. C., 72, 80, 81, 96 Lamb, R. S., 154, 213 Lamond, A. I., 3, 21, 28, 29, 35, 36, 41, 42 Lander, G., 109, 143 Lanfaloni, L., 73, 82, 88, 97 Lang, P., 185, 223 Lange, B. M., 128, 140 Lapeyre, B., 27, 40 Lapik, Y. R., 19, 40 Lau, L. F., 19, 40 Laurie, S., 170, 205 Lavi, U., 177, 219 Lawrence, R. J., 13, 14, 40 Le, N. T., 49, 50, 80, 94 Lea, P. J., 172, 213 LeBourdelles, J., 152, 205 Lecharny, A., 128, 136 Lee, C. Y., 106, 140 Lee, D.-K., 67, 74, 93 Lee, H., 122, 141 Lee, H. C., 103, 104, 106, 141

280 Lee, Lee, Lee, Lee, Lee, Lee, Lee, Lee, Lee, Lee, Lee,

AUTHOR INDEX

H. H., 69, 84, 95 H. J., 110, 145 I., 122, 141 J. H., 244, 265 J. K., 244, 264 J. M., 19, 39 J. S., 67, 74, 93, 122, 141 P. F., 106, 108, 114, 117, 118, 144 R. B., 190, 213 S., 114, 141, 216, 250, 268 S. H., 75, 76, 93, 106, 109, 126, 128, 129, 130, 131, 139, 144 Lee, S. M., 110, 111, 136 Lee, Y., 50, 51, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 65, 66, 67, 68, 74, 77, 78, 90, 93, 96 Lee, Y. H., 108, 137 Lee, Y. J., 170, 172, 202, 212 Leebens-Mack, J., 111, 112, 145 Leegood, R. C., 172, 208, 213, 214 Lefebvre, S., 26, 40 Legaz, F., 148, 157, 160, 162, 183, 187, 192, 200, 211, 218 Legeai, F., 153, 211 Legrand, D., 27, 36 Leitch, I. J., 104, 136 Leitinger, N., 27, 41 Lejeune, B., 248, 251, 265 Lejeune, P., 154, 204 Lelievre, F., 190, 209 Leonardi, T., 174, 222 Lepiniec, L., 30, 32, 45 Lerch-Gaggl, A., 19, 41 Lermontova, I., 23, 32, 41 Lerner, D. R., 114, 136 Leu, Y. L., 106, 109, 126, 128, 129, 130, 131, 139 Leung, A. K., 21, 41 Leung, A. K. L., 3, 35 Leuzzi, U., 181, 209 Levy, Y., 154, 213 Lewandowska, D., 3, 21, 28, 29, 42 Lewandowski, D. J., 192, 194, 196, 197, 202, 205, 216 Lewinsohn, E., 128, 138, 144, 208 Lewis, L.N., 194, 213 Lewis, M. S., 15, 41 Lewisohn, E., 177, 219 Li, C. Y., 139, 153, 213 Li, D. Z., 238, 250, 263, 264 Li, J., 19, 41, 216 Li, J. X., 177, 179, 206 Li, L. C., 51, 54, 59, 61, 62, 63, 68, 72, 75, 80, 82, 91, 93, 96, 97 Li, M., 167, 212 Li, S. H., 111, 141 Li, W., 194, 213 Li, Y., 51, 54, 55, 56, 57, 58, 59, 80, 82, 91, 93, 110, 174, 213

Li, Y. H., 104, 106, 108, 140 Li, Z.-C., 50, 63, 76, 91, 93 Liang, H., 247, 248, 249, 263, 264 Liang, J., 244, 264 Liao, S. C., 105, 145 Liau, C. H., 111, 141, 145 Lichtenthaler, H. K., 163, 213 Lichtner, M., 174, 204 Liew, C. F., 134, 141 Lim, S. H., 108, 134, 137, 141 Lin, C. C., 104, 106, 108, 140 Lin, C. S., 259, 264 Lin, C. Y., 39, 105, 110, 137 Lin, H. C., 105, 110, 137 Lin, I. P., 105, 110, 137 Lin, J., 175, 177, 207, 213 Lin, J. J., 108, 137 Lin, N. S., 110, 137 Lin, R., 250, 267 Lin, S., 103, 104, 106, 141 Lin, T. Y., 103, 104, 106, 140, 141 Lin, Y. S., 108, 137 Lin, Z., 71, 82, 93 Linder, C. R., 108, 142 Linder, H. P., 140, 248, 260 Linder, P., 28, 40 Lingrel, J. B., 26, 27, 28, 39 Litt, A., 109, 113, 139, 141 Litt, M., 243, 264 Little, B. D., 177, 213 Liu Cm, C. M., 30, 32, 41 Liu, J., 25, 29, 32, 44, 167, 212 Liu, N. T., 259, 264 Liu, R., 88, 97, 110 Liu, S. M., 105, 110, 137 Livak, K. J., 240, 268 Lliso, I., 148, 157, 159, 160, 162, 172, 183, 192, 200, 202, 203, 207, 211, 213 Lloyd, A., 29, 30, 32, 43 Lloyd, J., 187, 213 Lluch, Y., 186, 187, 219 Lo Leggio, L., 168, 216 Lo Piero, A. R., 182, 183, 206, 213 Lo´pez-Diaz, I., 159, 222 Locy, R. D., 185, 223 Loh, C. S., 120, 134, 141 Loh, J. P., 242, 264 Lois, L. M., 165, 213 Loizzo, M. R., 173, 213 Londono, X., 227, 233, 239, 243, 260, 264, 265 Long, J., 88, 97 Long, S. B., 229, 263 Lopez, C., 22, 43 Loppes, R., 6, 11, 42 Lorenzen, I., 190, 214 Loreto, F., 132, 142 Lou, Y., 67, 74, 93

AUTHOR INDEX Lou, Y. G., 177, 179, 206 Louvet, E., 17, 18, 35 Lovatt, C. J, 156, 216, 217 Lowe, T. M., 23, 36 Lowell, C. A., 169, 214 Lu, H. C., 110, 141 Lu, J. C., 111, 141, 145 Lu, W., 70, 85, 97 Lu, Z. X., 120, 141 Lu¨cker, J., 177, 178, 207, 214 Lucas, L., 6, 38 Lucchini, R., 13, 37 Ludewig, U., 190, 208 Ludidi, N. N., 57, 93 Lukacin, R., 182, 214 Lund, O. S., 109, 137 Luo, Y., 70, 85, 97 Lupton, J. R., 174, 222 Lurie, S., 185, 186, 187, 217, 218 Lurin, C., 190, 209, 214 Luro, F., 149, 166, 208, 216 Lutty, J. A., 243, 264 Lyon, C. E., 3, 35 Lyon, J., 194, 207 Lysenko, I., 227, 259, 261 M Ma, D. P., 248, 262 Ma, H., 111, 112, 119, 140, 145, 154, 214 Ma, N. X., 229, 263 Maas, E. V., 187, 214 Maccarone, A., 182, 214 Maccarone, E., 182, 214 MacFarlane, S. A., 109, 137 Macleod, W. D., 175, 214 Madjar, J. J., 25, 26, 36 Magoulas, C., 19, 43 Maissen, C., 56, 93 Majumdar, R., 235, 265 MakaroV, C. A., 29, 33, 39 Malcolm, S., 21, 39 Malcomber, S. T., 227, 258, 265 Maliga, P., 105, 136 Malinsky, J., 6, 18, 43 Malone, S., 172, 214 Mandadi, K. K., 174, 214 Mandel, T., 64, 73, 77, 82, 90, 91, 95 Mann, M., 3, 21, 28, 29, 35, 41, 42 Manning, K., 70, 92 Mansel, R. L., 179, 215 Mant, J., 125, 141 Mant, J. G., 125, 143 Manthey, J. A., 174, 222 Maraia, R. J., 30, 39 Marcos, J. F., 163, 164, 165, 217 Mariani, C., 72, 81, 94, 95 Markhart, A. H., 104, 141

281

Markl, C., 102, 138 Marquez, P., 243, 260, 265 Marsh, K. B, 170, 218 Marshall, C. R., 101, 142 Marshall, D. V., 21, 23, 36 Martens, S, 182, 214 Martıńez, V., 190, 208 Martin, C., 134, 141 Martıń, M., 6, 9, 11, 26, 41 Martin, S. F., 182, 218 Martin, W., 111, 142 Martıń-Trillo, M., 154, 216 Martinez, G., 191, 206 Martıńez, G. A., 69, 85, 91 Martinez-Fuentes, A., 193, 203 Martıńez-Zapater, J. M., 154, 216 Martino, G., 109, 139 Marulanda, M. L., 243, 260, 265 Maruyama, K., 168, 209 Masata, M., 6, 18, 43 Masci, T., 128, 132, 139 Masson, C, 20, 38, 40, 42 Masson, M., 27, 36 Massonneau, A., 65, 78, 94 Mastroianni, C. M., 174, 204 Masuda, M., 174, 216 Maszewski, J., 18, 40 Mateos, I., 66, 68, 78, 96 Matern, U., 182, 214 Mathews, S., 251, 265 Mathur, J., 75, 99 Matıá, I., 26, 27, 28, 29, 32, 43 Matsubara, K., 168, 216 Matsui, S., 133, 141 Matsumoto, H., 163, 164, 165, 166, 192, 212, 215 Matsumoto, R., 179, 181, 207 Matsuo, A., 72, 81, 97 Maul, P., 186, 187, 219 Maulbetsch, C., 30, 32, 45 Maurel, C., 190, 209, 214 Mayer, U., 25, 29, 30, 32, 38, 45 Mazurier, J., 27, 36 Mazzanti, G., 174, 204 Mbe´gui-A-Mbe´guie´, D., 70, 85, 86, 93 McCann, M., 19, 24, 32, 46 McCaskill, D., 128, 140 McClinton, R., 25, 29, 32, 38 McClure, F. A., 230, 231, 232, 233, 265 McCollum, G. T., 186, 187, 219 McCollum, T. G., 185, 186, 187, 217 McDaniel, C. M., 154, 215 McElver, J., 30, 32, 41 McGlasson, W. B., 161, 215 McGovern, J. H., 5, 40 McInnis, S., 74, 92 McIntosh, C. A., 179, 215 McLure, K. G., 26, 45 McManus, M. T., 84, 95

282

AUTHOR INDEX

McMurchie, E. J., 161, 215 McQueen-Mason, S., 49, 50, 51, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 68, 69, 70, 77, 78, 80, 82, 84, 88, 89, 90, 91, 94, 95 McRae, A. F., 244, 263 McStay, B., 16, 43 Me´le´se, T., 2, 42 Medina, F. J., 5, 6, 7, 8, 9, 10, 11, 13, 16, 17, 18, 19, 20, 22, 23, 26, 27, 36, 37, 38, 41, 42, 43 Meeley, R. B., 54, 55, 67, 74, 75, 97 Mehouachi, J., 157, 160, 169, 191, 192, 195, 209, 215, 221 Meinke, D., 30, 32, 41, 50, 94 Meir, S., 66, 94 Mella, R., 72, 79, 94 Mellerowicz, E. J., 69, 78, 91 Melzer, R., 120, 140 Men, S., 110, 141 Menager, M., 6, 11, 38, 42 Mengoni, F., 174, 204 Menichini, F., 173, 213 Menoni, H., 25, 35 Menzel, R., 102, 138 Meredith, F., 157, 223 Mesero, C., 193, 203 Meskiene, I., 26, 27, 36 Meyer-Gauen, G., 176, 204 Meyerowitz, E., 154, 206 Meyerowitz, E. M., 111, 144, 154, 168, 214, 218, 222 Mıńguez-Mosquera, M. I., 148, 157, 166, 183, 192, 203 Michaelidis, T. M., 17, 19, 39 Michaux-Ferriere, N., 24, 35 Michelmore, R. W., 242, 265 Mietton, F., 25, 35 Mii, M., 111, 136, 142 Milac, A., 56, 93 Milanesi, C., 72, 79, 80, 96, 97 Milioni, D., 19, 24, 32, 46 Miller, N., 109, 143 Ming, F., 117, 118, 133, 138, 139 Ming, X., 110, 141 Mıńguez, A., 26, 42 Mink, M., 26, 27, 36 Mishiba, K., 111, 142 Misof, B., 251, 267 Mita, S., 71, 85, 94 Mitchell, B. M., 25, 29, 37, 38 Miyake, M., 179, 180, 208, 210 Miyoshi, M., 114, 142 Mizutani, F., 191, 205 Mockler, T., 109, 142 Moisand, A., 25, 26, 43 Monachello, D., 190, 206 Mondello, L., 182, 207 Monerri, C., 153, 162, 163, 209, 210

Mongelard, F., 25, 35 Monselise, S. P, 152, 162, 163, 167, 207, 209, 215, 217 Montagut, G., 152, 205 Monteiro, L., 70, 71, 91 Montieri, S., 123, 142 Montoya, M., 109, 143 Moon, Y. H., 114, 141 Moore, G. A., 185, 186, 189, 192, 205, 218, 221 Moore, R. C., 51, 59, 68, 75, 82, 91 Morabito, D., 182, 207 Morcillo, G., 4, 37 Morelle, W., 27, 36 Moreno Dıáz de la Espina, S., 6, 7, 9, 11, 16, 26, 41, 42, 43 Moreno, A., 24, 38 Moreno, F. J., 22, 43 Moreno, S., 17, 35 Moretti, C., 73, 82, 88, 97 Moreuil, C., 152, 205 Moriguchi, T, 92, 162, 181, 207, 210, 212, 215 Moriguchi, T., 71, 85, 92, 169, 179, 181, 207, 210, 212, 215 Morillon, R., 148, 149, 157, 160, 162, 166, 183, 192, 200, 208, 211 Morimoto, H., 27, 42 Moritz, T., 154, 218 Morton, B. R., 248, 262 Moss, G. I., 152, 215 Moss, T., 18, 22, 39 Motte, P., 6, 11, 42 Mou, X. Y., 174, 216 Moya, J. L., 187, 190, 215, 216, 218 Mudalige-Jayawickrama, R. G., 132, 134, 139, 142 Mueller, L. A., 109, 143 Muller, B., 65, 78, 94 Munnich, A., 26, 40 Munns, R., 187, 210 Munro, W., 235, 265 Munster, T., 111, 112, 114, 144 Murakami, A, 174, 212, 219 Murakami, K., 168, 216 Murakoshi, M., 174, 216 Muramatsu, M., 109, 143 Murphy, M. E., 174, 222 Murtha, M., 251, 267 Muse, S. V., 248. 262 Muster, M., 77, 90 N Nadot, S., 248, 251, 265 Nagai, Y., 71, 85, 94 Nagao, A., 243, 263 Nagasawa, N., 114, 142 Nagata, T., 109, 140, 168, 216

AUTHOR INDEX Nagato, Y., 114, 142 Naim, M., 175, 213 Nairn, C. J., 192, 194, 196, 197, 202, 205, 216 Nakagawa, T., 26, 27, 28, 29, 32, 40 Nakai, K., 51, 52, 94 Nakai, T., 232, 265 Nakajima, M., 109, 143 Nakajima, N., 166, 215 Nakamura, K., 26, 27, 28, 29, 32, 40 Nakamura, M., 133, 141 Nakamura, T., 119, 143 Nakano, M., 161, 162, 208 Nakano, R., 69, 70, 71, 87, 92, 94, 170, 219 Nakayama, T., 178, 220 Nam, K. H., 128, 138 Naranjo, M. A., 148, 157, 160, 162, 172, 183, 192, 200, 211 Narusaka, M., 109, 143 Nasirudin, K. M., 26, 42 Nath, P., 70, 85, 86, 89, 96 Navarrete, M. H., 20, 44 Navarro, L., 149, 154, 159, 200, 207, 210, 216 Navascues, J., 3, 36 Navashin, M., 13, 42 Nayak, S., 243, 265 Nayak, S. G. R., 240, 265 Nebauer, S. G., 152, 216 Nees von Esenbeck, C. G. D., 235, 265 Negre, F., 128, 132, 139 Nelson, T. M., 26, 27, 28, 29, 32, 42 Nembaware, V., 57, 94 Nesumi, H., 176, 178, 211 Neves, N., 13, 14, 40 Newman, I. A., 190, 204 Newman, M. L., 244, 258, 260 Ng, J. H., 110, 111, 136 Ngo, Q. A., 25, 29, 32, 38, 42 Nguyen, J. T., 122, 140 Ni, Z., 71, 82, 93 Nicolaisen, M., 109, 137 Nicolosi, E., 149, 216 Nieuwland, J., 56, 81, 94, 95 Nilsson, O., 154, 155, 204, 216 Ning, G., 19, 41 Nishikawa, F., 155, 161, 162, 208, 216, 219 Nishino, H., 174, 216 Nixon, P. J., 105, 136 Noaillac-Depeyre, J., 6, 9, 11, 16, 26, 35, 41 Noel, B., 153, 211 Noel, J. P., 132, 142 Nogues, I., 132, 142 Nolte, K. D, 169, 216, 221 Noma, Y., 175, 209 Nomura, M., 22, 23, 42 No¨sberger, J., 77, 95 Nyyssonen, E., 57, 95

283

O O’Dell, M., 14, 38 Obara-Okeyo, P., 108, 142 Obayashi, K., 243, 260, 267 Obenland, D. M., 71, 94 Oberholster, R., 165, 216 OVringa, R., 30, 31, 33, 46 Oh, B. J., 134, 140 Oh, S. K., 110, 145 Ohdo, S., 72, 81, 97 Ohigashi, H., 174, 212, 219 Ohme-Takagi, M., 168, 209 Ohno, S., 117, 142 Okamura, H., 27, 42 Okamuro, J. K., 113, 139 Okuda, H., 192, 212 Oliveira, C. M., 86, 87, 91 Oliveira, R. P., 250, 265 Ollacarizqueta, M. A., 10, 11, 45 Ollitrault, P., 149, 166, 208, 216 Olmedilla, A., 10, 11, 45 Olmstead, R. G., 245, 265 Olsen, A. N., 168, 216 Olsen, J., 154, 218 Olson, M. O., 27, 40 Olson, M. O. J, 17, 21, 37, 42 Omura, M., 154, 155, 161, 162, 168, 169, 176, 178, 181, 207, 208, 210, 211, 212, 215, 216, 219, 220 Ong, S. E., 3, 35 Ongaro, V., 51, 54, 55, 57, 58, 59, 93 OnoDera, H., 243, 263 Onouchi, H., 122, 143, 155, 220 Ooka, H., 109, 140, 168, 216 Ookawara, R., 78, 94 Oono, Y., 109, 143 Oprins, J., 243, 262 Or, E., 170, 177, 218, 219 Orellana, J., 13, 40 Orlova, I., 130, 144 Ortunõ, A., 174, 182, 206, 216 Osaka, Y., 174, 216 Osato, N., 168, 216 Osheim, Y., 25, 29, 37, 38 Osipov, V., 190, 212 Otero, A., 160, 220 Otomo, Y., 168, 216 Otsuga, D., 29, 30, 32, 43 Overmars, H., 56, 93, 95 Owino, W. O., 69, 94 Ozga, J. A., 86, 95 P Pacher, T., 30, 32, 45 Padilla, C. M., 114, 136, 145 Padolina, J., 108, 142 Pages, M., 22, 24, 38 Pahnke, J., 111, 142

284

AUTHOR INDEX

Pais, M. S., 70, 71, 91 Pak, C. H., 108, 137 Pal, A., 241, 242, 252, 258, 259, 261 Pallares, C., 184, 207 Palmer, J. D., 245, 265 Paloheimo, M., 57, 95 Palys, J. M., 69, 70, 84, 85, 86, 88, 90, 92 Pan, Z. J., 106, 108, 112, 114, 117, 118, 144 Paran, I., 242, 265 Parcy, F., 154, 212, 216 Park, E., 122, 141 Park, J. G., 174, 206 Park, K. C., 244, 264, 265 Park, M. C., 67, 74, 96 Park, S. B., 229, 265 Park, Y.-I., 75, 76, 93 Parodi, L. R., 236, 266 Patil, B. S., 174, 214, 222 Patton, D., 30, 32, 41 Pavoncello, D., 185, 186, 187, 217 Pe´rez-Botella, J., 192, 204, 221 Peacock, W. J., 122, 123, 138, 143 Peakall, R., 102, 125, 142, 143 Pedersen, K. B., 113, 143 Pederson, G. A., 244, 258, 260 Pederson, K. B., 119, 143 Pederson, K. R., 101, 137 Pederson, T., 4, 42 Peeters, A. J. M., 63, 66, 67, 69, 74, 75, 78, 90, 96 Peeters, H., 243, 262 Pelaz, S., 120, 138, 142 Peleman, J., 242, 267 Pelletier, G., 29, 30, 32, 40 Penã, L., 154, 159, 200, 207, 216 Pendle, A. F., 3, 21, 28, 29, 42 Pendleton, J., 251, 267 Pennisi, E., 256, 266 Penttila, M., 57, 95 Pere, J., 57, 95 Perez-Botella, J., 159, 193, 204, 221 Perol, J., 189, 190, 191, 205 Perrini, G., 182, 214 Pestov, D. G., 19, 40 Peters, W. S., 66, 95 Petersen, A., 62, 80, 92, 96 Petitjean, A., 154, 204 Petrescu, A., 56, 93 Petricka, J. J., 26, 27, 28, 29, 32, 42 Petrone, G., 183, 213 Pezzotti, M., 72, 73, 81, 82, 88, 95, 97 Phillips, M., 177, 205 Phillips, R., 170, 205 Phinney, B. S., 170, 172, 202, 212, 213 Pichersky, E., 125, 127, 128, 130, 132, 137, 138, 139, 142, 144, 173, 216 Pien, S., 64, 65, 88, 95 Pierce, N. E., 101, 142 Piersanti, S., 25, 36

Pih, K. T., 23, 32, 42 Pikaard, C. S., 13, 14, 15, 22, 35, 36, 40, 41, 43, 44 Pike, L. M., 174, 222 Pillitteri, L. J., 156, 216, 217 Pina, L. A., 149, 154, 216 Pines, J., 18, 36 Ping, C. Z., 244, 264 Pinyopich, A., 120, 138 Pitman, M., 189, 217 Plank, C., 26, 27, 36 Platt, R. G., 184, 207 Plieth, C., 190, 214 Ploton, D., 6, 38, 45 Pohl, R. W., 229, 266 Poinar, G. O., 227, 266 Polena, I., 4, 42 Policriti, A., 153, 211 Politz, J. C. R., 4, 42 Pollard, H. B., 27, 45 Polzikov, M., 19, 43 Pons, A., 27, 36 Pontes, O., 13, 14, 40 Pontvianne, F., 19, 24, 26, 27, 28, 29, 32, 37, 43 Popeijus, H., 56, 93, 95 Poppelmann, M., 62, 92 Porat, R., 185, 186, 187, 217, 218, 219 Porceddu, A., 73, 82, 88, 97 Porras, I, 174, 216 Porter, J. M., 250, 251, 260 Portereiko, M. F., 29, 30, 32, 43 Porwancher, K. A., 25, 29, 37 Pot, J., 242, 268 Pouchnik, D., 128, 140 Powell, A. A., 160, 217 Powell, A. L. T., 69, 84, 88, 92, 95 Pozo, L., 194, 203, 217 Pozo, L. V., 192, 194, 196, 197, 202, 205 Pozueta-Romero, J., 169, 207 Prasad, V., 111, 141, 145 Prat, H., 236, 266 Preston, J. C., 227, 258, 265 Preuss, S., 13, 15, 43 Priester, B., 30, 32, 45 Prieto J. L., 16, 43 Primo-Millo, E., 148, 157, 159, 160, 162, 169, 183, 187, 190, 191, 192, 193, 195, 200, 203, 204, 209, 211, 215, 216, 218, 220, 221, 222, 223 Proctor, M. C. F., 102, 142 Proietti, P., 172, 208 Pruijn, G. J., 26, 38 Puglisi, I., 183, 213 Punwani, J. A., 29, 30, 32, 43 Purugganan, M. D., 111, 142 Putnam, N., 153, 221

AUTHOR INDEX Q Qin, L., 56, 93, 95 Qu, L. H., 23, 36 Quan, L. Y., 229, 263 Quint, A., 30, 31, 33, 46 R Rafalski, J. A., 240, 268 Ralph, S., 153, 221 Ramachandiran, V., 177, 205 Ramirez, S. R., 101, 142 Ramon-Laca, L., 174, 204 Randle, C. P., 249, 268 Rangaswamy, V., 192, 194, 196, 197, 202, 205 Rao, S. V., 27, 40 Rapisarda, P., 182, 183, 213, 214 Raschke, K., 190, 212 Raska, I., 6, 18, 37, 42, 43 Rasmussen, G. K., 197, 217 RatcliVe, R. G., 170, 205 Ratner, A., 197, 217 Rawlins, D. J., 10, 11, 12, 39, 43 Rayle, D. L., 49, 66, 95 Raymond, P., 170, 218 Reale, L., 73, 82, 88, 97 Recupero, G. R., 182, 206 Reddy, A., 134, 142 Redei,G. P., 153, 217 Reece, P. C., 149, 220 Reeder, R. H., 14, 43 Reeves, P. D., 122, 142 Reichler, S., 26, 27, 45 Reichler, S. A., 27, 43 Reichow, S. L., 21, 23, 43 Reid, D. S., 69, 88, 92 Reid, M. S., 66, 70, 73, 82, 85, 91, 94 Reidy, B., 65, 77, 78, 94, 95 Reig, C., 193, 203 Reijans, M., 242, 267 Reinecke, D. M., 86, 95 Reinhardt, D., 73, 82, 95 Reinheimer, R., 227, 258, 265 Rendon, M. C., 22, 43 Renvoize, S. A., 235, 236, 237, 241, 250, 261, 263, 266 Reski, R., 55, 58, 96 Reuther, W., 152, 217 Reynaldo, I., 174, 216 Rhee, S. Y., 109, 143 Rhodes, A. M., 149, 204 Richman, A., 128, 136 Richter, A., 6, 16, 43 Riera, M., 22, 43 Rieseberg, L. H., 242, 263 Ring, J., 72, 79, 89 Rıós, G., 148, 157, 160, 162, 183, 192, 200, 211

285

Rios-Castano, D., 152, 217 Riov, J., 161, 162, 194, 197, 210, 211, 212, 217, 218, 221, 223 Risuenõ, M. C., 5, 10, 11, 17, 18, 20, 41, 42, 43, 45 Rivas, F., 160, 217 Riviere, A., 230, 266 Riviere, C., 230, 266 Roan, J. G., 14, 43 Roberts, J. A., 65, 68, 78, 84, 89, 95 Roberts, K., 19, 24, 32, 46 Roberts, S. K., 189, 217 Robles, M., 198, 206 Robles, P., 120, 138 Robson, F., 155, 220 Roca, M., 148, 157, 166, 183, 192, 203 Rochange, S. F., 77, 95 Rodrigo, M. J., 163, 164, 165, 174, 177, 178, 179, 186, 192, 203, 205, 217, 219 Rodrigo, R. M., 22, 43 Rodriguez-Concepcion, M., 165, 176, 213, 217 Rodrı´guez-Garcıá, M. I., 6, 7, 8, 9, 10, 11, 13, 16, 17, 19, 20, 23, 41 Rodrı´guez-Vilarino, V., 4, 37 Roger, A. J., 56, 89 Roger, B., 25, 26, 38, 43 Rogowsky, P., 65, 78, 94 Rohde, A., 154, 218 Rohde, W., 134, 142 Rohmer, M., 163, 176, 213, 218 Rojas, C., 177, 205 Rolfe, B. G., 55, 80, 93 Rolland, G., 65, 78, 94 Rombauts, S., 153, 221 Romero-Aranda, R., 187, 218 Roose, M. L., 170, 218, 223 Rose, J. K., 148, 157, 166, 183, 192, 203 Rose, J. K. C., 50, 56, 57, 69, 70, 71, 77, 83, 84, 86, 87, 90, 92, 93, 94, 95, 96 Rose, K.M., 6, 16, 43, 44 Rosenbauer, H., 19, 22, 46 Roshevits, R. Y., 236, 266 Rota, M. L., 244, 268 Rothan, C., 170, 218 Rothblum, L. I., 6, 18, 22, 36, 39 Rounsley, S. D., 111, 142 Rouse, D. T., 122, 143 RouseV, R., 177, 207 RouseV, R. L., 166, 175, 177, 179, 182, 209, 210, 213, 215, 218 Roussel, P., 16, 17, 18, 19, 43, 44, 45 Rout, G. R., 243, 265 Roux, S. J., 26, 27, 43, 45 Roze, E., 56, 93 Roze, E. H., 56, 95 Rozenzvieg, D., 187, 218 Rudall, P. J., 101, 143 Ruf, S., 105, 138

286

AUTHOR INDEX

Ruiyang, C., 230, 266 Ruiz-Rivero, O., 148, 157, 160, 162, 174, 177, 178, 179, 183, 192, 200, 205, 211 Ruperti, B., 65, 68, 78, 84, 89 Russo, M. P., 182, 206 Rutto, K. L., 191, 205 Ryan, C. A., 13, 37 Ryburn, J. A., 252, 266 S Sańchez, G., 66, 67, 78, 140 Saab, A. M., 173, 213 Sabater, F., 174, 182, 206, 216 Sablowski, R. W. M., 154, 218, 222 Sachan, M. S., 258, 261 Sachse, S., 102, 138 Sacristań-Ga´rate, A., 20, 44 Sadka, A., 170, 212, 218, 219 Sadle, J., 248, 263 Saedler, H., 111, 112, 114, 134, 138, 142, 144 Saéz-Va´squez, J., 26, 27, 28, 29, 32, 40 Sagee, O., 194, 218 Sahin-Cevik, M., 185, 186, 192, 218 Saija, A., 174, 204 Saito, N., 133, 144 Saitoh, T., 229, 267 Sakai, H., 114, 142 Sakata, K., 174, 219 Sakurai, T., 109, 143 Salaj, J., 75, 89 Salamini, F., 134, 142 Salamov, A., 153, 221 Salinas, P., 22, 33, 44 Saloheimo, M., 57, 95 Samach, A., 122, 143, 186, 187, 218, 219 Samaj, J., 75, 89 Sampedro, J., 50, 51, 53, 54, 57, 58, 59, 72, 80, 81, 95, 96 Sanchez, C., 128, 140 Sańchez, M. A., 66, 68, 78, 96 Sanchez, R. A., 72, 79, 89 Sańchez, R., 72, 79, 94 Sanchez-Ballesta, M. T., 186, 187, 192, 219 Sańchez-Pina, M. A., 6, 7, 8, 9, 10, 11, 13, 16, 17, 19, 20, 23, 41, 45 Sanders, D., 189, 203 Sanderson, M. J., 250, 251, 260 Sane, A. P., 70, 85, 86, 89 Sane, V. A., 70, 85, 86, 89, 96 Sanjaya, M. T., 111, 143 Sano, Y., 114, 142 Santhamma, B., 176, 211 Santos, J., 86, 87, 91 Santos, J. L., 13, 40 Sapitnitskaya, M., 186, 187, 219 Sari-Gorla, M., 80, 96 Satoh, H., 114, 142

Satoh, K., 109, 140, 168, 216 Satoh, S., 26, 27, 28, 29, 32, 40 Satomi, Y., 174, 216 Satou, M., 109, 143 Sau-Na, T., 190, 222 Savidge, B., 124, 138 Savino, T. M., 17, 18, 35, 44 Saxena, A., 26, 40 Sayed, M., 57, 94 Sazanov, L. A., 105, 136 Scali, M., 80, 96 Schaap, P., 56, 91 Schaefer, D., 55, 58, 96 Scheer, U., 6, 16, 43, 44 Schein, J., 153, 221 Schepper, S. D., 244, 264 Schiefelbein, J., 75, 92 Schiestl, F. P., 125, 141, 143 Schipper, O., 51, 54, 55, 57, 58, 59, 93, 96 Schlaak, M., 62, 92 Schlauch, K. A., 85, 92 Schmidt, R. J., 111, 114, 136, 142, 145 Schnapp, A., 19, 22, 46 Schots, A., 56, 95 Schrader, J., 69, 78, 91 Schulman, A. H., 244, 267 Schulz, C., 125, 141, 143 Schulz, C. M., 125, 141 Schumacher, K., 190, 208 Schuurink, R. C., 130, 144 Schwab, W., 177, 178, 214 Schwappach, B., 190, 210 Schwartz, E., 30, 39 Schwarz, H., 30, 32, 45 Schwarz-Sommer, Z., 122, 143 Schwender, J., 163, 213 Scofield, S. R., 109, 143 Scolnik, P. A., 165, 209 Scora, R. W., 149, 219 Scurlock, J. M. O., 229, 266 Scwab, W., 178, 207 Seadler, H., 134, 141 Seelanan, T., 252, 266 Seither, P., 22, 44 Seki, H., 133, 144 Seki, M., 109, 143 Sellos, D., 57, 97 Semir, J., 102, 136 Seoighe, C., 57, 93, 94 Seon, J. H., 110, 145 Serin, G., 26, 44 Serna, D., 157, 169, 215 Set, O., 242, 264 Settlage, R. E., 25, 29, 37 Shabanowitz, J., 25, 29, 37 Shalit, M., 177, 219 Sharma, A., 229, 264 Sharon-Asa, L., 177, 219 Sharrock, R. A., 251, 265

AUTHOR INDEX Shaw, P., 21, 23, 36 Shaw, P. E., 175, 177, 219 Shaw, P. J., 3, 4, 5, 10, 11, 12, 14, 19, 21, 28, 29, 38, 39, 42, 43, 44, 45 Shchennikova, A., 112, 138 Shcherban, T. Y., 50, 96 Sheldon, C. C., 122, 143 Shelp, B. J., 170, 205 Shen, D., 117, 118, 138 Shen, D. L., 133, 139 Sheng, W., 244, 264 Sherman, D., 130, 144 Shi, D. Q., 25, 29, 32, 44 Shi, J., 50, 96, 117, 118, 138 Shieh, M., 50, 96 Shimada, S. H., 178, 220 Shimada, T., 71, 85, 86, 92, 154, 155, 161, 162, 168, 170, 176, 178, 179, 181, 207, 208, 211, 216, 219 Shimada,K., 243, 264 Shimbo, K, 162, 168, 219 Shimizu, T., 155, 161, 162, 176, 178, 208, 211, 216, 219 Shin, B., 134, 140 Shin, D. H., 110, 145 Shin, J. H., 67, 74, 96 Shin, Y. B., 244, 265 Shinozaki, K., 28, 33, 39, 168, 209 Shirley, B. W., 181, 220 Shukla, V. K., 122, 140 Shulaev, V., 170, 219 Sibarita, J.-B., 17, 18, 44 Sicard, H., 25, 26, 38 Silva, M., 13, 14, 40 Simon, J. E., 128, 138 Simpson, B. B., 108, 142 Simpson, G. G., 122, 143 Sinclair, W. B., 175, 220 Singer, P. B., 74, 92 Singer, R. B., 101, 142 Singer, S. R., 154, 215 Singh, K., 258, 261 Singh, L. B., 258, 261 Singh, M. B., 72, 79, 80, 81, 89, 96, 97 Singh, N. K., 185, 223 Singh, S., 196, 203 Sipes, D. L., 194, 207 Sirri, V., 16, 17, 18, 19, 44, 45 Skerrett, M., 189, 220, 221 Skipper, M., 113, 119, 143 Skriver, K., 168, 216 Sliwa-Tomczok, W., 72, 79, 89 Small, R. L., 252, 266 Smant, G., 56, 93, 95 Smart, C., 64, 65, 88, 95 Smith, D. L., 71, 92 Smith, S. M. E., 154, 215 Snow, P., 251, 267 So, I. S., 108, 137

287

Soderstrom, T. R., 227, 229, 230, 232, 235, 236, 238, 266 Sogo, J. M., 13, 37 Solanilla, E. L., 6, 7, 8, 9, 10, 11, 13, 16, 17, 19, 20, 23, 41 Soler, G., 148, 157, 160, 162, 167, 168, 169, 170, 171, 172, 183, 191, 192, 197, 198, 200, 205, 211, 221 Soltis, D. E., 119, 140 Soltis, P. S., 119, 140 Somerville, C. R., 123, 145 Sommer, H., 112, 134, 138, 141 Song, I. J., 119, 143 Song, S.-K., 67, 74, 93 Soong, B. C., 240, 261 Soost, R. K., 159, 220 Sopory, S. K., 26, 42 Soreng, R. J., 245, 247, 251, 261 Sorrells, M. E., 244, 268 Southwick, S. M., 152, 220 Speghini, A., 73, 82, 88, 97 Spiegel-Roy, P., 152, 183, 220 Srivastava, M., 27, 40, 45 Staal, M., 63, 66, 69, 78, 96 Staals, R. H. J., 63, 66, 69, 78, 96 StaV, I. A., 72, 79, 96 Standardi, A., 172, 208 Stapleton, C., 227, 259, 261 Stapleton, C. M. A., 231, 232, 233, 235, 236, 239, 241, 242, 250, 263, 266, 267 Statti, G. A., 173, 213 Stauber, E. J., 128, 140 Steele, C. L., 177, 220 SteYen, J. G., 29, 30, 32, 43 Steinbaker, C. R., 54, 91 Steinborn, K., 30, 32, 45 Steinmetz, F., 23, 32, 35 Steinmeyer, K., 190, 210 Stenseth, N. C., 229, 267 Stephenson, A. G., 72, 80, 96 Sterck, L., 149, 221 Sterky, F., 69, 78, 91 Stern, M. J., 227, 233, 264 Stewart, G. R., 170, 205 Stierhof, Y. D., 30, 32, 45, 90 Storey, R., 187, 190, 220 Stoughton, R. B., 109, 143 Strauss, S. H., 155, 204 Strittmatter, L. I., 29, 33, 39 Strosberg, A. D., 191, 204 Struijs, K., 69, 88, 92 Studitsky, V. M., 25, 35 Stunnenberg,, H. G., 19, 22, 46 Su, H. J., 110, 141 Su, V., 134, 143 Sua’rez-Lo’pez, P., 155, 220 Sugii, N., 110, 140 Sugiura, M., 163, 164, 165, 166, 212 Sugiyama, A., 161, 162, 208

288

AUTHOR INDEX

Suh, S. S., 122, 141 Sun, C. W., 259, 264 Sun, Q., 71, 82, 93 Sun, Y., 242, 250, 267 Sundaresan, V., 25, 29, 32, 38, 42, 43 Sundberg, B., 69, 78, 91 Surendrarao, A., 25, 29, 32, 38 Suyama, Y., 243, 260, 267 Suzuki, K., 109, 145, 168, 216 Suzuki, T., 26, 27, 28, 29, 32, 40 Suzuki, Y., 178, 220 Svab, Z., 105, 136 Swain, S., 154, 155, 156, 221 Swanson, A. K., 128, 136 Swanson, B., 57, 95 Swingle, W. T., 149, 220 Swoboda, I., 26, 27, 36, 89, 90 Sykes, S. R., 189, 220 Syvertsen, J. P., 160, 190, 220 Szarejko, I., 68, 76, 93 Szick-Miranda, K., 29, 35 Szopa, J., 6, 16, 43 T Tabata, S., 26, 27, 28, 29, 32, 40 Taberlet, P., 249, 263 Tabuchi, A., 54, 59, 61, 62, 63, 97 Taddeo, S. S., 174, 222 Tadege, M., 122, 143 Tadeo, F., 191, 197, 198, 200, 221 Tadeo, F. R., 148, 157, 159, 160, 162, 172, 183, 187, 192, 193, 195, 200, 202, 203, 204, 207, 209, 211, 213, 215, 218, 221 Takada, S., 168, 220 Takagi, M., 26, 45, 209 Takahashi, N., 159, 193, 210 Takano, T., 63, 71, 78, 81, 92 Takayasu, J., 174, 216 Talon, M., 148, 153, 157, 159, 192, 193, 200, 203, 204, 206, 207, 209, 211, 215, 216, 218, 220, 221, 222 Tan, F. Ch., 154, 155, 156, 221 Tan, H. T. W., 240, 261 Tan, J., 106, 144 Tan, Q. K. G., 154, 213 Tanaka, N., 243, 263 Tanaka, T., 149, 221 Tandler, C. J., 10, 11, 42, 45 Tang, C. S., 133, 144 Tao, N. G., 166, 221 Tao, W., 22, 45 Tardieu, F., 65, 78, 94 Tasaka, M., 168, 203, 220 Tashpulatov, A. S., 72, 81, 92 Tateoka, T., 237, 267 Tatsuzawa, F., 133, 144 Taylor, J., 250, 268

Taylor, P. E., 72, 79, 96 Teeri, T. T., 69, 78, 91 Teo, L. L., 113, 114, 117, 118, 119, 120, 145 Terashima, S., 185, 186, 192, 210 Terol, J., 191, 197, 198, 200, 206, 221 Tester, M., 190, 209 Testillano, P. S., 10, 11, 45 Thammasiri, K., 133, 144 Thannhauser, T. W., 148, 157, 166, 183, 192, 203 Theissen, G., 111, 112, 114, 120, 140, 142, 144, 145 Tholl, D., 130, 137, 144 Thomasson, J. R., 227, 267 Thomine, S., 190, 206 Thompson, W. F., 10, 14, 38, 45 Tillet, R. L., 85, 92 Timmer, L. W., 157, 194, 195, 206, 212, 213, 221 Tingey, S. V., 240, 268 Tisserat, B., 174, 212 To, H., 72, 81, 97 Tokuda, H., 174, 216 Tollervey, D., 30, 37 Tollsten, L., 125, 140 Tomczok, J., 72, 79, 89 Tomlinson, P. T., 169, 214, 221 Tong, C. G., 26, 27, 45 Tornielli, G. B., 73, 82, 88, 97 Torreblanca, J., 22, 43 Toscano, G., 181, 209 Toth, G., 165, 216 Touraev, A., 72, 81, 92 Tourmente, S., 26, 27, 28, 29, 32, 43 Tozlu, I., 189, 221 Traas, J., 26, 27, 36 Trainotti, L., 71, 86, 96 Traktman, P., 19, 41 Tramier, M., 17, 18, 35 Tran, L. S. P., 168, 209 Trask, I., 4, 42 Trautmann, S., 30, 32, 45 Trebitsh, T, 162, 221 Tribulato, E., 149, 216 Trinkle-Mulcahy, L., 21, 41 Triplett, J. K., 239, 267 Trivedi, P. K., 70, 85, 96 Tsai, R. C., 251, 265 Tsai, W. C., 106, 108, 109, 110, 112, 114, 117, 118, 126, 128, 129, 130, 131, 137, 139, 141, 144 Tsay, H. S., 259, 264 Tseng, I. C., 108, 137 Tsui-Hung, P., 190, 222 Tucker, D. P. H., 190, 220 Tudela, D., 157, 195, 221 Tundis, R., 173, 213 Tung, C. W., 106, 109, 144

AUTHOR INDEX Turnbull, C. G. N., 159, 193, 221 Turner, N. D., 174, 222 Tuskan, G. A., 153, 221 Tuteja, N., 26, 27, 42, 45 Tuteja, R., 26, 27, 42, 45 Tyerman, S. D., 189, 220, 221 Tyrrel, C. D., 239, 261 Tzafrir, I., 30, 32, 41 Tzvelev, N., 236, 267 U Ueda, T., 178, 219 Ueng, P. P., 194, 195, 212 Umbach, I., 196, 203 Usui, H., 231, 267 V Vainstein, A., 127, 128, 132, 139, 144 Valdivia, E. R., 72, 80, 81, 96 Valente, P., 229, 262 Valiente, J. I., 152, 222 Valverde, F., 155, 220 van de Lee, T., 242, 267 Van der Pijl, L., 125, 126, 144 van der Plas, L. H., 177, 178, 214 Van Driel, R., 6, 36 van Hautum, B., 178, 214 van Huizen, R., 86, 95 Van Lammeren, A. A., 30, 32, 41 Van Lijsebettens, M., 28, 32, 33, 45 Van Montagu, M, 28, 32, 33, 45, 113, 139, 154, 218 van Tunen, A. J., 114, 118, 130, 144, 178, 207 Vanamala, J., 174, 222 Vandercook, C., 174, 212 Vanderhaeghen, R., 28, 32, 33, 45 Vassilev, A., 30, 39 Vaz, A. P. A., 123, 144 Vazquez, N., 191, 206 Vencken, R. J., 30, 31, 33, 46 Venrooij, W. J., 26, 38 Vente, A., 17, 19, 39 Verbelen, J. P., 229, 262 Verdon, C. P., 179, 210 Verdonk, J. C., 130, 144 Vereecken, N. J., 125, 141 Verhoeven, H. A., 177, 178, 207, 214 Verkaart, S., 26, 38 Verschure, P. J., 6, 36 Verstappen, F. W. A., 177, 178, 207 Vezzi, A., 153, 207 Vicient, C. M., 244, 267 Vicuna, R., 177, 205 Vidal, A. M., 159, 193, 200, 208, 222 Viegas, W., 13, 14, 40 Vignani, R., 80, 96

289

Vignols, F., 19, 24, 32, 37 Viljugrein, H., 229, 267 Villarroel, R., 28, 32, 33, 45 Vincente, A. R., 83, 96 Viollet, L., 26, 40 Visser, P. B., 109, 136 Vitulo, N., 153, 211 Voesenek, L. A. C. J., 50, 56, 57, 63, 66, 67, 69, 74, 75, 78, 90, 93, 96 Vogler, H., 77, 90 Voit, R., 17, 19, 22, 39, 46 Volkmann, D., 75, 89 Voragen, A. G. J., 178, 207 Vos, P., 242, 267 Vreeburg, R. A. M., 63, 66, 69, 78, 96 Vriezen, W. H., 67, 74, 75, 90 Vullo, V., 174, 204 W Wada, S., 174, 216 Wagemaker, C. A. M., 67, 74, 75, 90 Wagner, A., 251, 267 Wagner, D., 154, 222 Wagner, E., 26, 27, 36 Wagner, G. P., 251, 267 Walker, R. R., 172, 187, 190, 220 Wallerstein, I., 160, 222 Walling, L. L., 156, 216, 217 Walter, C., 71, 82, 89 Wang, C. M., 173, 222 Wang, D. P., 106, 109, 144 Wang, H. C., 106, 109, 126, 128, 129, 130, 131, 139, 144 Wang, H. L., 106, 144 Wang, J., 128, 138, 139, 206 Wang, J. W., 133, 139 Wang, L. J., 177, 179, 206 Wang, M. L., 244, 258, 260 Wang, S., 229, 263 Wang, W., 72, 79, 80, 96, 97, 133, 139 Wang, Y., 70, 85, 97, 110, 141 Wang, Z. P., 234, 264 Wang,C. P., 240, 267 Ward, M., 57, 95 Warner, J. R., 28, 46 Watanabe, S., 243, 263 Watson, L., 229, 236, 252, 264, 267 Weakley, A. S., 239, 267 Webber, H. J., 149, 222 Wegner, L. H., 190, 212 Wehner, K. A., 25, 29, 37 Wehrli, E., 64, 91 Wei, C., 110, 141 Wei, W. J., 106, 108, 114, 117, 118, 144 Wei, Y., 167, 212 Weigel, D., 111, 122, 123, 136, 140, 144, 154, 205, 216 Weijers, D., 30, 31, 33, 46

290

AUTHOR INDEX

Weinman, J. J., 55, 80, 93 Weiss, B., 186, 187, 219 Weiss, D., 128, 132, 139, 144, 153, 161, 212, 213 Weiss, E. A., 175, 222 Wellmann, F., 182, 214 Wells, B., 10, 14, 38, 45 Wen, T. H., 235, 267 Wendel, J. F., 229, 246, 248, 250, 251, 252, 260, 261, 262, 266, 268 Went-Siefert, I., 56, 89 Wenzel, C. L., 77, 95 Wesierska-Gadek, J., 27, 41 Wessler, S. R., 244, 268 Wheatley, K., 155, 220 Wheatley, M. D., 85, 92 Whipple, C. J., 114, 145 White, P. J., 83, 97, 189, 222 White, S., 244, 268 White, T. J., 250, 268 Whitelaw, C. A., 84, 95 Whitlock, B. A., 114, 136 Whitman, S. C., 174, 222 Whitten, W. M., 104, 137 Widjaja, E. A., 236, 238, 262 Widmer, A., 102, 108, 136, 137 Widmer, R. M., 13, 37 Wildung, M., 177, 206 Wildung, M. R., 128, 140 Wilkerson, C. G., 172, 202, 213 Williams, J. G. K., 240, 268 Williams, N. H., 104, 137 Wilson, C. W., 175, 177, 219 Wilson, R. N., 123, 145 Wing-Yen, F. L., 190, 222 Winter, K. U., 112, 114, 144 Winzell, A., 69, 78, 91 Wisman, E., 120, 142 Wittich, P., 30, 32, 41 Wittwer, F., 73, 82, 95 Wojciechowski, M. F., 250, 251, 260 Wolfe, A. D., 249, 268 Won, S.K., 75, 76, 93 Wong, K. M., 230, 232, 268 Wong, S. M., 108, 137 WoodruV. K., 27, 43 Wormsley, S., 25, 29, 37 Wu, H., 71, 82, 93 Wu, J., 54, 55, 67, 74, 75, 97 Wu, J. T., 110, 137 Wu, M., 120, 141 Wu, T., 88, 97 Wu, T. F., 112, 144 Wu, T. S., 106, 109, 126, 128, 129, 130, 131, 139 Wu, W. L., 106, 140 Wu, X., 88, 97 Wu, Y., 72, 80, 96 Wu, Z., 194, 196, 197, 222, 223

Wyrzykowska, J., 64, 65, 88, 95 X Xia, M., 29, 33, 39 Xia, N., 242, 250, 267 Xiang, C. Y., 177, 179, 206 Xiang, Y. H., 25, 29, 32, 44 Xu, B., 57, 97 Xu, H., 72, 80, 97 Xu, J., 166, 221 Xu, X., 167, 212 Xu, Y., 113, 114, 117, 118, 119, 120, 145 Xu, Z., 167, 212 Xue, H. W., 67, 74, 93 Xue, Z., 2, 42 Xun, N. M. A., 244, 264 Y Yam, T. W., 108, 137 Yamada, H., 109, 140 Yamada, Y., 174, 219 Yamaguchi-Shinozaki, K., 168, 209 Yamamoto, H. Y., 133, 144 Yamane, H., 159, 193, 210 Yamanishi, O. K., 160, 222 Yamasaki, Y., 176, 178, 222 Yan, H., 167, 212 Yan, L. Q., 244, 264 Yang, C. Q., 177, 179, 206 Yang, J., 110, 145 Yang, J. B., 250, 263 Yang, N. S., 111, 141 Yang, W. C., 25, 29, 32, 44 Yano, M., 163, 164, 165, 166, 174, 192, 212, 216 Yanofsky, M. F., 111, 114, 120, 122, 124, 136, 138, 142, 143, 154, 214 Yao, Y., 71, 82, 93 Yazaki, J., 109, 162, 168, 219 Ye, D., 25, 29, 32, 42, 44 Ye, G.H., 240, 267 Ye, M. M., 133, 139 Yeh, H. H., 110, 141 Yeh, K. W., 106, 144 Yelenosky, G., 184, 185, 223 Yennawar, N. H., 54, 59, 61, 62, 63, 97 Yeo, P., 102, 142 Yeong, C. Y., 120, 141 Yi, H., 134, 140 Yin, Q., 88, 97 Yin, X. Z., 244, 264 Yogosawa, S., 174, 216 Yokoi, M., 133, 144 Yokoyama, J., 119, 143 Yoo, M. J., 119, 140 Yoshida, K., 174, 219 Yoshida, M., 72, 81, 97

AUTHOR INDEX Yoshida, T., 166, 215 Yoshikuni, Y., 177, 223 Yoshioka, H., 71, 86, 92 Yoshioka, T., 78, 94 You, S. J., 111, 141, 145 Youn, B., 176, 211 Young, R., 157, 162, 223 Youtsey, C. O., 182, 218 Yu, H., 102, 110, 111, 113, 114, 117, 118, 119, 120, 123, 124, 136, 145 Yu, J. K., 244, 268 Yu, Z. H., 240, 267 Yuan, R., 193, 194, 195, 196, 210, 212, 213, 217, 223 Yun, S. H., 174, 206 Z Zabeau, M., 242, 267 Zacarias, L., 163, 164, 165, 186, 187, 192, 193, 217, 219, 220, 221, 223 Zahn, L. M., 111, 145 Zamboni, A., 73, 82, 88, 97 Zanin, D., 71, 86, 96

291

Zapater, M., 192, 195, 203, 216 Zaragoza, S., 157, 159, 160, 169, 192, 203, 215 Zararıás, L., 174, 177, 178, 179, 205 Zatsepina, O., 19, 43 Zeevaart, J. A. D., 159, 220, 221, 223 Zeng, W. H., 105, 145 Zenoni, S., 73, 82, 88, 97 Zhang, C., 109, 145 Zhang, C. K., 185, 223 Zhang, N., 75, 97 Zhang, T., 117, 118, 138 Zhang, W., 227, 229, 246, 248, 249, 251, 261, 268 Zhang, X. Y., 229, 263 Zhang, Y., 71, 82, 88, 93, 97 Zhao, Y., 30, 39, 176, 211 Zheng, H. J., 229, 263 Zheng, R. L., 173, 222 Zhong, G., 194, 196, 217, 223 Zhou, C. P., 229, 263 Zhou, J., 113, 114, 117, 118, 119, 120, 145 Zimmer, E. A., 248, 263 Zografidis, A., 19, 24, 32, 46 Zucol, A. F., 227, 261

SUBJECT INDEX

A ABCDE model and orchid floral development, 111–2 Acid growth theory, 66 and plant development, 49 Acorus americanus, 106 Actinocladum verticillatum, fodder resource, 229 Allium cepa microspores nucleolus, 8 root meristematic cell nucleoli, 4 N-(Aminocarbonyl)–2chlorobenzenesulfonamide (2-CBSU) and growth response, 63 Anacamptis palustris, 108 Anther and postmitotic nucleologenesis, 7–9 APETALA1/FRUITFULL (AP1/FUL) MADS-box gene lineage and floral development, 113 AP3-like (CitMADS8) from C. sinensis, 156 Arabidopsis thaliana AYmetrix GeneChip ATH1 database, 87 AGL6-like OMADS1, ectopic expression in, 123–4 amino acid sequences of expansin comparisons with rice, 52 APETALA1 (AP1) genes, 154 AtEXP10 gene, leaf development, 65, 106, 153 A. thaliana RNase III-like protein (AtRTL2), 24 cauliflower mosaic virus 35S promoter in, 117 CBF5 activity and, 23 CO and FT gens, 155 DNA-binding domain in, 154 EARLY BOLTING IN SHORT DAYS (EBS), 155 EXPA genes and root growth, 75 flowers development genes in, 121 integration genes, 153 genome, 22 sequencing programmes, 21 K-box in, 154 MADS-box in, 154 NCBI public databases, 130 nuclear factor D (NFD), 25 nucleolin mutants, 28–9 Pescadillo (AtPES) proteins, 24

RD26 gene, 168 root meristematic cell nucleoli, 4 TERMINAL FLOWER 1 (TFL1), 154 vernalization eVect of, 154 Arundinaria spp., 230–232, 235, 238, 248 Asparagus oVcinalis, 106 AtEXP10 gene, 63 B Bamboo Bambusa spp., 233, 240–2, 244, 257 Ac-like sequences, 244 ebracteate grass panicle, 233 gregarious flowering, 233 Bambuseae, pseudo-spikelets and spikelets, 233 Bambusoideae, spikelet and lemma, 233 B. arundinacea, microsatellites from, 243 B. striata, somatic mutant, 241–2 B. tulda bent culms and compressed swollen internodes, 252–3 isolated populations, 242 striated culms, 252 B. ventricosa, cultivated variety, 240–1 B. vulgaris, Ac-like transposon elements, 244 candidate genes and spacers, 256 chloroplast genes in, 248 chromosome number and genome size, 229–30 comparative genomics, future scope of, 258–9 DNA fingerprinting-based methods, markers in amplified fragment length polymorphism (AFLP), 242–3 expressed sequence tag derived microsatellites (EST-SSR), 244 microsatellites (SSRS), 243–4 miniature inverted-repeat transposable elements (MITEs), 244–5 51 PHYB sequences, nuclear, 251 randomly amplified polymorphic DNA(RAPD), 240–2 restriction fragment length polymorphism(RFLP), 240 sequence characterized amplified regions (SCARs), 242

294

SUBJECT INDEX

Bamboo (cont. )

transposon, 244–5 DNA sequence-based methods, genes nuclear, 249–52 organellar, 245–9 and floral genes, 258–9 flower and fruit characters in, 233–5 genera of, 238–9 genetic diversity and ecology of, 259–60 geographical distribution of, 227–8 molecular phylogeny of, 245–7 molecular taxonomy in, 239–40 morphological features in, 227 branching, bud and leaf characters, 231–3 classification and, 235–7 rhizome types, 230–1 systematics and identification, 252–6 origin, systematic position, 226–7 phylogenetic relationships of, 252–3 polyploidization in, 251–2 rhizome types in, 231 ribosomal protein S4 (rps4) in, 248 spacers and 5.8S rRNA sequences variations, 257 species characterization, 252–5 systematics and identification, molecular aspects, 256–8 UPGMA cluster analysis, 253 use of, 229 Web-resources for, 259 woody bamboo classification of, 238–9 world distribution of, 228 Bam HI, bacterial artificial chromosome (BAC) libraries, 107 Banana fruit, MaEXPA4 genes, 86 Barley EXPB gene and root hair initiation, 76 wound induced (bawin) protein, 57 N-Alpha-Benzoyl-arginine-p-nitroanalide (BAPNA) hydrolysis, 62 Brassica oleracea RNA pol I, 21 Bromoheadia finlaysoniana, 133 C CArG-box motif and DNA-binding core sequences (TGAC) of class 1 knox genes, 124 Carpel development, AGAMOUS (AG) gene, 154 Chick pea (Cicer arietinum) and CaEXPA2 transcripts, 66 Chimonobambusa spp., sporadic/irregular flowering, 233 Chinese bamboos, Flora Reipublicae Popularis Sinicae study, 233–5 Chlamydomonas reinhardtii, 58

CHS gene and phenylpropanoid biosynthesis, 133 Chusquea culeou, bud-scale, 232 CiFT genes, 155 Citrus abscission auxins, role of, 159, 193–4 CMNP, eVect of, 196–7 and EST, 197–8 ethylene-induced, 199 organ, 195 promoters/inhibitors, regulation by, 193–5 proteins encoded by cDNA, 201–2 reproductive organs of, 157 water stress-induced, 196 acidless and acidic varieties, 169–70 AP3-like (CitMADS8), 156 bitterness/tastelessness, 179–83 biosynthetic genes, 182 flavanone-based, 181–3 limonid-based, 179–81 carrizo citrange and gene expression in, 151, 154, 159, 187–8 Citrus genus CitSUS1,CitSUS2 and CitSUSA, 169 CsETR1, 161 as health food, 172–83 Citrus jambhiri, monoterpene synthesis, 176 CsETR1 genes and ethylene production, 161 cultivated species, nomenclature and agronomic characteristics, 151 flowers breeding process, 155–6 cloning and functional character, 156 FT gene (CiFT1), role of, 155–6 seasonal and climatic conditions, 152–3 fruits acid metabolism, model, 170–1 anatomical and physiological ripening, 160–1 anthocyanins and, 182 carotenoid and chlorophyll biosynthesis in flavedo, 164 chloride homeostasis, 188–90 chlorophyllase expression in, 162–3 -cryptoxanthin in, 166 developmental stages of, 157 and environmental conditions, 183–5 ethylene treatment, degreening and regreening, 162 flavanone naringenin 7-Oglucoside, 182 flavonoids and bitterness, 181 freezing eVect, 185–7 gene expression in, 161–2

SUBJECT INDEX gibberellins (GAs) and development, 153, 157, 159, 193 growth and sugars, 160 limonid-based bitterness, 180 maturity index, 152 MEP pathway, 163–4 nan mutant and, 166 nutrition and fertilization, 184 parthenocarpic setting in, 159 physiological and biochemical events, 158 pigment substitution and, 164 quality environment, 183–5 salinity and water shortage, 187–91 transcriptome profiling, 167–8 gamma-aminobutyrate (GABA) shunt and acid metabolism in, 170–1 as health food anti-cancer agents, 173 bioactive compounds, 173–4 flavonoids source, 174 gene transcription, 176 oil glands, 175–6 in markets, 152 NCED and Crcor15 genes, 192 origination and production areas, 149–50 phenotypical variability of, 150–2 physiology of, 150–2 production areas of, 149–50 proteomic study for, 172 stress-responsive genes, 191 system I and system II, ethylene synthesis, 161 taxonomy of, 148–9 vegetative and reproductive physiology of, 150, 152 Clarkia breweri, 127–8 CONSTANS (CO) transcription factor and photoperiod pathway, 122–3 Crepis and nucleolar dominance, 13 C. sinensis, flowering genes, 156 Cymbium, 125–6 D Dendrobium spp. cloned genes from, 113–4 Den-CHS-4 genes in, 133 DFR genes, 134 Dendrocalamus spp., 233, 235, 242 Den-CHS-4 genes, 133 DOMADS2 and DthyrFL genes, 113 E-class genes, 120 gregarious flowering, 233 sporadic/irregular flowering, 233 Deoxy-D-xylulose-5-phosphate (DXP) pathway in orchid flowers, 128 DfEXPA1 expression and gibberellin signallings in Datura ferox, 79

295

Dictyostelium discoideum, 56 DOMADS1 expression in wild-type orchid plants, 124 DOMADS2 gene, in shoot apical meristem (SAM), 113 Doritis pulcherrima, 108 E Expansin proteins action mechanism and, 61 amino acid sequences of, 52 angiosperm expansin genes, 58 cellular localization and, 59 cellulose hydrolysis of, 61 cell wall-binding properties of, 61–2 EXPA gene families and, 51 EXPA and EXPB genes, GA treatment, 55 families of expansin-like A (EXLA), 50, 55 expansin-like B (EXLB), 50, 55–6 expansin-like X (EXLX), 54, 56–7 features of, 51 in fruit development climacteric ripening, 84–5 fruits and cell wall proteins, 83 hormones and, 85–6 non-climacteric ripening, 85 stages of, 83–4 function and agricultural application, 87–8 genes expression in plant organs and developmental stages, 67–73 origin and evolution, 57–9 and phylogenetic tree, 54 plant species from, 53 leaf development, 64–6 nomenclature of, 50–1 OsEXPB3 protein and, 59 plants growth rate and environmental factors, 78 vegetative growth and, 76–7 pollen-type EXPBS hydrolytic activity, 62–3 reproductive growth and development floral development, 82–3 male gametophytic development, 79–81 seed formation and embryogenesis, 81–2 root growth, 66, 74–5 hair development, 75–6 seed germination, 78–9 growth, 63–4

296

SUBJECT INDEX

Expansin proteins (cont. )

stems and petioles elongation, 66 three-dimensional structure of, 59–61 xylem tissues diVerentiation, 77–8 ZmEXPB1 proteins, 59

F Filgueirasia spp., as fodder resource, 229 FLOWERING LOCUS C and T, orchid flowering, 122–3 Fortunella genus, 149 Fruit development and expansins, 83–7 NAC transcription factor and, 168 G GA20-oxidase, over expression in tobacco, 159 Genome survey sequences (GSS) in bamboo, 229–30, 259 Gibberellin (GA) pathway and orchid flowering, 122–3 Globodera rostochiensis, 56 GmEXP1 over expression in tobacco plants, 74 Granule bound starch synthase gene (GBSSI), 250 Guadua spp., 227, 243, 260 Gypsy/DIRS1 and Ty1/Copia, LTR retrotransposons, 244–5 H Hesperidium berry, 160 Hin dIII,bacterial artificial chromosome (BAC) libraries, 107 Hordeum vulgare, 107–8 I Indocalamus wightianus,annual flowering, 233 Internal transcribed spacer regions (ITS1, 5.8S rDNA and ITS2), 108 ITS and trnD-T sequence data, 250–1 K Kiyomi tangor plants, 155 L Leaf abscission process and, 66 development and expansins, 64 primordia, 65 (See also Expansin proteins)

(S)-Linalool synthase gene and orchid flowers, 127–8 M MADS-box genes ABCDE class, phylogenetic relationship of, 115–6 B-class, 125 OMADS1 in Oncidium orchid and floral initiation, 113 Phalaenopsis, tepal formation, 120 Maize, MA16 and ZmDRH1 proteins, 24–5 Marsilea quadrifolia, 58 Medicago sativa, root meristematic cell nucleoli, 4 Melocanna, monopodial rhizome, 230 Miniature inverted-repeat transposable elements (MITEs), 244–5 Mirabilis jalapa, 66, 82 MITE-transposon display (MITETD), 244–5 Monoterpene synthases genes, 178 N Nicotiana tabacum, 81 Nucleologenesis, 7. See also Plant nucleolus O Ochlandra sp., annual flowering, 233 Ocimum basilicum, 128 Oncidium hybrids, 108 OMADS1gene in, 113 Onion meristematic cell nucleoli transcription, 9–10 nucleolin-like protein NopA64, 26 Orchid flowers ABCDE model, 111–2 bacterial artificial chromosome (BAC) libraries, 107 colour presentation, molecular biology of biosynthesis genes for, 132–4 DFR gene in, 134 E-class DOMADS1 promoter fragment, 124 evolutionary trends in, 100–2 expressed sequence tags in, 106–7 flowering regulation of floral meristem identity gene LFY, 123 floral repressor and flowering-time genes, 122 gene regulation for, 123–4 genetic transformation system for

SUBJECT INDEX Agrobacterium tumefaciensmediated, 111 biolistic bombardment and, 110–1 genome-size estimation, 103–4 GLO/PI-like gene and age estimation, 118–9 karyotype determination for, 104–5 microarray analysis for, 109 molecular biology development for, 111, 115 A-class genes, 112–3 B-class genes, 114–9 C- and D-class genes, 119–20 CymMV vector and, 110 DOMADS1 promoter, 124 E-class genes, 120–2 MADS box transcription factors, 112 molecular markers in, 107–8 Orchid Tepal Model, 121 Orchis italica, 123 paleo AP3 genes, 114, 118 pollination strategies of, 102–3 scent production components investigation for, 125–6 enzymes in, 130–2 molecular aspects of, 127–8 P. bellina metabolism in, 128–32 Phalaenopsis spp.in, 126 research diYculties in, 127 significance of, 125 virus-induced gene silencing in RNA interference (RNAi), 109–10 T-DNA insertion, 110 Oryza sativa, 79, 106–7 OsEXPA4 proteins, 63 OsEXPB9 and OsEXPB13, cDNA microarray analysis, 81 OsEXPB3 protein, 59, 62 rice immuno cytochemical localization and, 60 OsTPS3 gene, 177 Ostreococcus lucimarinus, 58 Oxytenanthera genus, 235 P Peach fruit, PpEXP2 and PpEXP3 genes, 86 PeMADS1, C-class gene from P. equestris, 119 PeMADS6 gene, 110 Pepper ferredoxin-like protein (pflp), 111 Pescadillo a-conserved protein SURF-6, 19 Petals and sepals, APETALA1 (AP1) gene expression, 154 Petunia hybrida, EXPA gene, 82 Phalaenopsis spp., 103–4, 133 chloroplast genome of, 105

297

DXP-geraniol-linalool pathway, 129 metabolic pathway and scent synthesis, 131 scented species of, 126 scent metabolism pathway of, 129 Pheophorbide a oxygenase (PaO) gene and chlorophyll disappearance, 166 Phyllostachys spp., 240–4 Ac-like transposon elements, 244 genome size, 229–30 Pinus taeda, 57 Pistil Pollen Allergen-Like (PPAL) gene, 81 Plant natriuretic peptide (PNP), 57. See also Expansin proteins Plant nucleolus Arabidopsis La protein 1and, 29 CDK/cyclin complexes, 18–9 cell cycle interphase, 19–20 mitosis, 15–8 DOMINO role in, 30 nucleolar-processing complex and, 17 nucleolin-like proteins, 25–8 protein factors, 21–5 proteins and plant development, 28–31 rRNA gene expression, regulation of, 11–5 structural components, 2 dense fibrillar component (DFC) and granular component (GC), 6–7 fibrillar centres (FCs), 4–6 nucleolar organizer region (NOR), 3 transcription and, 7–11 Pollen-type EXPB genes, 81 Poncirus spp., 149 fruit diameter, 150 FT gene expression, 155 Populus trichocarpa, 153 Post-bloom fruit drop (PFD), 195 Postmitotic nucleologenesis, 7–9. See also Plant nucleolus Potato cyst nematode Globodera rostochiensis and expansin-like X proteins, 56 P. pubescens, genetic variations, 242 Pseudoxytenanthera genus, 235 Psygmorchis pusilla, 123 PttEXP1 and PttEXP5 in xylem tissues, 78 R rbcL gene, 248 Regnelidium diphyllum, 58 RlemispF gene, plastid transit peptide, 176 RlemTPS1 and RlemTPS2 genes, 178 RNA interference technique, 87 Root growth, expansins role in, 66, 74–6

298

SUBJECT INDEX

RPS13B and RPS18A gene mutations, 28–9 Rumex palustris, 63, 75 S Sambucus nigra,SniEXP2 and SniEXP4 genes expression, 65 Sasa spp., 229, 260 Ac-like transposon elements, 244 dwarf bamboo, 243 higher clonal variation, 260 SGR gene, in chlorophyll degradation, 166–7 Shoot apical meristem (SAM) and leaf development, 64–6 SLOW WALKER1 (SWA1) and TORMOZ (TOZ) genes, 29 SniEXP2 and SniEXP4 genes in ethylene-treated leaf abscission, 65 SOC-like (CsSL1 and CsSL2) genes, 156 Spyrogyra nucleolar structure, 11 Stamen development, AGAMOUS (AG) and APETALA3 (AP3) gene, 154 Stems and petioles growth, expansins role in, 66 Stevia rivaudiana, 128 Streptochaeta, pseudo-spikelet, 235

T Thamnocalamus spp., 232, 250–2, 259 Tomato LeEXP1 fruit-specific expansin gene in, 84 LeEXP8 mRNA, root elongation zone, 74 Triticim aestivum, 107–8 V Virus-induced gene silencing (VIGS), plant loss-of-function assay, 109–10 Vitis vinifera, 85, 153 Voltage-dependent Cl channel (CLC) gene family, 190 W WUSCHEL-like (CsWUS) genes from C. sinensis, 156 Z Zea mays, 107–8 ZeEXP genes in Zinnia, 78 Zinnia elegans, 77–8 ZmEXPB1 (Zea m 1) proteins, 59, 61–2

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