Horticultural Reviews, Volume 27

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HORTICULTURAL REVIEWS Volume 27

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Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, Volume 27 Nigel H. Banks Frederick T. Davies Susan Lurie

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HORTICULTURAL REVIEWS Volume 27

edited by

Jules Janick Purdue University

NEW YORK

John Wiley & Sons, Inc. / CHICHESTER / WEINHEIM / BRISBANE / SINGAPORE / TORONTO

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Copyright © 2001 by John Wiley & Sons, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: PERMREQ @ WILEY.COM. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. This title is also available in print as ISBN 0-471-38790-8. Some content that appears in the print version of this book may not be available in this electronic edition. For more information about Wiley products, visit our web site at www.Wiley.com

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Contents

Contributors

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Dedication: John V. Possingham

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Keith Boardman

1. The Molecular Biology of Flowering

1

Steve van Nocker I. II. III. IV. V. VI. VII.

Introduction Arabidopsis as a Model for Flowering-Time Studies Floral Inductive Pathways Role of Carbohydrates Control of Meristem Identity Competency Conclusion and Perspectives Literature Cited

2. Floral Homeotic Gene Regulation

1 2 3 20 21 25 28 30

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Robert G. Franks and Zhongchi Liu I. II. III. IV. V.

Introduction Conservation of the ABC Functions in Angiosperms Positive Regulators of Floral Organ Identity Genes Negative Regulators of Floral Organ Identity Genes Summary Literature Cited

42 51 56 63 69 71 v

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CONTENTS

3. Lingonberry: Botany and Horticulture

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Inger Hjalmarsson and Rodomiro Ortiz I. II. III. IV. V. VI.

Introduction History Botany Management of Natural Stands Horticulture Summary and Future Prospects Literature Cited

4. Caper Bush: Botany and Horticulture

80 81 87 93 99 111 114

125

Gabriel O. Sozzi I. II. III. IV. V. VI. VII. VIII.

Introduction Botany Ecophysiology Horticulture Postharvest Technology Composition and Utilization International Trade Concluding Remarks Literature Cited

126 132 137 140 156 159 170 172 173

5. Water Relations and Irrigation Scheduling in Grapevine

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M. H. Behboudian and Zora Singh I. II. III. IV. V. VI.

Introduction Phenology Aspects of Water Relations Irrigation of Vineyards Quality Attributes for Wine, Dried, Table, and Juice Grapes Future Prospects Literature Cited

6. Physiology and Biochemistry of Superficial Scald of Apples and Pears

190 191 193 207 215 218 219

227

Morris Ingle I. II.

Introduction Scald Symptoms and Cell Changes

228 228

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CONTENTS

III. IV. V. VI.

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Biochemistry of Scald Physiology of Scald A Model of Scald Development Prospects Literature Cited

7. Health Functional Phytochemicals of Fruit

229 245 253 259 262

269

Wilhelmina Kalt I. II. III. IV. V. VI.

Introduction Citrus Grapes and Wine Vaccinium Other Fruits Conclusions Literature Cited

8. Producing Sods over Plastic in Soilless Media

270 282 291 298 303 307 308

317

Henry F. Decker I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction Producing Sods in Soilless Media Development of the Concept Producing Mature Sods over Plastic Producing Sods for Golf Greens Solving the Problem of a Stable Continuum Subsequent Proposals in the Genre Manufacturing Sods New Machinery Future Potential Summary Literature Cited

318 319 321 327 330 331 333 338 340 342 345 346

Subject Index

353

Cumulative Subject Index

355

Cumulative Contributor Index

377

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Contributors M. H. Behboudian, Institute of Natural Resources, College of Sciences, Massey University, Private Bag 11222, Palmerston North, New Zealand Keith Boardman, 6 Somers Crescent, Canberra ACT 2603, Australia Henry F. Decker, Buckeye Bluegrass Farms, Inc., Box 176, Ostrander, OH 43061 Robert G. Franks, Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 Inger Hjalmarsson, The Nordic Gene Bank, Smedjevägen 3, PO Box 41, S-230 53 Alnarp, Sweden Morris Ingle, Division of Plant and Soil Sciences, 1090 AG SCI BD, PO Box 6108, West Virginia University, Morgantown, WV 26506 Wilhelmina Kalt, Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, Kentville, Nova Scotia B4N 1J5, Canada Zhongchi Liu, Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 Steve van Nocker, Department of Horticulture, Michigan State University, 390 Plant and Soil Science Building, East Lansing, MI 48824 Rodomiro Ortiz, IITA c/o Lambourn & Co., Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, UK Zora Singh, Department of Horticulture, Muresk Institute of Agriculture, Curtin University of Technology, GPO Box U 1987, Perth, WA 6845, Australia Gabriel O. Sozzi, Departamento de Biología Aplicada y Alimentos, Facultad de Agronomía, Universidad de Buenos Aires, Avda. San Martín 4453, C 1417 DSE Buenos Aires, Argentina

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John V. Possingham

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Dedication: John V. Possingham This volume is dedicated to Dr. John Possingham, an outstanding scientist and administrator, in recognition of his outstanding contribution to Australian horticulture and for his international efforts in horticultural science and viticulture. John was born in 1929 and grew up on a small horticultural property in rural South Australia. He studied agriculture and plant physiology at the Universities of Adelaide in Australia and at Oxford in England. He initially worked in the field of plant nutrition at the Laboratories of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Canberra establishing that manganese was essential for the photochemical reactions of higher plant chloroplasts. In 1962 he accepted responsibility for CSIRO’s program of research in horticulture that was mainly concerned with grapevines and centered at Merbein Victoria. He subsequently established horticultural laboratories at Adelaide, Darwin, and Brisbane that, together with an existing postharvest horticulture group at Sydney, enabled CSIRO’s Division of Horticulture to carry out a wide-ranging program of basic and applied horticultural research concentrating mainly on wine and raisin grape vines and on a range of selected tropical and subtropical fruit crops. At Merbein, Dr. Possingham established a major grapevine improvement program that covered virus-tested introductions from overseas and the breeding of new cultivars suited to the warm irrigated conditions of inland Australia. This program also included the development of grapevine rootstocks that were tolerant of Australian plant parasitic nematodes. New cultivars released from the grape breeding program and adopted by the viticultural industries include ‘Tarrango’ for light red wine; ‘Carina’, a black seedless raisin; and the table grape ‘Marroo Seedless’. A number of other potential cultivars are being evaluated by industry. Dr. Possingham also obtained major government funding and established a vineyard mechanization program based on importing prototype grape harvesting machines from the University of California, Davis and Cornell University. Vine training and management systems were developed for use in mechanically harvested vineyards. These included minimal pruning, which is now used extensively in warm to hot grape growing regions in both Australia and California. xi

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DEDICATION: JOHN V. POSSINGHAM

The work of the Division of Horticulture assisted the recent rapid expansion of the Australian wine industry and enabled it to become a major player in world markets. It provided the industry with high-quality vine planting material and developed training systems that enabled Australian wine-grape vineyards to become highly mechanized. Together with other members of the Division he was awarded both a CSIRO Medal for his contributions to vine improvement and an Ian McLennan Award from Industry for his foresight in developing a program of work on vineyard mechanization for Australia. Dr. Possingham was involved with other scientists in many aspects of the Division’s program including the minimal pruning of grapevines and the evaluation of introduced grape cultivars. He contributed to the understanding of how the waxy layers of raisin grapes control water loss and the elucidation of a major role of endotrophic mycorrhizae in the uptake of phosphorus by grapevines. Research concerned with grapevine physiology, biotechnology, and molecular biology was developed at the Division’s laboratory located on the Waite Institute Campus. Studies on grapevines included the features of and factors affecting flowering, fruit set, photosynthesis, response to salinity, and root/shoot hormonal interactions. Biotechnology investigations were aimed at better methods for grapevine propagation and systems for virus elimination. Recent work has been concerned with methods for the DNA finger-printing of grapevines and techniques for their genetic modification. Some genetically modified grapevines are currently undergoing field trials. Throughout his career, Dr. Possingham has maintained a personal research program concerned with factors involved with the division of higher plant chloroplasts. This work included both structural and biochemical studies and provided support for the long-held hypothesis that the plastids of higher plants arise from the division of pre-existing plastids and cannot be formed de novo. He showed that chloroplasts of spinach are highly polyploid and that division can be temporally separated from the synthesis of chloroplast located DNA. Using barley mutants he demonstrated that the polymerase used for c-DNA replication is nuclear coded and a component in the complex control exerted by the nucleus over plastic division. Dr. Possingham has contributed to international horticulture via a number of FAO/United Nations Development Program (UNDP) missions, but more importantly through his work with the International Society for Horticultural Science (ISHS). He established the Viticulture Section within ISHS and has been a Council member for Australia for a number of years. He is in his second term as a member of the ISHS Board and is currently the Society’s Vice President. In Australia he was directly

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involved in setting up the Australian Society for Horticultural Science and was its second President. He is a fellow of the Australian Institute of Agricultural Science and Technology, the Australian Academy of Technological Science and Engineering, the Russian Academy of Agricultural Sciences, and holds a DSc from the University of Oxford. In his “retirement” he grows wine grapes and makes wine on highly mechanized vineyards near Adelaide. John is known throughout Australia and the world for his generosity and warmth, for his dedication to high standards, and for his love of good fellowship and fine wines. Keith Boardman Formerly Chairman and Chief Executive CSIRO, Canberra, Australia

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1 The Molecular Biology of Flowering Steve van Nocker Department of Horticulture Michigan State University East Lansing, MI 48824

I. INTRODUCTION II. ARABIDOPSIS AS A MODEL FOR FLOWERING-TIME STUDIES III. FLORAL INDUCTIVE PATHWAYS A. Photoperiodic Induction 1. Light Effects on Flowering in Arabidopsis 2. The Endogenous Clock 3. Entrainment of the Clock by Light 4. Other Photoperiodic Pathway Genes B. Non-Photoperiodic Induction: The Autonomous Pathway C. Vernalization D. Induction by Gibberellins IV. ROLE OF CARBOHYDRATES V. CONTROL OF MERISTEM IDENTITY A. Meristem Identity Genes B. Integration of Flowering Pathways and Activation of Meristem Identity Genes VI. COMPETENCY VII. CONCLUSION AND PERSPECTIVES LITERATURE CITED

I. INTRODUCTION Of the myriads of developmental processes that define plant form and function, flowering is of exceptional interest to horticulturalists. The vast majority of horticulturally important crops are in some way dependent upon flowering, whether the flower is the primary goal of production, or is simply required for a crop to be produced. Much effort is currently being put into regulating the timing of flowering. In floriculture crops, the interest is in abbreviating or extending the vegetative Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 1

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phase in order to create an aesthetically pleasing balance between leaves and flowers, or to conveniently induce or repress flowering to take advantage of market potential. In ornamental foliage plants, and agronomically important plants that are grown for their leaf tissues (e.g., lettuce, spinach, and other greens), it is highly desirable to suppress flowering as long as possible. Also, in woody plants, there is a great deal of interest in finding means to abbreviate the vegetative phase, which in some species can last ten or more years and is probably the single most limiting factor for germplasm improvement through traditional breeding techniques. Most efforts at controlling flowering time have involved manipulation of environmental conditions or the application of synthetic growth regulators. However, these approaches can increase production costs and labor requirements. In addition, the use of many traditionally utilized chemical compounds is becoming restricted. Alternative approaches to manipulate flowering—including biotechnology—will require a better understanding of the associated molecular mechanisms. The physiology and phenomenology of the developmental transition from vegetative growth to reproductive growth—flowering—has been studied for many years, but only in approximately the last 10 years have the molecular mechanisms begun to be addressed. Flowering is ultimately determined by genes that govern the identity of the meristem, promoting or repressing floral fate versus shoot fate. When and how these genes are activated, in response to environmental cues and/or developmental progression, is a fascinating question. As might be expected from the incredible diversity of flowering strategies employed in nature, it is now becoming apparent that flowering at the molecular level involves an extraordinarily complex web of interactive pathways. Here we review the current knowledge about the genetics and molecular biology of flowering in Arabidopsis thaliana, the only plant in which these aspects of flowering have been extensively studied.

II. ARABIDOPSIS AS A MODEL FOR FLOWERING-TIME STUDIES Arabidopsis thaliana is an herbaceous weed of the mustard family with a natural distribution throughout the Northern Hemisphere (Meyerowitz 1989; Meinke et al. 1998). In addition to its many qualities that make it a superior model for plant biology in general (i.e., small size, rapid life cycle, and well-characterized genome), Arabidopsis is especially attractive as a subject for flowering-time studies because the timing of flow-

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ering can be strongly influenced by environmental conditions (e.g., light and temperature), thus permitting the molecular analyses of the associated input pathways. In this species, at least fifty genes have been identified that act directly or indirectly either to promote or to repress flowering (Levy and Dean 1998). Many of these genes have been identified through a traditional genetic approach. Delayed flowering can result from loss of function of genes that presumably act to promote flowering, whereas accelerated flowering can result from loss of function of flowering-repressor genes. Although several repressor genes have been identified, most mutagenic approaches have targeted genes that act to promote flowering (Redei 1962; Koornneef et al. 1991). This is in part because the genotypes commonly used in the laboratory flower soon after germination in photoperiodically inductive conditions (long-day photoperiods), and mutants that flower even earlier, are difficult to discern in large populations. Screens designed to find early-flowering mutants among late-flowering genetic backgrounds, or employing photoperiodically noninductive conditions, should result in the identification of additional repressor genes.

III. FLORAL INDUCTIVE PATHWAYS An interesting finding coming from genetic analyses is that no single mutation completely eliminates flowering. This was an early indication that flowering is promoted by at least two pathways that can operate in a parallel, or partially redundant, manner. That such redundancy should have evolved makes sense, given the crucial importance of flowering in maintaining the species. The promotive genes identified through genetic analyses have traditionally been assigned into distinct groups based on the sensitivity of the mutant phenotype to environmental conditions, and these groups have formerly been considered to define the pathways (Martinez-Zapater et al. 1994; Coupland 1995). Mutations in a subset of flowering-time genes predominately affect the photoperiodic control of flowering, such that the flowering habit of the corresponding mutant tends toward day-neutrality. Mutations in another subset of floweringtime genes result in delayed flowering without a significant loss of photoperiodic sensitivity—i.e., these mutants flower later than wild-type plants under both photoperiodically inductive and noninductive conditions. Because mechanisms for sensing daylength are evidently intact in the latter mutants, the corresponding genes are supposed to function in an environmentally “autonomous” pathway that acts in parallel with the “photoperiodic” pathway to eventually initiate flowering, even

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under unfavorable conditions (Martinez-Zapater et al. 1994; Coupland 1995; Amasino 1996). Another characteristic of mutants in the autonomous pathway is that they exhibit a significant vernalization response—i.e., the late-flowering phenotype can be fully “rescued” by a long-term cold treatment given to the imbibed seed or young plant. In contrast, cold is largely ineffective to accelerate flowering of the photoperiodic pathway mutants (Martinez-Zapater et al. 1994). Koornneef et al. (1991, 1998a) used double-mutant analysis to examine the epistatic relationships between the commonly studied floweringpromoting genes in an attempt to better define such pathways. The rationale for this type of approach is as follows: if two genes operate in a more-or-less linear pathway, then loss of both genes’ function should confer a phenotype that is similar to that of the single mutant (i.e., the double mutant should flower no later than either single mutant). However, if genes operate in parallel pathways, a significant enhancement of the late flowering might be conferred by combining the mutant alleles. A caveat to this type of genetic approach is that it is only valid when using complete loss-of-function alleles, as enhancement of the phenotype should be expected when partially functional alleles operating in the same pathway are combined. In general, the results of these experiments were inconsistent with the simple assignment of flowering-time genes to independent pathways. This suggests that there is significant interaction (“crosstalk”) between pathways. Another finding from these studies was that flowering was not prevented even when combining mutations in genes considered to act in the photoperiodic and autonomous pathway. Thus, the redundancy of flowering pathways is more extensive than was previously thought. A. Photoperiodic Induction 1. Light Effects on Flowering in Arabidopsis. As in many other plants, both light quality (wavelength) and photoperiod strongly influence flowering time in Arabidopsis. In general, flowering in this species is delayed by red light and accelerated by blue light (Brown and Klein 1971; Eskins 1992). The molecular biology of the major photoreceptors in plants, the red/blue-sensitive phytochromes and green/blue/UV-A-sensitive cryptochromes, has been extensively reviewed and will not be discussed here (Barnes et al. 1997; Cashmore 1998; Whitelam and Devlin 1998; Ahmad 1999; Cashmore et al. 1999; Deng and Quail 1999; Briggs and Huala 1999). Mutations that abrogate synthesis of the phytochrome chromophore and therefore result in an absence of functional phytochrome, or mutations that specifically result in loss of the major light-stable phy-

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tochrome, PHYB, confer early flowering, suggesting that the negative effect of red light is mediated by PHYB. Mutants lacking function of the CRYPTOCHROME1 (CRY1) gene exhibit delayed flowering that is evident in both long and short days (King and Bagnall 1996; Coupland 1997). This phenotype is especially striking when plants are grown under blue light (Bagnall et al. 1996), suggesting that CRY1 mediates blue-light promotion of flowering. In contrast, mutants lacking function of CRY2 (allelic to the previously described flowering time gene FHA) exhibit a much-reduced photoperiodic response, flowering much later than wild type in long days and slightly earlier than wild type in short days (Koornneef et al. 1991; Guo et al. 1998). In addition, constitutive expression of the CRY2 gene in transgenic plants accelerates flowering in short days, but not long days. Unlike in cry1 mutants, flowering in plants lacking CRY2 is accelerated by blue light (Guo et al. 1998). Given the delay in flowering in white light conferred by loss of CRY2 activity, one interpretation of this data is that CRY2 normally acts not as a direct positive regulator under blue, but as a negative regulator of the repression of flowering imposed by PHYB (Guo et al. 1998). 2. The Endogenous Clock. In plants, as in other organisms, one or more molecular mechanisms sustain oscillations with periods of approximately 24 h. The circadian rhythms generated by these endogenous “clocks” allow plants to anticipate daily variations in environmental conditions and thereby optimize their responses to them. One example is the family of LHC genes encoding light-harvesting chlorophyll a/b-binding (CAB) proteins, which are upregulated in a diurnal manner before the expected onset of illumination (Piechulla 1988; Nagy et al. 1988). A large body of physiological evidence implicates the clock in mediating the effects of photoperiod on flowering. Evidence is also accumulating that light quality as well influences flowering time by virtue of its effects on the clock. Thus, the clock has a central and very important role in flowering. How might a self-sustaining oscillatory mechanism in plants be composed at the molecular level? Some clues come from research on the Drosophila (fruit fly) clock mechanism that controls eclosion (emergence from the pupae) and locomotor activity. This clock is essentially comprised of an oscillatory mechanism set up through the interactions between two proteins, TIMELESS (TIM) and PERIOD (PER). Transcription of both the PER and TIM genes increases during the subjective day, from a minimum rate near the onset of illumination (referred to as Zeitgeiber time 0, or Zt0) and reaching a maximum rate at approximately Zt12 (Hardin et al. 1992). Maximal accumulation of PER and

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TIM mRNA is offset 2 to 4 hours, whereas maximal accumulation of the proteins is offset another 2 to 4 hours (So and Rosbash 1997). Thus, PER and TIM protein levels reach a maximum at Zt16-20, a point during which transcription of the genes is rapidly decreasing. In fact, transcriptional repression of the PER and TIM genes is a direct result of the increase in protein levels. Heterodimerization between PER and TIM allow the proteins to gain entrance into the nucleus, where they block the transcription of their own genes by the CLOCK and BMAL1 transcription factors (Vosshall et al. 1994; Gekakis et al. 1995; Darlington et al. 1998). The inhibition of transcription by PER/TIM allows the circadian cycle to begin anew. Constant turnover of the mRNAs and proteins is necessary for the oscillations to continue. The TIM protein is thought to be destabilized through phosphorylation by the product of the DOUBLETIME gene, which is structurally related to the kinase domain of human casein kinase Iε (Kloss et al. 1998; Price et al. 1998). In addition, in the absence of TIM, PER protein fails to accumulate, suggesting that TIM functions directly or indirectly to stabilize PER (Price et al. 1995). Although great progress has been made in understanding the basics of this Drosophila clock mechanism, how the clock operates in plants is mostly unknown. PER, TIM, and other components of the fly clock were discovered through traditional genetic analysis. Arabidopsis displays numerous visible phenotypes that cycle in a circadian manner [e.g., movements of cotyledons and primary leaves (Engelmann et al. 1992), alterations in the rate of hypocotyl elongation (Dowson-Day and Millar 1999), and changes in stomatal aperture (Somers et al. 1998b)], but in all cases these phenotypes are subtle and thus not useful for mutant screening. Millar et al. (1995) generated a synthetic circadian phenotype by expressing the firefly luciferase gene under the control of an LHC gene promoter in transgenic plants. Screens using this LHC:LUC genetic background yielded numerous mutants. The best-characterized, designated toc1-1, exhibits a slightly shorter period length of LHC mRNA expression in both constant light and constant darkness (Millar et al. 1995; Somers et al. 1998b). In addition, the mRNA expression of members of at least one other circadian-cycling nuclear gene family, GRP7/8 (see below), is altered in a similar manner (Kreps and Simon 1997). Although toc1-1 plants were originally reported to be phenotypically indistinguishable from wild-type plants, more careful observations revealed that toc1-1 plants were disrupted in multiple circadian cycling phenotypes. In addition, toc1-1 plants exhibited aberrant floral initiation, flowering earlier than wild-type plants under short photoperiods and later than wild-type plants under long photoperiods (Somers et al. 1998b). These findings suggest that the multiple circadian processes

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and the timing of flowering are controlled either by a single clock, or by multiple related clocks sharing the TOC1 component. The TOC1 gene was recently cloned and found to encode a protein with homology to the receiver domain of response regulators from two-component signal transduction systems (Strayer et al. 2000). A dominant mutation in the LATE ELONGATED HYPOCOTYL (LHY) gene leads to loss of rhythmic mRNA expression of clock-regulated genes and defects in multiple clock-influenced phenotypes, including flowering time and circadian leaf movements (Schaffer et al. 1998). In wild-type plants, LHY mRNA levels oscillate in a circadian manner, whereas LHY mRNA is expressed at a constitutive high level in the lhy mutant (Schaffer et al. 1998). In addition, in transgenic plants containing a singly copy of a lhy mutant allele, cycling of the endogenous wildtype LHY mRNA is suppressed. These findings indicate that LHY is part of a feedback circuit that regulates its own mRNA expression. The LHY gene product is a member of a large family of proteins structurally related to the vertebrate proto-oncogenic transcription factor c-Myb (Martin and Paz-Ares 1997). In Arabidopsis, this family also includes the product of the CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) gene, originally identified as a factor that bound a LHC promoter element essential for its regulation by light and the clock (Wang et al. 1997). Like LHY mRNA, CCA1 mRNA and protein also cycle with a circadian rhythm (Wang and Tobin 1998). Constitutive expression of CCA1 mRNA under control of the strong, viral CaMV 35S promoter (35S:CCA1) in transgenic Arabidopsis, like constitutive expression of LHY mRNA from the mutant lhy allele, leads to the disruption of the circadian mRNA expression patterns of various clock-regulated genes, including LHY, and such plants exhibit delayed flowering in long-day conditions (Wang and Tobin 1998). These findings suggest that both LHY and CCA1 are potential key components of a central clock mechanism. That the clock defect conferred by loss of CCA1 function is apparent even in the presence of LHY activity indicates that, despite their structural similarities and similar effects by constitutive expression, LHY and CCA1 do not have strictly redundant roles (Green and Tobin 1999). Like that of TIM, the activity of the CCA1 and LHY proteins may be negatively regulated by phosphorylation. Both proteins are substrates for the protein kinase CK2 in vitro (Sugano et al. 1998, 1999). Constitutive expression of CKB3 mRNA, encoding a regulatory subunit of CK2, in Arabidopsis mimics the effects of loss of CCA1 function, substantially shortening the rhythm periods of multiple clock-regulated genes. However, in contrast to the delay of flowering conferred by loss of CCA1, flowering is accelerated in 35S:CKB3 plants (Sugano et al. 1999).

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GIGANTEA (GI) is another member of the set of genes involved in the promotion of flowering by long days. In plants grown in a regular lightdark photoperiod, mRNA levels for GI oscillate in a diurnal pattern, and studies in which plants were kept in constant light or darkness indicate that GI is under clock control (Fowler et al. 1999; Park et al. 1999). In 35S:CCA1 or 35S:LHY plants, this rhythmic expression pattern is disrupted, indicating that GI is regulated by these genes (Fowler et al. 1999). However, disruption of GI function also affects expression of CCA1 and LHY mRNA. In some gi mutant backgrounds, the amplitude of CCA1 and LHY oscillations are diminished and periodicity becomes less obvious (Fowler et al. 1999; Park et al. 1999). The finding that GI may act both upstream and downstream of the clock genes CCA1 and LHY suggests that GI is intimately associated with the clock. Mutants lacking GI function also exhibit reduced seedling deetiolation under red light, suggesting that GI could be involved in PHYB signaling (Huq et al. 2000). How GI might carry out its function has not been determined. GI was recently cloned and encodes a large protein that is predicted by computer modeling to contain transmembrane domains (Fowler et al. 1999; Park et al. 1999). However, more recent evidence indicates that the GI protein is localized to the nucleus (Huq et al. 2000). The GI transcript was detected in both very young seedlings and mature plants, and is apparently not restricted to any specific tissue type (Fowler et al. 1999). Open reading frames have been identified from both rice and maize that would encode proteins with significant amino acid sequence identity to the GI protein (Fowler et al. 1999; J. Liu and S. van Nocker, unpublished data). Because structural homology is often associated with functional homology, it is possible that these monocot GI orthologs are also involved in flowering. The current efforts to better characterize the rice genome, and determine gene function in maize through reversed genetics approaches, will allow this idea to be tested (Goff 1999; Martienssen 1998; Walbot, 2000). Other potential clock genes include members of the GRP7/8 (also called CCR1/2) family. These genes encode small proteins containing an interesting bipartite structure (van Nocker and Vierstra 1993; Carpenter et al. 1994). The amino-terminal domain contains a specific RNA-binding consensus sequence termed the RRM motif (found also in the flowering-time genes FCA and FPA; below), whereas the carboxyl-terminal region is greatly enriched in glycine residues, a configuration seen in many plant cell wall proteins (Showalter 1993; Cassab 1998). These genes are expressed to high levels in meristematic tissues, and, in addition to being regulated in a circadian pattern, are upregulated by lowered temperatures

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(Heintzen et al. 1994; Kreps and Simon 1997). The protein products of these genes also oscillate with a circadian period, and are localized to the nucleus (Heintzen et al. 1994). Constitutive expression of the GRP7 gene in transgenic Arabidopsis suppresses the circadian oscillations of mRNAs for both the endogenous GRP7 gene and for GRP8, suggesting that the respective proteins are involved in a mutual, autoregulatory feedback loop. This effect of GRP7 on the oscillations of its own transcript is not mediated entirely through its promoter, suggesting that at least some regulation occurs at the posttranscriptional level (Staiger and Apel 1999). As previously mentioned, GRP7/8 expression is affected by impairment of TOC1 function. Interestingly, however, unlike in toc1 mutants, the rhythmic expression patterns of specific clock-regulated genes were not affected in 35S:GRP7 plants (Heintzen et al. 1997). This suggests both that the GRP7/8 clock acts downstream from TOC1, and that the output of the AtGRP7/8 oscillator is limited. The function of the so-called AtGRP7/8 “slave” oscillator is not known, as phenotypic abnormalities associated with constitutive expression of GRP7 have not been reported. In light of the intimate relationship between circadian rhythms and flowering, it is reasonable to hypothesize that these genes are somehow involved in the regulation of flowering time. On the other hand, genes encoding small proteins exhibiting the RRM motif/glycine-rich bipartite structure have also been identified in mammals, amphibians, ascidians, and cyanobacteria (Nishiyama et al. 1997; Danno et al. 1997; Uochi and Asashima 1998; Tanaka et al. 2000; Maruyama et al. 1999). At least a subset of these genes cycle in a diurnal manner and/or are inducible by lowered temperatures (Nishiyama et al. 1997, 1998; Sato and Maruyama 1997; Maruyama et al. 1999; Danno et al. 1997). Thus, these RRM-GRP proteins may carry out a function that is conserved among kingdoms. 3. Entrainment of the Clock by Light. A notable feature of clocks in all organisms yet studied is that the innate period is somewhat longer or shorter than 24 h. In order to cycle with a precise daily rhythm, the clock must be entrained, or synchronized, each day. In Drosophila, light serves to entrain the clock by initiating the phosphorylation and rapid degradation of the TIM protein (Hunter-Ensor et al. 1996; Myers et al. 1996; Zeng et al. 1996). This results in a phase delay in the evening, when TIM is being continually resynthesized, and a phase advance in the morning, when TIM is not effectively replaced. The light signals are perceived by cryptochrome, a protein that is similar in amino acid sequence to the CRY proteins in plants. Upon illumination, CRY undergoes a photochemical change that allows a physical interaction with the TIM protein and presumably initiates the degradation process (Ceriani et al. 1999).

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As in other organisms, under constant illumination, the period of the clock can be modulated by light in an intensity-dependent manner (Somers et al. 1998a,b). Although this phenomenon may not be physiologically relevant, it has proven useful for determining the potential role of specific genes in the light entrainment of the clock. Millar et al. (1995) found that both red and blue wavelengths were effective in shortening the period of LHC:LUC expression, suggesting the involvement of phytochromes and potentially also cryptochromes. By examining the period length of LHC:LUC expression in phyA, phyB, cry1, or cry2 mutant plants, Somers et al. (1998a) concluded that red light signals are transmitted to the clock by phytochromes A and B, with PHYA acting under low intensities and PHYB acting under high intensities, whereas blue light inputs are provided by PHYA and cryptochromes. Interestingly, loss of CRY2 shortened the period under low-fluence blue light, but had little effect under the higher fluences where photoperiodic timing of flowering is affected. Unlike in Drosophila, no direct interactions between photoreceptors and clock components have been demonstrated in plants, and clock function may depend on a mechanism to transmit information from photoreceptors to the clock. The EARLY FLOWERING 3 (ELF3) gene may play such a role. Plants lacking ELF3 activity display phenotypes that mimic phyB mutations (i.e., increased hypocotyl elongation in red light and petiole length). However, mutations in ELF3 and PHYB have additive effects when combined, suggesting that ELF3 is not simply a component of a PHYB signal transduction pathway (Reed et al. 2000). Unlike mutations in PHYB, which alter the periodicity of the clock (above), mutations in ELF3 abolish rhythmicity, and do so in a light-dependent manner (Hicks et al. 1996). The CCA1 gene is rapidly and transiently upregulated in response to light, specifically red light (Wang et al. 1997), suggesting that CCA1 could also be involved in transmitting signals to the clock from phytochrome. Whether or not the phosphorylation of CCA1 or LHY is associated with their turnover, as is the case with TIM, has not been reported. However, as in the Drosophila clock, protein degradation is an important process of the plant clock mechanism. Two homologous genes have been identified in Arabidopsis that might play a role in the turnover of clock components. These genes, FKF1 and ZEITLUPE (ZTL), encode proteins containing an F-box motif (Nelson et al. 2000; Somers et al. 2000). Where studied in other organisms, F-box-containing proteins act in the recognition of degradation substrates for the ubiquitin proteolytic pathway (Patton et al. 1998; Kornitzer and Ciechanover 2000). FKF1 transcripts oscillate in a circadian manner, whereas ZTL mRNA expression

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is apparently not under clock control. However, mutations in both genes confer a similar phenotype, with flowering delayed primarily under photoinductive conditions. In addition to the F-box, the FKF1/ZTL proteins contain a segment similar to the flavin-binding domain in the bluelight receptor NPH1 involved in phototropism. At least ztl mutants exhibit a period-lengthening phenotype that is strongly light-dependent, and at least the FKF1 promoter is selectively activated under white or blue light. Taken together, these observations indicate that these proteins may function as light-dependent clock regulators (Nelson et al. 2000; Somers et al. 2000). 4. Other Photoperiodic Pathway Genes. CONSTANS (CO), another of the promotive photoperiod pathway genes, was one of the first of the flowering-time genes to be cloned, and thus has been the most extensively studied (Putterill et al. 1995). The CO protein contains zincfinger-type DNA-binding domains common to the GATA1 family of transcription factors, and thus likely acts as a component of the transcriptional apparatus. Known mutations in CO are semidominant. Where a mutation results in complete loss of function of the gene, semidominance is an indicator that the respective gene product is limiting for the respective process (i.e., that relative levels of the gene product are important). Consistent with this, increasing CO activity, either constitutively through adding extra copies of the gene in transgenic plants (Putterill et al. 1995), or transiently by activating the protein in an inducible system (Simon et al. 1996), is sufficient to trigger flowering. In addition, consistent with its role in promoting flowering under photoinductive conditions, the mRNA levels of CO are elevated in long-day grown Arabidopsis plants relative to short-day grown plants (Putterill et al. 1995), and this regulation is accomplished at least in part by transcriptional upregulation of the gene (Suarez-Lopez et al. 1998). CO was found to be expressed in both leaf and stem tissue, but the very low abundance of the mRNA complicated a more thorough analysis of spatial expression patterns (Putterill et al. 1995). Its likely function as a transcription factor and regulation at the transcriptional level suggests that CO is an intermediate in a cascade of transcriptional events. What could be upstream regulators and downstream targets of CO? CRY2 and PHYB likely act upstream of CO as indirect positive and negative regulators, respectively, because mutations in CRY2 decrease CO mRNA expression (Guo et al. 1998), and the early-flowering seen in phyB mutants in short days is alleviated in a genetic background compromised for CO activity (Putterill et al. 1995). Onouchi et al. (1998) clarified the relationship between CO and several other photoperiodic

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pathway genes by examining the effect of 35S:CO expression in photoperiodic pathway-mutant backgrounds. The premise of this experiment was that if CO acted in a genetic pathway downstream from GENE X, then removal of GENE X function should have no effect on the phenotype conferred by constitutive expression of CO (i.e., constitutive expression of CO would be epistatic to loss of GENE X function). In contrast, if CO acted upstream of GENE X, then adding CO activity should make no difference to the phenotype conferred by loss of GENE X function. In this case, the 35S:CO transgene was completely epistatic to gi and lhy, suggesting that CO acts downstream from these two genes. In contrast, 35S:CO had only a small effect in genetic backgrounds in which expression of the FT or FWA flowering-time genes was disrupted (see below), suggesting that these two genes function downstream from CO. Extending this experimental approach, Onouchi et al. (2000) discovered a target of CO by searching for mutations that suppressed the earlyflowering phenotype conferred by constitutive CO expression. This gene, designated SUPPRESSOR OF CONSTITUTIVE EXPRESSION OF CO1 (SOC1), encodes a protein containing a domain designated the MADS box. This motif is present in other proteins known to bind DNA as homo- or heterodimeric complexes (Trobner et al. 1992; Riechmann and Meyerowitz 1997). SOC1 is expressed in the shoot and inflorescence apical meristem as well as the leaf primordia in response to inductive photoperiods (Samach et al. 2000). Consistent with the results of Onouchi et al. (1998), Samach et al. (2000) identified the FT gene (see below) as a very early downstream target of CO activity. In the approach used here, the CO coding sequence was translationally fused to the ligand-binding domain of the rat glucocorticoid receptor. This CO-GR fusion protein was expressed constitutively in transgenic plants, and could be directed to the nucleus and thus “activated” by application of the synthetic glucocorticoid hormone dexamethasone. In this case, FT transcript accumulation was seen within two hours of dexamethasone application. This experimental approach also resulted in the identification of two other early downstream targets of CO, AtP5CS2 involved in proline biosynthesis, and ACS10, encoding a potential 1-aminocyclopropane-1-carboxylic acid (ACC) synthase involved in the production of ethylene. AtP5CS2 is apparently essential for the elongation of the internodes that occurs upon flowering in Arabidopsis (bolting), as reduction in AtP5CS2 expression in transgenic plants eliminated this response (Nanjo et al. 1999). Although ethylene plays an obvious role in flowering in other species (e.g., Bromeliads), the precise role of ethylene in flowering in Arabidopsis is not known (Bernier et al. 1981). Mutants insensitive to ethylene exhibit slightly

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delayed flowering, but the molecular mechanism of this effect has not been explored (Guzman and Ecker 1990). CO exists as a member of a gene “family,” or group of genes that encode structurally related proteins (Ledger et al. 1996). It is possible that these CO-like (COL) genes also have a role in flowering, however Putterill et al. (1997) have noted that at least one of these genes, COL1, does not seem to be expressed at higher levels in inductive photoperiods, and to date has not been identified as important in flowering by traditional genetic analyses. In other species studied [i.e., apple (Hoon et al. 1999) and Brassica napus (Robert et al. 1998)], families of CO homologs also exist. It has been suggested that the function of CO in apple could be different from that in Arabidopsis based on the apparent abundance of mRNAs of two of the apple genes in the developing flower and fruit (Hoon et al. 1999), but such studies are complicated by the ambiguity of the evolutionary relationships between the identified apple genes and the Arabidopsis CO and COL genes, and the fact that the spatial pattern of CO expression in Arabidopsis has not been fully established. In Brassica napus, CO homologs are found at genomic positions corresponding to quantitative trait loci (QTL) affecting flowering time, and at least one of the Brassica CO homologs is functionally homologous to its Arabidopsis counterpart, because it is able to complement the flowering-time defect conferred by the co-2 mutation when expressed in transgenic Arabidopsis (Robert et al. 1998). A gene encoding a protein closely related to CO has recently been cloned from the short-day plant Pharbitis nil through an assay designed to identify genes that are upregulated in response to inductive photoperiods (i.e., short days; J. Liu and H. Kende, pers. commun). This is an important finding because the fact that CO is upregulated in both species in response to inductive photoperiods, even though the plants are of opposite flowering habits, suggests that the molecular mechanisms that are distinct between plants of varying photoperiodic responses lie genetically upstream of CO. In maize, ancestrally a short-day plant, the INDETERMINATE1 (ID1) gene promotes flowering in response to inductive photoperiods (Singleton 1946; Galinat and Naylor 1951). This is the only gene cloned to date that has unequivocally been demonstrated to be involved in flowering time in species other than Arabidopsis. ID1 encodes a protein containing zinc-finger motifs, suggesting that it binds DNA. It is expressed predominately in the leaf and influences flowering in a noncell-autonomous manner, and thus possibly regulates the production of a transmissible signal (Colasanti et al. 1998). There is no strong structural homology between ID1 and any of the Arabidopsis flowering-time genes that have been cloned to date, and the family of ID1-like genes that

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do exist in Arabidopsis (Colasanti et al. 1998) have not yet been reported to be involved in flowering. This could indicate a significant divergence in flowering mechanisms between Arabidopsis and maize. B. Non-photoperiodic Induction: The Autonomous Pathway As mentioned previously, loss of function of photoperiodic pathway genes does not prevent flowering but merely delays it, suggesting that at least one redundant pathway exists. Numerous flowering-time genes have been identified that are presumed to work outside of the photoperiodic control of flowering in the so-called autonomous pathway. LUMINIDEPENDENS (LD) was one of the first flowering-time genes identified (Redei 1962), and one of the first plant genes cloned through T-DNA mutagenesis (Lee et al. 1994a). In this technique, segments of DNA of known sequence are transferred into a plant by Agrobacterium, where they integrate into the genome at random locations. Interruption of a gene by the T-DNA often results in loss of gene function, and the corresponding gene sequence can be easily cloned by simple molecular techniques (Azpiroz-Leehan and Feldman 1997). The LD gene encodes a large protein containing two interesting structural features. First, a homeodomain—a nucleic acid-binding motif found in developmentally important proteins from yeast, plants, and animals—is found near the amino terminus. The homeodomain in LD is highly homologous to that found within the Drosophila Distal-less protein, which functions as a developmental switch to initiate limb formation (Cohen 1990). It also closely resembles the homeodomain found in Mata1, a yeast protein that acts as one component of a heterodimeric factor that represses expression of haploid-specific genes (Johnson and Herskowitz 1985). The other interesting structural feature is an acidic carboxyl-terminal region enriched in glutamine residues and containing short, homopolymeric glutamine stretches. These structural features are common to the activation domains of known transcriptional activators such as Drosophila Antennapedia and herpes virus VP16 (Gerber et al. 1994; Triezenberg 1995). Thus, it is possible that LD acts as a transcriptional regulator. Consistent with this proposed role, the LD protein contains nuclear localization signals and is localized to the nucleus. LD is expressed ubiquitously throughout the plant, with a concentration of mRNA expression in proliferating tissues, including the shoot, root, and floral apices (Aukerman et al. 1999). The function of LD may have diverged through evolution. An orthologous gene has been characterized from maize (van Nocker et al. 2000). The maize LD gene is highly homologous to its Arabidopsis counterpart,

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containing both the homeodomain and the potential transcriptional activator region, and exhibits an analogous mRNA expression pattern in the maize plant. However, when expressed in transgenic Arabidopsis containing an ld mutation, it does not complement the flowering-time defect, but instead causes developmental abnormalities associated with the shoot and floral meristems (van Nocker et al. 2000). What function this gene has been recruited for in maize is not presently known, but should be revealed by analyses of transposon-tagged lines. Although the activity of most plant genes studied to date seems to be controlled predominately at the transcriptional level, recent evidence suggests that posttranscriptional control may be an important factor in the regulation of flowering. The FCA gene was cloned and found to encode a large protein containing RRM motifs thought to mediate binding to RNA (Macknight et al. 1997). In support of the structural suggestion of function, the FCA protein binds to RNA in vitro, with a preference for G- and U-rich sequences (Macknight et al. 1997). The FCA gene produces multiple transcripts as a result of alternative splicing and transcriptional termination. Only one of these, which is a minority of FCA transcripts, would encode the presumed, full-length protein, and splicing to produce the full-length “active” FCA mRNA is likely to be regulated, as high-level expression of the genomic FCA sequence in transgenic plants resulted in only a minor increase in the amount of active mRNA (Macknight et al. 1997). Interestingly, it appears that FCA is able to promote flowering in a cell non-autonomous manner, because flowering is not delayed in periclinal chimaeras that express FCA only in the epidermal cell layers (Furner et al. 1996). In addition to the RNA-binding motifs, the presumed active form of the FCA protein contains a region designated the WW motif that contains two closely spaced tryptophan residues (Bork and Sudol 1994). This is potentially an essential component of the FCA protein, as it is excluded from the protein encoded by the strong fca-1 allele (Macknight et al. 1997). In other systems, WW motifs mediate interactions with protein partners containing proline-rich regions (Kay et al. 2000). Proteins or RNAs that interact with FCA have not been identified. One possibility is the protein encoded by FY. Mutations in FY do not further enhance the late flowering conferred by loss of FCA function (Koornneef et al. 1998a), suggesting that the two gene products operate in close proximity. In contrast, mutations in two other autonomous pathway genes, FPA and FVE, greatly enhance the lateness of fca (and fy) mutants. This suggests some redundancy in the mechanism of the autonomous pathway. However, as previously cautioned, this type of genetic analysis is contingent on mutations creating a complete loss of function, and even

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in cases where the gene has been cloned, this is difficult to demonstrate. FPA was recently cloned and, like FCA, encodes a protein containing RRM-type RNA-binding motifs (R. Amasino, pers. commun.). The cloning of FY, FVE, and another autonomous pathway gene, FLD, have not yet been reported. Semidominant mutations in the SHORT VEGETATIVE PHASE (SVP) gene confer photoperiod-sensitive early flowering (Hartmann et al. 2000). SVP encodes a MADS-box transcription factor that is expressed in the apical meristem during the vegetative phase, but apparently not in the inflorescence apical meristem. This expression pattern is consistent with its role as a repressor of flowering. SVP mRNA is also expressed in the early floral meristem, suggesting a role in flower development. However, loss of SVP function confers no gross floral defects, indicating that a function at this stage is redundant or minor (Hartmann et al. 2000). The genetic relationships between SVP and other autonomous pathway genes have not been characterized. C. Vernalization In many plants, flowering can be accelerated or induced by exposure to a long period of near-freezing temperatures. This is a commonly employed reproductive strategy that allows for flowering and seed production in the environmentally favorably period following natural winter. This phenomenon, termed vernalization, has been studied for decades at the physiological level but only recently at the molecular level. The lack of molecular work addressing vernalization is partly due to the fact that in Arabidopsis thaliana, the commonly utilized laboratory strains flower soon after germination, and extended cold treatments do little to further abbreviate the vegetative phase (Koornneef et al. 1998b). However, most natural ecotypes of Arabidopsis behave as winter annuals, flowering extremely late in the absence of cold, but very early when exposed to cold for extended periods. The flowering habit among natural ecotypes is largely determined by allelic variation at two loci, designated FRIGIDA (FRI) and FLOWERING LOCUS C (FLC); (Lee et al. 1993; Koornneef et al. 1998b). “Early” alleles at either loci behave similarly to presumed null alleles created by induced mutation, suggesting that natural early alleles have lost function (Michaels and Amasino 1999). FLC is expressed predominately in the vegetative apex and roots, but is absent from the inflorescence apex. Expression of FLC mRNA is apparently not significantly decreased as the plant proceeds through the vegetative phase, suggesting that repression of flowering by FLC can be overcome by developmental progression (Sheldon et al.

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1999). FLC encodes a MADS-box-containing protein (Michaels and Amasino 1999; Sheldon et al. 1999). Because other MADS-box proteins are known to work as heterodimers (Trobner et al. 1992), it is possible that FLC has a DNA-binding partner. One possibility is SVP, as both genes are expressed in the shoot apical meristem specifically during the vegetative phase. However, unlike those of FLC, mRNA levels of SVP are not diminished after extended cold treatments (Hartmann et al. 2000). The activity of FLC is semidominant, and transgenic plants containing extra copies of the FLC genomic sequence never flower without cold, acting in essence as biennials (Michaels and Amasino 1999; Sheldon et al. 1999). Importantly, these findings suggest that the difference in flowering habit between winter-annual plants and biennial plants could be quantitative rather than qualitative. The cloning of FRI has recently been reported; this gene encodes a protein that does not exhibit significant sequence identity to any other protein of known function (Johanson et al. 2000). In Arabidopsis, a genotype conferring the winter-annual habit can also be synthesized by impairing the function of the promotive autonomous pathway genes (Koornneef et al. 1991), and, like repression of flowering imposed by FRI, the block to flowering resulting from the loss of autonomous-pathway gene function is also dependent on FLC activity (Lee et al. 1994b; Koornneef et al. 1994; Sanda and Amasino 1996a,b). These data suggest that the flowering-repressive activity of FLC is both positively regulated by FRI and negatively regulated by autonomous pathway genes. Consistent with this idea, FLC mRNA expression is increased both in genotypes containing late FRI alleles, and in autonomous-pathway gene mutants (Michaels and Amasino 1999; Sheldon et al. 1999). FLC mRNA expression is decreased after extended cold exposures (Michaels and Amasino 1999), suggesting that vernalization involves molecular events “upstream” from FLC. The winterannual habit conferred by loss of autonomous-pathway gene function is not dependent on FRI, indicating that neither the activity of FRI nor the autonomous pathway genes is necessary for the vernalization response. Thus, although these genes set up a requirement for cold for flowering, they are unlikely to be directly involved in the associated cold signal transduction. Specific components involved in transmitting the signal from the cold stimulus to FLC expression have not yet been identified. Using a genetic approach, Chandler et al. (1996) identified at least two loci, designated VRN1 and VRN2, that could play such a role. These mutants were isolated in an fca mutant background based on a lack of vernalization response. FLC mRNA levels are only partially decreased after

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cold treatment in these mutants, consistent with the idea that VRN1 and VRN2 function upstream to regulate FLC expression (Sheldon et al. 2000). Ishitani et al. (1998) identified a recessive mutation, hos1-1, that constitutively activated gene expression from a cold-responsive promoter. hos1-1 plants exhibited accelerated flowering, suggesting that HOS1 might normally act as a negative regulator of vernalization. However, these results were difficult to interpret because hos1-1 conferred pleiotropic effects on growth, and the specific genetic background utilized (C24) is normally early flowering due to an “early” FLC allele (Sanda and Amasino 1996a). The well characterized cold-regulated (COR) genes involved in the process of acclimation probably have little or no role in vernalization, as freezing tolerance is not affected in the vrn mutants (Chandler et al. 1996), and constitutive expression of members of the CBF family of transcriptional activators upregulates COR gene expression in a winter annual line in the absence of cold, but has no effect on flowering time (J. Liu and S. van Nocker, manuscript submitted). Some characteristics of vernalization, including the requirement for cell division for the vernalized state to be attained and the stability of the vernalized state through mitosis, suggests an epigenetic mechanism (Wellensiek 1964). One possibility is the covalent modification of DNA through cytosine methylation. Evidence for the involvement of DNA methylation in the vernalization response has been presented by Burn et al. (1993) and Brock and Davidson (1994), who found that the promotion of flowering by extended cold in Arabidopsis and wheat, respectively, could be partially substituted for by exposure of plants to the ribonucleotide analog 5-azacytidine (5-azaC). Treatment with this compound results in demethylation of DNA. In the study by Burn et al. (1993), flowering was reportedly accelerated only in genotypes that are known to exhibit a strong vernalization response. Thus, the partial substitution for cold treatment conferred by 5-azaC apparently acted specifically upon the vernalization pathway. It was hypothesized that extended cold results in the selective demethylation and transcriptional activation of floral-promotive genes (Finnegan 1998). A further possible link between DNA methylation and vernalization was hypothesized by Finnegan et al. (1996), who reported that antisense expression of the METHYLTRANSFERASE1 (MET1) gene in transgenic Arabidopsis resulted in both decreased genomic DNA methylation levels and early flowering. This early flowering was apparently associated with decreased FLC mRNA abundance (Sheldon et al. 1999), again suggesting specificity for the vernalization pathway. In contrast to these results, Ronemus et al. (1996) found that MET1 antisense expression

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conferred highly pleiotropic effects, including slightly delayed flowering, in transgenic Arabidopsis. The apparent contradictions between these two reports could reflect differences in environmental conditions or genetic backgrounds used. However, neither group utilized a genetic background that exhibits a strong vernalization response. Other aspects of these results should also be interpreted with caution. Goto and Hamada (1988) and Chandler and Dean (1994) demonstrated that growth of plants on the nucleotide analog 5-bromodeoxyuridine, which does not result in reduced DNA methylation, could also accelerate flowering. D. Induction by Gibberellins The influence of GAs on flowering in many plants is well known (Lang 1965; Zeevaart 1983). In long-day rosette plants such as Arabidopsis, GAs generally have an inductive effect, and this is especially striking in Arabidopsis where flowering is delayed by growth in short days, or in winter-annual genotypes grown in the absence of cold. Consistent with this, flowering is delayed in the ga1 mutant that is defective in GA biosynthesis, and in the gai mutant, which is insensitive to GAs (Koornneef and van der Veen 1980; Koornneef et al. 1985). In addition, plants carrying mutations in the SPINDLY (SPY) gene, which exhibit a constitutive GA response, flower early (Jacobsen and Olszewki 1993). Exogenously applied GA is able to promote flowering in all late mutants studied, and mutations in GA biosynthesis or perception are interactive with all flowering-promotive genes studied, especially those grouped into the photoperiodic pathway (Putterill et al. 1995; Simpson et al. 1999; our independent observations). Consistent with the strongly interactive effect with photoperiod pathway genes, ga mutant plants are apparently unable to flower when grown in short days, and gai plants flower extremely late under such conditions (Wilson et al. 1992). Thus, it appears that the production of GAs represents an additional pathway to flowering that operates in parallel with the photoperiodic pathway, and, to some extent, the autonomous pathway as well. The role of GAs in vernalization is unclear. Although GAs are able to promote flowering in winter-annual genotypes (thereby bypassing the requirement for cold), winter-annual genotypes containing the ga or gai mutations still exhibit a normal vernalization response (R. Amasino, pers. commun.; Chandler et al. 2000). This would suggest that GAs are not necessary for vernalization. However, as GA production is not completely eliminated in the ga mutant (J.A.D. Zeevaart, pers. commun.), such results should be interpreted with caution. In addition, although GAI has an obvious role in GA signal transduction during vegetative

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growth, other yet unidentified GA-signaling components could be involved in the flowering response.

IV. ROLE OF CARBOHYDRATES Carbohydrates have long been known to play a key role in flowering (Bernier et al. 1993). The concentration of sucrose, the major translocated sugar in most plants, increases dramatically in phloem exudates upon photoinduction in both short-day and long-day plants, even when the photoinductive treatment does not result in a net increase in photosynthesis (Bodson and Outlaw 1985; Houssa et al. 1991; Corbesier et al. 1998). One of the earliest biochemically detectable changes in the shoot meristem upon photoinduction is the accumulation of sucrose (Bodson and Outlaw 1985) and labeling experiments suggest that this sucrose originates not from increased photosynthesis, but from mobilization of sugars from reserve carbohydrates such as starch in the leaves and stem (Bodson et al. 1977). Arabidopsis will flower in complete darkness if the aerial portion of the plant is supplied with sucrose or glucose (Redei et al. 1974; Goto 1982; Araki and Komeda 1993). Under such conditions, the lateflowering phenotype conferred by mutations in GI, CO, FCA, FPA, and FVE was complemented or nearly complemented. In contrast, flowering was not promoted by these conditions in plants carrying mutations in FWA or FT (Araki and Komeda 1993; Roldan et al. 1999). These surprising results suggest that the fundamental mechanism of both the photoperiodic and autonomous pathways could be the delivery of sugars to the shoot apex! Sucrose is synthesized in the cytosol from the products of photosynthesis or starch degradation, transported to and loaded into the phloem, translocated throughout the plant, unloaded from the phloem, and then transported from cell to cell. This complicated routing provides many opportunities for control of sugar transport, and thus it is likely that many genes are involved. Interestingly, mutations in the GI gene are pleiotropic in that mutants accumulate excess levels of starch in the leaves and stem (Eimert et al. 1995). At least one other Arabidopsis mutant that was originally identified as a starch accumulator, carbohydrate accumulation mutant1 (cam1), was found to flower late relative to wild-type plants, especially when grown under continuous light (Eimert et al. 1995). High starch content per se does not seem to be the direct cause of the flowering-time defect, because the flowering-time defect conferred by gi and cam1 was not rescued in genetic backgrounds where starch synthesis was dis-

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rupted (Eimert et al. 1995). Other mutants that lack starch, ADP-glucose pyrophosphorylase1 (adg1) and phosphoglucomutase1 (pgm1), and at least one other mutant that accumulates starch, starch-in-excess1 (sex1), also exhibit delayed flowering, but only under photoperiods of less than 16 h (Lin et al. 1988; Caspar et al. 1985; Caspar et al. 1991; Corbesier et al. 1998). The observation that both the overabundance of starch, and lack of starch, can affect flowering in a similar manner further suggests that flowering is not directly affected by starch content. In fact, the lack or excess of starch in the pgm1 and sex1 mutants, respectively, seems to disrupt carbohydrate metabolism in a similar manner, as in both mutants soluble sugars (including sucrose) accumulate to abnormally high levels (Caspar et al. 1985; Caspar et al. 1991). Thus, it seems probable that the predominant effector of flowering in these mutants is the levels of sugars. Arabidopsis plants grown at low temperature also accumulate soluble sugars, and this may be related to the delayed flowering seen under these conditions.

V. CONTROL OF MERISTEM IDENTITY The shoot and flower are, in spite of their radical difference in morphology, essentially analogous structures produced by the meristem. The fate of meristems—to generate flowers rather than shoots—is governed by a group of meristem identity genes, which are activated during the transition to flowering. This group of genes in turn controls expression both of the floral organ identity genes, which control the development of the floral organs, and cadastral genes, which regulate the boundaries of expression of the organ identity genes. The molecular biology of flower development is beyond the scope of this review, and the reader is referred to recent excellent discussions on this topic (Bowman 1997; Sessions et al. 1998). A. Meristem Identity Genes As with genes influencing the timing of flowering, genes involved in influencing meristem identity have been identified by screening for mutants in which meristem identity is altered. Such screens have identified genes that both positively and negatively regulate the shoot-toflower transition. In Arabidopsis plants homozygous for the recessive terminal flower 1 (tfl1) mutation, the normally indeterminate inflorescence terminates in a single flower, and lateral shoots develop as solitary flowers (Shannon and Meeks-Wagner 1991; Alvarez et al. 1992).

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Thus, a presumed function of TFL1 is to keep the inflorescence meristem in an indeterminate state. Plants lacking TFL1 activity also flower slightly early, suggesting that TFL1 functions during the vegetative phase as a repressor of the shoot-to-inflorescence transition (Shannon and Meeks-Wagner 1991; Schultz and Haughn 1993). TFL encodes a member of a small protein family exhibiting limited homology to mammalian Raf kinase inhibitor protein (RKIP). RKIP is a membrane-associated protein that regulates Raf-1 kinase, which is intimately involved in signal transduction cascades controlling cell proliferation and differentiation in mammals (Ferrell 1996). The amino-terminus of RKIP is cleaved off to form a small peptide hormone, leading to the speculation that TFL may in a similar manner be the progenitor of a small signaling peptide involved in flowering (Bradley et al. 1997). That intercellular signaling should be involved in flowering is expected, as the meristem must function as a unit to organize flower primordia even though it is composed of clonally unrelated cells (see below). Constitutive expression of the TFL1 gene in transgenic Arabidopsis confers a phenotype that is essentially opposite to that seen in tfl1 mutants—such plants exhibit delayed flowering, and produce secondary inflorescences that are not subtended by cauline leaves (Ratcliffe et al. 1998). Because Arabidopsis flowers are not normally found in association with leaves (bracts), such structures can be interpreted as a conversion of flowers to inflorescence shoots. Conversion of flowers to shoots is also seen in plants carrying loss-offunction mutations in a group of genes best typified by LEAFY (LFY). In plants carrying strong lfy alleles, early-arising (basal) flowers are completely transformed into shoots, whereas those that develop in more apical positions exhibit partial floral character. In plants carrying very weak lfy alleles, secondary shoots subtended by cauline leaves develop at the first few positions normally occupied by flowers (Schultz and Haughn 1991). That flowers eventually do develop even in the absence of LFY activity indicates that other genes function in a partially redundant manner to promote the inflorescence-to-floral switch. One of these is APETALA1 (AP1). Loss of AP1 function phenocopies very weak lfy alleles with respect to inflorescence structure, but dramatically enhances the phenotype of lfy plants, such that in lfy/ap1 double mutants, even the most apical nodes produce structures with strong shoot characteristics (Bowman et al. 1993; Huala and Sussex 1992; Weigel et al. 1992). Strong ap1 alleles also confer a striking floral phenotype—sepals found in the outer whorl of the flower exhibit leaf-like characteristics, and often subtend secondary flowers (Irish and Sussex 1990). This phenotype can be interpreted as a partial reversion of the flower into a shoot, further implicat-

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ing AP1 in meristem identity (Mandel et al. 1992). Strong constitutive expression of both LFY and AP1 in transgenic plants results in premature transformation of the shoot into a flower, mimicking loss of TFL function (Weigel and Nilsson 1995; Mandel and Yanofsky 1995). Incredibly, Arabidopsis LFY is able to accomplish this even in a divergent tree species, aspen, suggesting a high degree of conservation of meristem identity function during evolution (Weigel and Nilsson 1995). That loss-of-function mutations in genes such as LFY and AP1 exhibit additive phenotypic effects when combined is evidence that at least two pathways are normally involved in establishing the floral meristem (Shannon and Meeks-Wagner 1993). The genetic evidence indicating that these pathways are partially redundant is reinforced by experiments showing that the shoot-to-flower conversion conferred by 35S:AP1 is not dependent on LFY, and that mutations in AP1 cannot fully suppress this effect in 35S:LFY plants (Mandel and Yanofsky 1995; Weigel and Nilsson 1995). However, these two pathways are strongly interactive. In primordia destined to become flowers, LFY mRNA expression precedes that of AP1, and AP1 upregulation is delayed in lfy plants (Simon et al. 1996; Hempel et al. 1997; Liljegren et al. 1999). In addition, ectopic activation of LFY activity results in premature AP1 expression (Parcy et al. 1998). These findings suggest that LFY acts as a positive regulator of AP1. Conversely, LFY is expressed prematurely in primordia of 35S:AP1 plants, suggesting a reciprocal positive regulation between the two genes (Liljegren et al. 1999). The protein encoded by LFY does not resemble any other known protein (Weigel et al. 1992), but numerous lines of evidence indicate that it acts as a transcription factor. LFY protein is localized to the nucleus and is able to mediate transcriptional activation in yeast when fused to a suitable activation domain (Parcy et al. 1998). Consistent with the genetic evidence that LFY positively regulates AP1 expression, the LFY protein binds to a potential AP1 promoter element in vitro (Parcy et al. 1998). Moreover, Wagner et al. (1999) demonstrated through the use of an inducible LFY-GR fusion protein that LFY is able to rapidly activate AP1 expression even in the presence of cycloheximide, suggesting a direct interaction. Another gene with a promotive role in floral identity is CAULIFLOWER (CAL). The function of this gene is apparently completely redundant with that of AP1, such that in an otherwise wild-type genetic background, plants lacking CAL activity appear normal. However, in plants lacking both CAL and AP1 activity, meristems never develop determinate floral character and continue to proliferate, and inflorescences develop into structures that resemble tiny versions of the garden

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vegetable for which the gene is named (Bowman et al. 1993). The phenotypic similarity between Arabidopsis cal/ap1 double mutants and cauliflower led Kempin et al. (1995) to investigate functional conservation of CAL genes in the two species. In both Brassica oleracea, and its cauliflower derivative (Brassica oleracea, var. botrytis) CAL is expressed in a spatial and temporal pattern similar to that seen with CAL in Arabidopsis. However, the gene from var. botrytis encodes a protein that is significantly truncated and is probably nonfunctional (Kempin et al. 1995), suggesting a molecular explanation for what is probably the most popularly recognized natural inflorescence variation. Both AP1 and CAL encode proteins containing a MADS-box domain, consistent with roles as transcription factors (Mandel et al. 1992; Kempin et al. 1995). The essentially opposite phenotypes conferred by loss of TFL and LFY/AP1/CAL function indicates that these genes operate antagonistically, and molecular studies support this conclusion. Expression of both TFL and LFY mRNAs is maintained at a low level in the shoot apex during the vegetative phase, and is upregulated upon the transition to flowering. However, their expression is spatially separated, with TFL mRNA present in the center of the meristem, and LFY mRNA present only in emerging primordia (Bradley et al. 1997). In plants lacking LFY, AP1, or CAL activity, TFL expression extends into the lateral primordia (Ratcliffe et al. 1999), whereas in plants constitutively expressing LFY, TFL expression is greatly decreased (Ratcliffe et al. 1999; Liljegren et al. 1999). Like AP1 and CAL1, at least two other genes are known to have redundant roles in promoting floral identity. Mutations in APETALA2 (AP2), for example, do not confer strong flower-to-shoot conversion, but instead enhance both lfy and ap1 phenotypes (Shannon and Meeks-Wagner 1993). Plants carrying mutations in the UNIDENTIFIED FLORAL ORGANS (UFO) gene resemble weak lfy mutants in that basal inflorescence nodes exhibit some shoot identity (Levin and Meyerowitz 1995; Wilkinson and Haughn 1995). In addition, in short-day-grown ufo plants, the transition to an inflorescence meristem is incomplete, and reversion to a vegetative meristem can occur (Wilkinson and Haughn 1995). Like the aforementioned FKF1 and ZTL, UFO encodes an F-boxcontaining protein, implicating this factor in the selective elimination of other, possibly regulatory proteins (Ingram et al. 1995). The AGAMOUS gene, better known for its role in governing floral organ identity in the inner whorls, also functions to promote floral meristem identity by regulating meristem determinacy. The normally determinate floral meristem becomes indeterminate in ag mutant plants, and under short-day conditions can even completely revert to an inflorescence meristem (Yanofsky et al. 1990; Mizukami and Ma 1995, 1997).

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B. Integration of Flowering Pathways and Activation of Meristem Identity Genes Genes affecting the timing of flowering are obvious candidates for direct regulators of meristem identity genes, and recent genetic and molecular studies have demonstrated numerous interactions between the two classes of genes. Simon et al. (1996) utilized the inducible CO-GR expression system described above to examine the response of the LFY, AP1, and TFL1 genes to increased CO activity. Within 24 h of CO:GR activation in plants grown in inductive photoperiods, LFY and TFL1 mRNAs accumulated to detectable levels, and detectable AP1 mRNA upregulation occurred after ~72 h. The delayed expression of AP1 relative to that of LFY is in accordance with genetic evidence that LFY acts upstream of AP1 (above). These kinetics were similar to those seen upon the transfer of short-day-grown plants to inductive photoperiods (Simon et al. 1996). In contrast, when CO-GR was activated in short-day-grown plants, LFY and TFL1 were again activated within 24 h, but the delay in the upregulation of expression of AP1 was extended to ~120 h. The authors concluded that since inductive photoperiods were more effective than CO to activate AP1, an additional, unknown mechanism operating in inductive photoperiods is involved in the upregulation of AP1 (Simon et al. 1996). In fact, the flowering time genes FT and FWA (see below) may play a role that is redundant with that of LFY in activating AP1. Evidence for this was presented by Ruiz-Garcia et al. (1997), who showed that the phenotype conferred by a strong allele of lfy is greatly enhanced in an ft or fwa mutant background. It is known that GAs promote flowering at least in part through upregulation of LFY, because in the ga1 mutant, LFY promoter activity is reduced, and its upregulation in response to inductive photoperiods is delayed relative to wild-type plants. In addition, 35S:LFY expression can partially complement the flowering defect conferred by ga1 in short day conditions (Blazquez et al. 1998). Recently, Blazquez and Weigel (2000) demonstrated that distinct cis elements in the LFY promoter mediate the induction of LFY in response to GAs or inductive photoperiods. Thus, it appears that LFY represents an integration point of at least the photoperiod pathway and the GA pathway.

VI. COMPETENCY A multitude of physiological studies has indicated that flowering is dependent not only on the ability of the leaf to produce the floral stimulus, but also on the ability of the shoot apical meristem to respond to

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it (Lang 1965). The shoot apical meristem is a group of specialized cells found within the apex at the growing tip of shoots. In Arabidopsis, as in other plants, the meristem displays typical tunica-corpus organization, recognizable as early as the torpedo stage of embryogenesis (Long et al. 1996). The tunica layers (L1 and L2) are propogated through anticlinal cell divisions, and thus the L1, L2, and L3 tend to be clonally unrelated. The meristem can display an additional level of organization that is superimposed on the tunica-corpus structure. This consists of radially symmetric “zones” that are often distinguished by mitotic activity and cell size and density (Vaughan 1952; Steeves and Sussex 1989). The central initiation zone, at the summit of the meristem, is characterized by a group of large cells with prominent vacuoles that apparently divide very slowly. Flanking the central initiation zone is a ring of smaller, more densely cytoplasmic, proliferative cells termed the peripheral zone. In the peripheral zone, groups of cells are recruited into leaf or flower primordia where they may soon assume specialized roles. Immediately subtending the central initiation zone is a group of proliferative cells referred to as the rib, or file, meristem that produces the internal tissues of the plant stem. When given inductive photoperiods, the commonly studied annual genotypes of Arabidopsis flower soon after germination. Before the transition to flowering in such young plants, the apical meristem is small and without easily recognized cytological zonation. However, when the vegetative phase is extended (i.e., through growth in short-day photoperiods, or in winter-annual genotypes lacking cold treatment) the zonal pattern described above becomes more apparent (Vaughan 1952; Besnard-Wibaut 1981). The appearance of well-defined zonal organization in Arabidopsis has been correlated with the ability of the plant to exhibit a significant flowering response to applied GAs or long days (Besnard-Wibaut 1981), suggesting that appropriate meristem structure could constitute the morphological basis of competency. The molecular determinants of competency remain unknown. It is important to note that although transgenic Arabidopsis expressing LFY in a constitutive manner flower very early, they still progress through a short vegetative phase (Weigel and Nilsson 1995). That LFY is insufficient to force early-arising primordia into a floral fate suggests that genes controlling meristem competence act genetically downstream, or in a separate pathway, from LFY. Two candidates are FWA and FT. Mutations in these genes cause delayed flowering, and are epistatic to a 35S:LFY transgene [i.e., constitutive expression of LFY is unable to rescue that late-flowering phenotype conferred by fwa and ft mutations (Nilsson et al. 1998)].

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All known mutations in FWA are dominant. Dominance of mutations often indicates that the mutant gene product has gained activity. This could happen if the gene product were increased in abundance, or if it usually existed in the “off” position and were turned “on” by the mutation in a manner similar to the action of an upstream signaling molecule. Indeed, FWA mRNA levels were found to be increased in fwa mutants relative to wild-type plants, suggesting that the dominance of the mutation indeed results from increased FWA activity. Interestingly, it appears that the known mutant alleles of fwa result from epimutation, a class of mutation that does not disrupt the DNA sequence. In the case of fwa mutations, constitutive expression of the gene is associated with a reduction in methylation of DNA residues found in the promoter region of the gene (W. Soppe, pers. commun.). The FWA gene was recently cloned and found to encode a transcription-factor-like protein (M. Koornneef and W. Soppe, pers. commun,). Because a gain in function of FWA results in later flowering, the function of the wild-type FWA gene is likely to repress flowering. Alleles of FT conferring late flowering are recessive and likely result from decreased function, suggesting that FWA acts in a manner opposite that of FT. The Arabidopsis FT gene was recently cloned by activation tagging (Kobayashi et al. 1999; Kardailsky et al. 1999). In this approach, random plant genes are transcriptionally activated by the nearby insertion of T-DNAs containing strong enhancer sequences, and function of the activated gene is surmised based on the resulting phenotype. Activation tagging has become a powerful technique for the identification of genes whose products are normally limiting in a pathway affecting a given phenotype (Lindsey et al. 1998). The product of the FT gene is structurally related to that of TFL, suggesting that like TFL, the FT protein may function in cell-to-cell signaling (Kardailsky et al. 1999). FT mRNA is expressed throughout the aerial tissues of the plant, and is not localized to any specific domain within the shoot apex (Kobayashi et al. 1999; Kardailsky et al. 1999). This expression becomes evident only near the floral transition, and, consistent with being regulated by CO (see above), this upregulation of expression is delayed in the co mutant and in shortday conditions. However, upregulation of expression still occurs in plants lacking CO activity, indicating that FT is also regulated by another mechanism. As might be expected from a gene controlling meristem competence, constitutive expression of FT in transgenic plants results in a nearly complete elimination of the vegetative phase, as such plants form a flower after only ~2 leaves, which are embryonic in origin. Other genes that have been postulated to play a role in competence include EMBRYONIC FLOWER (EMF) 1 and 2. Plants carrying mutant

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alleles of these genes produce a modified flower upon germination (Sung et al. 1992; Bai and Sung 1995; Yang et al. 1995; Chen et al. 1997). Plants carrying strong emf1 alleles develop no leaves at all, indicating that the vegetative phase has been completely bypassed. Mutations in EMF2 confer a milder but similar phenotype, with a few small leaves produced on a modified inflorescence. The very early flowering associated with loss of EMF function suggests that the EMF genes normally act as strong repressors of reproductive development. It is possible that the two genes operate in distinct genetic pathways, as combining strong emf1 and emf2 alleles leads to severe developmental defects that could be considered an additive phenotype (Yang et al. 1995). Although cloning of the EMF genes has not been reported, some indication of their mode of action comes from examining interactions of emf mutations with other mutations affecting the timing of flowering. Mutations in GI or CO, for example, have no effect on emf1 or emf2 phenotype; that CO and GI are not required for the expression of the emf mutant phenotype suggests that CO and GI act as upstream regulators of the EMF genes. However, mutations in other flowering-time genes, including FCA, LD, FVE, FY, FHA, FPA, FWA, and FT resulted in a partial rescue of the early flowering emf phenotype, with mutations in FWA and FT having the greatest effect (Huang and Yang 1998; Page et al. 1999). These results suggest that the EMF genes, rather than having a general repressive effect on flowering, act specifically as downstream negative regulators of the photoperiodic pathway. Finally, it is interesting to note that constitutive expression of LFY was not sufficient to fully rescue the flowering defect conferred by the ga mutation in short days (Blazquez et al. 1998). This suggests that in addition to playing a role in LFY activation, gibberellins play a role in promoting competency. VII. CONCLUSION AND PERSPECTIVES Research into the molecular mechanisms of flowering is now entering its second decade. Many flowering genes have been identified in the model plant Arabidopsis thaliana, and most of these have now been cloned. In addition, the functions of most of the known flowering-time genes have been assigned within one of the multiple parallel pathways that promote flowering in this plant (Fig. 1.1). However, the research accomplished to date should be considered merely as a foundation for future work, as many aspects of flowering in this plant remain unexplored. For example, in many cases, the relationships and interactions among the genes in these pathways have been surmised based solely on

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florigen

COLD vernalization

molecular events at the shoot apex

sugars

VRN1 VRN2 LD, FCA, FPA, FVE, FLD

FT FWA FLC SOC1

FRI

AP1 LFY

GA molecular events at the leaf ATP5CS2

endogenous clock

LIGHT

TOC1, LHY, CCA1, ELF3, FKF1, ZTL, GI

phytochromes cryptochromes

starch

internode elongation

CO

sugars

ACS10

florigen ethylene production

Fig. 1.1. Schematic diagram of molecular pathways leading to flowering presumably operating in the leaf and in the shoot apex. Assignment of gene function to the leaf or apex is based predominantly on the reported mRNA expression pattern of the genes. Relationships among genes depicted here are based on genetic and/or molecular evidence. Arrows indicate a generally positive regulation, whereas lines with blocks indicate a repressive regulation. Not all of the genes referred to in the text are shown. The relationship between the CO gene, the hypothetical substance florigen, and genes acting downstream of florigen is especially speculative.

genetic evidence, and need to be confirmed with molecular and biochemical data. Some more general questions remain to be answered as well. For example, in spite of the wealth of physiological data suggesting that flowering is the result of a transmissible signaling substance (“florigen”), produced in the leaves and acting at the shoot apex, the molecular identity of this signal is still unknown. The possibility that flowering can be manipulated in species other than Arabidopsis through molecular methods is largely dependent on the degree to which flowering-time mechanisms have been conserved through evolution. In order to prove their utility in solving horticultural

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problems, the models proposed to describe floral initiation based on genetic and molecular studies in Arabidopsis will likely need to be evaluated in plants with dissimilar flowering habits. With very few exceptions (see above), this is an area of research that has remained largely unexplored. Probably the most attractive opportunity in this regard is maize, which has traditionally been the most popular monocot model for developmental studies, and has diverged significantly from Arabidopsis in terms of genetics, physiology, and anatomy. Current efforts underway to discover maize gene function by high-throughput, reversed-genetics approaches should greatly simplify this task (Martienssen 1998; Walbot, 2000). The apparent conservation of function of meristem identity genes among species offers some indication that the function of flowering-time genes may be similarly conserved. As the intriguingly complex pathways that constitute flowering become more completely characterized in Arabidopsis, this area holds much promise for future research.

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2 Floral Homeotic Gene Regulation Robert G. Franks and Zhongchi Liu* Dept. of Cell Biology and Molecular Genetics University of Maryland College Park, MD 20742

I. INTRODUCTION A. The ABC Model B. The MADS-domain Multi-gene Family C. The A Class Genes: AP2 and AP1 D. The B Class Genes: AP3 and PI E. The C Class Genes: AG, HUA1 and HUA2 F. Novel Class Genes: SEP1, SEP2, and SEP3 II. CONSERVATION OF THE ABC FUNCTIONS IN ANGIOSPERMS A. Dicotyledonous Species B. Monocotyledonous Species III. POSITIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Meristem Identity Genes: LFY and AP1 B. LFY, a Direct Activator of AP1 C. LFY, a Direct Activator of AG D. Two Phases of Regulation: Initiation and Maintenance of B Gene Expression E. UFO, a Coregulator of B Gene Expression IV. NEGATIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Temporal and Spatial Regulators of AG B. Repression of AP1 Expression in Floral Whorls 3–4 C. Restriction of B Gene Expression to Floral Whorls 2–3 V. SUMMARY LITERATURE CITED

*We thank Beth Krizek for photographs used for Figures 2.2E and 2.2F. Our research on Arabidopsis flower development is supported by the U.S. Department of Agriculture 9835304-6714 (Z.L.), U.S. Department of Energy 02-00ER20281 (Z.L.), and a NIH postdoctoral fellowship GM20426-01 (R.G.F.).

Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 41

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R. G. FRANKS AND Z. LIU

I. INTRODUCTION In the past decade, a major milestone in plant developmental biology has been the elucidation of the molecular genetic basis underlying floral organ specification. An elegant ABC model explains how three classes of genes (A, B, C classes) direct the development of four types of floral organs (Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994). This model enables one to design and engineer the structure of flowers in a predictable manner by altering the expression of these ABC class floral homeotic genes (Jack et al. 1994; Krizek and Meyerowitz 1996a). Although the function of the ABC genes has been studied extensively, only more recently has the regulatory mechanism of their expression been elucidated. Both positive and negative regulators of ABC gene expression have been identified and, in some cases, significant progress has been made toward understanding the molecular mechanisms underlying the regulation of their expression. This review will focus on the regulation of ABC gene expression in Arabidopsis but will highlight similarities and differences among the ABC genes found in other plant species. For simplicity and uniformity, standard Arabidopsis nomenclature is used throughout this review even if a gene is from a different species. Specifically, uppercase letters identify wild-type genes or gene products and lowercase letters identify mutant genes or mutants. Additionally, the names of genes and mutants are shown in italic type, while the names of proteins are not italicized. For example, AGAMOUS (AG) refers to the wild type gene, agamous (ag) refers to the mutation or mutant, and AGAMOUS (AG) refers to the protein. A. The ABC Model The structure of the Arabidopsis flower is typical of many angiosperm flowers (Fig. 2.1A; Fig. 2.2A). It is made up of four types of floral organs arranged in four concentric circles or whorls. The sterile perianth organs, sepals and petals, comprise the outer two whorls while the reproductive organs, stamens and carpels, make up the inner two whorls. Despite dramatic variation in the number, color, and shape of floral organs in different species this arrangement of sepal, petal, stamen, and carpel, from the outermost whorl to the innermost whorl, is fixed in the majority of the angiosperm species. This cross-species similarity suggests that the molecular genetic systems responsible for patterning of floral organs are similar in the majority of flowering plants.

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2. FLORAL HOMEOTIC GENE REGULATION

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A: A diagram of an Arabidopsis flower

B: Wild-type Whorls: 1

Whorls Sepal (Se)

1

Petal (Pe)

2

Se

2

3

4

Pe

Sta

Ca

A

Stamen (Sta) 3 Carpel (Ca)

B

4

C C: Class A mutants Whorls: 1

Ca

A B C

-

2 Sta

D: Class B mutants

3

4

1

2

Sta

Ca

Se

Se

A B C

3 Ca

E: Class C mutants

4 Ca

1

2

3

Se

Pe

Pe

4 Se

A

-

B C

-

Fig. 2.1. The ABC model. (A) A diagram of an Arabidopsis flower showing four sepals in whorl 1, four petals in whorl 2, six stamens in whorl 3, and two fused carpels in whorl 4. (B) In wild-type, the domains of the A, B, and C activities are indicated by the filled boxes. A class activity is only present in whorls 1–2; B class activity is in whorls 2–3; and C class activity is in whorls 3–4. (C) In A class mutants, such as ap2 or ap1, the A activity is absent. This results in the expansion of the C activity into all four whorls and the homeotic transformation of first whorl sepals to carpels and second whorl petals to stamens. (D) In B class mutants such as ap3 or pi, the B activity is absent. As a result, second whorl organs develop into sepals while third whorl organs develop into carpels. (E) In C class mutants, such as ag, the C activity is absent. This results in the expansion of the A activity into all four whorls and the homeotic transformation of third whorl organs into petals and forth whorl organs into sepals and another flower.

The analysis of mutations that alter the pattern of organ identity within the flower has generated a greater understanding of the molecular genetics of floral pattern specification. Floral homeotic mutations result in the substitution or replacement of one organ type by another organ type. (Fig. 2.2B–D; Bateson 1894; Acquaah et al. 1992). The isolation of floral homeotic mutations provided the initial clues to the genetic basis of floral patterning (Meyerowitz et al. 1989). It was the analyses of these floral homeotic mutants and the genetic interactions among them in Arabidopsis thaliana and Antirrhinum majus that led to

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Fig. 2.2. Microscopic photography of Arabidopsis flowers. (A) A wild-type flower with four sepals in whorl 1, four petals in whorl 2, six stamens in whorl 3, and two fused carpels in whorl 4. (B) A strong class A floral homeotic mutant, ap2-2. Two whorl 1 organs are carpel-like (arrow). The other two whorl 1 organs are still sepal-like. All whorl 2 organs are absent, and the whorl 3 consists of a single stamen. The whorl 4 carpels are similar to wild-type. (C) A class B floral homeotic mutant, pi-1. Organs in the outer two whorls are all sepals (arrowhead indicates a whorl 2 sepal). Organs in the inner two whorls are all carpels (arrow indicates a whorl 3 carpel). (D) A class C homeotic mutant, ag-1. Whorl 1 consists of four sepals; whorl 2 consists of four petals; whorl 3, however, is converted into six petals; and whorl 4 is a new flower with similar sepal, petal, petal arrangement (not shown in this picture). (E) A flower from a transgenic plant that ectopically expresses both B and C class genes: AP3, PI, and AG (Krizek and Meyerowitz 1996a). (F) A flower from a transgenic plant that ectopically expresses AP3 and PI and, at the same time, carries the ag-3 mutation (Krizek and Meyerowitz 1996a). Photos of (E) and (F) are gifts of Beth Krizek.

the establishment of the ABC model (Bowman et al. 1991b; Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994). The ABC model places floral homeotic mutants into one of the three classes: A, B, or C and thus defines three classes of gene activities (Fig. 2.1B). In the outermost (first) whorl, the activity of the A class genes specifies the development of sepals. In whorl 2, where both A and B class genes are active, petal development is specified. In whorl 3, B and

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C activities together specify stamen identity, and in the innermost (fourth) whorl, C activity alone specifies carpel development. Thus, the A and B activities are predicted to overlap in whorl 2, while B and C activities overlap in whorl 3. In contrast, the A and C activities are predicted to be present in their respective domains without overlap. To account for this, the model predicts that the A and C activities are antagonistic to each other. The A activity in the first two whorls inhibits expression of C genes and vice versa. This tenant of the model is supported by the altered organ identity displayed in the A class homeotic mutants in which the pattern of organs in whorls one through four is carpel, stamen, stamen, carpel, respectively. This is consistent with the postulate that the C activity has spread to all four whorls (Fig. 2.1C). In C class mutants, the A activity is proposed to spread throughout all four whorls (Fig. 2.1E). Since the primary function of the ABC genes is to specify floral organ identity, the ABC class floral homeotic genes are also termed the “organ identity genes.” Molecular genetic analyses of homeotic mutations in the fruit fly Drosophila melanogaster indicated that homeotic genes encode master regulatory proteins. These transcriptional regulators control developmental programs that cooperate to generate a particular organ type (Gehring and Hiromi 1986). The molecular isolation of members of the ABC class floral homeotic genes indicates that these genes also function as master regulators of organ specific developmental programs (Weigel and Meyerowitz 1994). The ABC genes all encode DNA-binding transcription factors. Furthermore, in situ examination of their RNA expression patterns have largely supported the basic tenets of the ABC model. Their expression domains largely coincide with the domains of their activity as predicted by the ABC model. For example, the A class gene APETALA1 (AP1) is expressed in the first two whorls, while the C class gene AGAMOUS (AG) is expressed in the third and fourth whorls (Drews et al. 1991; Mandel et al. 1992b). Furthermore, the expression of the C class gene AG is expanded throughout all four whorls in the floral meristems of some A class mutants (Drews et al. 1991). Hence, the spatially restricted function of the ABC genes is largely regulated at the RNA level. There are, however, exceptions. For example, the A class gene APETALA2 (AP2) is expressed in all floral whorls although its activity is limited to whorls 1–2 (Jofuku et al. 1994). In this case, AP2 activity is likely regulated by post-transcriptional mechanisms. Clearly, one of the next major challenges in the field of flower pattern formation is to elucidate how the spatially and temporally restricted ABC activities are regulated. Currently, most investigations in this area of research are conducted in Arabidopsis, which will be the focus of this review.

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B. The MADS-domain Multi-gene Family With the exception of AP2, all other ABC genes encode a highly conserved 56 amino acid domain called the MADS-domain and thus are members of a multi-gene family (Riechmann and Meyerowitz 1997a). The name of the MADS-domain was derived from the four founding members: MCM1, yeast; AG, Arabidopsis; DEFICIENS (DEF), Antirrhinum; and SRF, human. The basic N-terminal half of the MADS domain is essential for DNA binding and the C-terminal half of the MADS domain is required for dimerization (Riechmann et al. 1996b). In the majority of plant MADS domain-containing proteins, a second conserved domain, the K box, was identified because of its similarity to the coiled-coil domain of keratin (Ma et al. 1991). The distinctive feature of the K box is the disposition of hydrophobic residues with a spacing that permits the formation of amphipathic α-helices (Ma et al. 1991; Pnueli et al. 1991). Between the MADS domain and the K box is a less strictly conserved linker (L) region. Amino acids in the L region and the K box have been shown to be important for the partner specificity in dimer formation (Riechmann et al. 1996b). MADS-domain proteins function as dimers and bind to a core consensus site CC(A/T)6GG, which is known as the CArG-box (SchwarzSommer et al. 1992; Wynne and Treisman 1992; Huang et al. 1993; Shiraishi et al. 1993). However, different MADS-domain family members can possess related but distinct DNA-binding specificity (Nurrish and Treisman 1995). Nevertheless, functional specificity (i.e. distinct organ identity activity) of the MADS-domain proteins is independent of their DNA-binding specificity. For example, hybrid genes were generated by swapping the amino terminal half of the MADS domain of the Arabidopsis proteins AP1, AP3, PI, and AG with the corresponding portion of human MEF2A or SRF proteins. Such hybrid proteins, having acquired the in vitro binding specificity of MEF2A or SRF, are able to perform the specific functions of the corresponding Arabidopsis genes in transgenic plants (Riechmann and Meyerowitz 1997b; Krizek and Meyerowitz 1996b). Thus, interactions between these MADS proteins with additional cofactors are probably crucial for the specific organ identity functions. Although AP3 and PI can each dimerize with AG or AP1 in vitro, these complexes do not bind to CArG boxes (Riechmann et al. 1996b). Only AP3/PI heterodimers can bind to the CArG boxes. This partner specificity of AP3 and PI suggests that the combinatorial mode of action between A and B genes in whorl 2 and between B and C in whorl 3 is not achieved through direct interactions between A and B proteins in

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whorl 2 or interaction between B and C proteins in whorl 3 (Riechmann et al. 1996a). For example, petal specific gene expression in whorl 2 does not appear to result from the activity of AP1/PI or AP1/AP3 heterodimers. Rather petal identity in whorl 2 is specified by the interaction of genes regulated by AP1 homodimers and genes regulated by AP3/PI heterodimers. C. The A Class Genes: AP2 and AP1 A class genes are defined as those required to specify sepal and petal identity. Interestingly, all A class genes appear to have other functions in addition to organ identity specification. AP2 has at least two functions: the specification of sepal and petal identity and the repression of AG RNA expression in whorls 1–2 (Bowman et al. 1991a; Drews et al. 1991). In strong loss-of-function ap2 mutants, when both functions of AP2 are defective, AG expression is extended to whorls 1–2, causing carpelloid structures in whorl 1 and staminoid petals or loss of petals in whorl 2 (Fig. 2.1C; Fig. 2.2B). Further, AG was found to be expressed precociously at earlier stages and at elevated levels, which may be responsible for the loss of floral organs in strong ap2 mutants (Fig. 2.2B). Unlike other ABC genes, AP2 is unique in that it encodes a novel, putative nuclear protein with two 68 amino acid repeat sequences, dubbed the AP2 domain. The AP2 domain has been predicted to perform functions of protein-protein dimerization and DNA-binding (Jofuku et al. 1994). AP2 is a member of a multi-gene family (Riechmann and Meyerowitz 1998). Studies with other family members such as the ethyleneresponsive element binding proteins (EREBPs) demonstrated that the AP2-domain recognizes and binds to DNA specifically in an 11-bp sequence (TAAGAGCCGCC), the GCC box (Ohme-Takagi and Shinshi 1995). Hence, AP2-domain containing proteins define a novel class of plant transcription factors. AP2 mRNA is detected in all floral whorls as well as in vegetative tissues, indicating that the spatially restricted activity of AP2 in whorls 1–2 must depend on post-transcriptional regulation. AP1 is another A function floral organ identity gene. Unlike AP2, AP1 does not negatively regulate AG expression in whorls 1–2 (GustafsonBrown et al. 1994). In ap1 mutants, whorl 1 organs are bract/leaf-like; whorl 2 organs are usually absent; whorls 3–4 are usually normal (Irish and Sussex 1990). Thus, AP1 activity is required for the development of sepals and petals. Unique to ap1 mutants is the formation of secondary flowers in the axils of first whorl bract/leaf-like organs, suggesting that ap1 mutant flowers partially adopt the fate of inflorescence shoots (Irish and Sussex 1990). This second defect of ap1 in flower/shoot

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transition will be discussed in a later section of this review. AP1 encodes a MADS box protein and AP1 mRNA is initially expressed throughout the floral meristem and later becomes restricted to whorls 1–2 (Mandel et al. 1992b). The spatially-restricted expression of AP1 at later stages is consistent with its role as an A class gene (Bowman et al. 1993; Mandel et al. 1992b; Gustafson-Brown et al. 1994). D. The B Class Genes: AP3 and PI APETALA3 (AP3) and PISTILLATA (PI) are the Arabidopsis B class genes. Mutations in either AP3 or PI cause similar homeotic transformations in whorls 2–3 such that second whorl organs develop as sepals and third whorl organs develop as carpels (Fig. 2.1D; Fig. 2.2C; Bowman et al. 1989; Hill and Lord 1989). AP3 and PI both encode MADS domain proteins that have been shown to bind DNA only as AP3/PI heterodimers (Jack et al. 1992; Goto and Meyerowitz 1994; Riechmann et al. 1996a; Riechmann et al. 1996b; Hill et al. 1998; Tilly et al. 1998). Obligatory heterodimer formation explains why both AP3 and PI are required to specify petal and stamen identity. Ectopic expression studies involve artificially expressing a gene in a new spatial or temporal domain, in which the gene is normally not expressed. The 35S promoter from the cauliflower mosaic virus (CaMV) is a constitutive plant promoter that is frequently used to drive the expression of genes in all tissues and at all developmental stages. If AP3 and PI together are sufficient to confer the B class activity, then ectopically expressing AP3 and PI in all four floral whorls would result in a flower of petals in the outer two whorls and stamens in the inner two whorls. Transgenic plants that constitutively and simultaneously express both AP3 and PI under the direction of the 35S promoter develop flowers that have petals in whorls 1–2 and stamens in whorls 3–4 (Jack et al. 1994; Krizek and Meyerowitz 1996a). Ectopic expression of both B class and C class genes led to the production of Arabidopsis flowers with stamens in all four whorls (Fig. 2.2E; Krizek and Meyerowitz 1996a). Thus, AP3 and PI together are both necessary and sufficient for the B activity within the context of a flower. AP3 and PI initially are not expressed in identical domains. AP3 mRNA is detected in whorls 2–3 plus in a small number of cells at the base of the first whorl (Weigel and Meyerowitz 1993; Tilly et al. 1998), while PI RNA is detected in whorls 2–4 (Goto and Meyerowitz 1994). At later stages of flower development, the expression of both genes is restricted to petals and stamens. Maintenance of this later expression in petals and stamens requires the functional activity of both AP3 and PI,

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suggesting a system of autoregulation (Jack et al. 1992; Goto and Meyerowitz 1994). Presumably, auto- and cross-regulation of AP3 and PI are responsible for the similar expression domains of AP3 and PI in later stages of flower development. E. The C Class Genes: AG, HUA1, and HUA2 AG was the first C class gene identified and isolated (Bowman et al. 1989; Bowman et al. 1991b; Yanofsky et al. 1990). AG plays a key role in specifying stamen and carpel identity. In ag loss-of-function mutants (Fig. 2.1E; Fig. 2.2D), the A activity is expanded into whorls 3–4, where stamens are replaced by petals, and carpels are replaced by a new flower. This results in a floral pattern of sepal, petal, petal, (sepal, petal, petal)n. The generation of flowers within a flower reveals a second role of AG: to maintain the determinacy of the floral meristem. AG encodes a MADSdomain containing protein (Yanofsky et al. 1990). As predicted by the ABC model, AG mRNA is detected in the inner two whorls during early floral stages. AG mRNA continues to be expressed in stamens and carpels during later stages and eventually becomes restricted to specific cell types within the stamens and carpels (Bowman et al. 1991a; Drews et al. 1991). Ectopic expression of AG under the 35S promoter in transgenic Arabidopsis or tobacco plants causes homeotic conversion from sepals into carpels and from petals into stamens (Mizukami and Ma 1992; Mandel et al. 1992a). Thus, AG appears not only necessary but also sufficient to specify stamen and carpel identity within the context of a flower. In summary, AG has at least three functions: repressing A class gene activity in whorls 3–4, specifying stamen and carpel organ identity, and maintaining the determinacy of floral meristems. For many years, it was thought that AG was the only C class gene because AG alone appeared sufficient to specify C activity, and several mutageneses only yielded additional ag alleles without identifying mutations in other genes that confer similar phenotypes. However, genetic redundancy and/or embryonic or seedling lethality may have prevented the isolation of mutations in additional C class genes. Chen and Meyerowitz (1999) searched for new C class genes by looking for mutations that enhanced the phenotype of the weak ag-4 allele. Flowers of ag-4 plants can still make stamens in the third whorl because of residual activity of the mutant ag gene product (Sieburth et al. 1995; Chen and Meyerowitz 1999). Mutations that enhance the ag-4 phenotype are expected to convert the third whorl stamens into petals, as is seen in the stronger ag alleles. Chen and Meyerowitz (1999) successfully

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isolated two new C class genes, HUA1 and HUA2 (Hua means “flower” in Chinese). Either one of the hua1 or hua2 mutations weakly enhances ag-4, while hua1 hua2 double mutants strongly enhance the ag-4 phenotype. Both hua1 and hua2 single mutant flowers are phenotypically normal. Flowers of the hua1 hua2 double mutants show a weak stamen and carpel phenotype. In the first few flowers, lateral stamens are petaloid, and the gynoecia are enlarged toward their tips and constricted along their sides. The lack of phenotype in hua1 and hua2 single mutants explains why they were only recovered in genetic screens when AG activity is compromised. Genetic analyses indicated that HUA1 and HUA2 share a redundant role with AG in all aspects of AG function: repression of A class gene expression, stamen and carpel identity specification, and regulation of floral determinacy. HUA1 and HUA2 act in parallel or together with AG in the specification of C activity because AG mRNA expression is not affected in the hua1 or hua2 mutants. Similarly, HUA2 expression is not altered in ag mutants. Since HUA2 encodes a novel protein that contains multiple nuclear localization signals and additional motifs, it is likely that HUA2 is a transcription factor and acts as a cofactor of the AG gene. F. Novel Class Genes: SEP1, SEP2, and SEP3 Recently, another class of floral homeotic mutants has been described (Pelaz et al. 2000). This new class is encoded by three genes: SEPALLATA1 (SEP1), SEP2, and SEP3. All three genes are MADS-box containing genes and were isolated based on their sequence similarity to AG. In fact, SEP1, SEP2, and SEP3 were previously named AGAMOUS-LIKE2 (AGL2), AGL4, and AGL9, respectively (Ma et al. 1991; Mandel and Yanofsky 1998). A reverse genetic approach was used to identify mutations in SEP1, SEP2, and SEP3. Reverse genetics refers to a variety of techniques that can be used to generate mutations in a particular gene whose sequence is known. Since we have now entered an era of genomics, the large number of DNA sequences available in Arabidopsis, rice, tomato, and a few other plant species allow the discovery of many genes and gene families. However, in a majority of the cases, the function of these genes remains unknown. Thus, reverse genetic approaches are now more frequently employed and are crucial to illuminate gene function. These approaches are labeled “reverse” because they lead from gene sequence to mutation in the opposite direction of the typical “forward” genetic approach that leads from mutation to gene sequence.

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In the reverse genetic approach employed by Pelaz et al. (2000), polymerase chain reaction (PCR) amplification was used to identify transposable element insertions within the SEP gene of interest. After generating individual mutations in each of the SEP genes, phenotypic analyses indicated that mutations in any one of the SEP genes alone cause only subtle phenotypes. However, sep1 sep2 sep3 triple mutants displayed a striking phenotype in which all floral organs in the first three whorls were sepals or sepal-like organs. The fourth whorl was converted into a new flower that repeats this same floral pattern (Pelaz et al. 2000). The phenotype displayed by the sep1 sep2 sep3 triple mutant is very similar to “bc” double mutants (such as pi ag or ap3 ag), suggesting that SEP1, SEP2, and SEP3 are required for B and C class gene expression or for their activity. Because of redundancy among SEP1, SEP2, and SEP3, removing one of these SEP genes by mutations normally would not reveal such a requirement. SEP1, SEP2, and SEP3 are all expressed just prior to the expression of the B and C class genes and are expressed throughout whorls 2–4 (SEP1 and SEP2 are also expressed in whorl 1 in young flowers) (Pelaz et al. 2000). The initial patterns of B and C class gene expression are not altered in the sep1 sep2 sep3 triple mutants, suggesting that SEP1, SEP2, and SEP3 are not required for the initiation of B or C class gene expression. Thus, it has been suggested that SEP1, SEP2, and SEP3 regulate B and C class genes post-transcriptionally. One possible mechanism for this post-transcriptional regulation is by a direct interaction between the SEP1, SEP2, and SEP3 gene products and the B and C class gene products. Yeast two-hybrid assays have revealed a number of interactions between SEP proteins and B and C gene products (Fan et al. 1997).

II. CONSERVATION OF THE ABC FUNCTIONS IN ANGIOSPERMS The ABC genes provide an excellent opportunity to understand the evolutionary conservation and divergence of floral development in angiosperms. Studies carried out in both dicot and monocot plants suggest that the basic genetic mechanisms that determine floral organ identity are conserved across angiosperms. For most of the ABC genes, cognate homologs (orthologs) can be identified in diverse angiosperm species. For some of these homologs, functional analogy to corresponding Arabidopsis genes has been confirmed (see Table 2.1) by studying loss-of-function mutants, transgenic plants ectopically expressing these

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Table 2.1. ABC genes in different plants. Only those genes are included that exhibit functional homology with corresponding Arabidopsis genes except ZAP1 and ZMM2 in Maize and BLIND in Petunia. Class

Arabidopsis

Antirrhinum

Petunia

Maize

Rice

A

APETALA1 APETALA2 APETALA3 PISTILLATA AGAMOUS

SQUAMOSA — DEFICIENS GLOBOSA PLENA

— BLINDy GREEN PETAL FBP1, PMADS2 pMADS3, FBP6

ZAPz — SILKY1 — ZAG1, ZMM2z

— — OsMADS16 OsMADS4 OsMADS3

SEP1, 2, 3

—

FBP2

—

LHS1

B C Novel (BC) z

Only sequence and expression data are available (Mena et al., 1995). Only genetic data are available (de Vlaming et al., 1984; Tsuchimoto et al., 1993).

y

homologs, transgenic plants expressing antisense genes to corresponding homologs, or functional complementation of Arabidopsis mutants with homologs isolated from different species. One major theme from these studies (mostly on the B and C class genes) is that organ identity genes in different species differ both in the number of genes involved (due to gene duplication) and in the distribution of functional roles among these duplicated genes. Because dicots and monocots are on separate branches of the angiosperm phylogenetic tree, the conservation of the ABC model in dicot and monocot species suggests that the ABC model represents an ancient regulatory network that in all likelihood is generally applicable to most angiosperms. In this review, we will focus on studies where functional data on ABC gene homologs are available. For a more extensive review of angiosperm flower development, see Irish and Kramer (1998). For reviews on grass species, see Schmidt and Ambrose (1998) and Ma and dePamphilis (2000). A. Dicotyledonous Species Like Arabidopsis, Antirrhinum majus is a dicot plant that has been extensively studied and has contributed greatly to the establishment of the ABC model. Although Arabidopsis (Brassicaceae) and Antirrhinum (Scrophulariaceae) are widely divergent dicot species and belong to different subclasses, the phenotype of the corresponding Antirrhinum mutants (Carpenter and Coen 1990; Schwarz-Sommer et al. 1990) are quite similar to Arabidopsis mutants (Coen and Meyerowitz 1991). DEF and GLO, orthologs of AP3 and PI, respectively, are both required for B function in Antirrhinum (Sommer et al. 1990; Trobner et al. 1992). How-

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ever, the expression domains of these orthologs are switched. In Arabidopsis, PI is expressed early in whorls 2–4, while AP3 is expressed in whorls 1–3. In Antirrhinum, DEF is expressed early in whorls 2–4, while GLO is expressed largely in whorls 2–3 (Schwarz-Sommer et al. 1992; Trobner et al. 1992). Despite the difference in expression, AP3 and DEF are functionally conserved as demonstrated by cross species complementation (Irish and Yamamoto 1995; Samach et al. 1997). Similar expression studies in several other species showed that the DEF expression pattern represents an ancestral condition, while the AP3 expression pattern represents an exception rather than the rule (Irish and Kramer 1998). Based on sequence similarity, there are two petunia PI-like genes, FBP1 and pMADS2 (Angenent et al. 1992, 1994; Kush et al. 1993). Consistent with it being a B function gene, FBP1 was shown to be required for petal and stamen development in co-suppression experiments (Angenent et al. 1992, 1994). However, pMADS2 appears to encode a redundant function, since loss of pMADS2 function has no phenotypic effect (van der Krol et al. 1993). The petunia AP3 homolog, pMADS1 is also called GREEN PETAL (GP), because mutations in this gene cause a homeotic conversion from petals to green sepals but do not affect stamen development (van der Krol et al. 1993). Furthermore, ectopic expression of pMADS1/GP under the control of the 35S promoter in wild-type plants resulted in partial conversion of sepals into petaloid organs, but had no effect on stamen identity (Halfter et al. 1994). The fact that pMADS1/GP is not required for stamen development suggests that petunia may have another, as yet unidentified, AP3 homolog specifying the B function in whorl 3. Thus, petunia differs from Arabidopsis and Antirrhinum both in an increased number of B class genes and in the limitation of B class gene activity to only one whorl. AG orthologs from various species are more conserved than are the B class genes, both with respect to DNA sequence and gene function. In Antirrhinum, PLENA (PLE) is considered the functional ortholog of AG (Table 2.1), as both the expression pattern and the function of PLE are similar to AG in Arabidopsis (Bradley et al. 1993). Petunia has two AGlike genes, pMADS3 and FBP6, both of which appear to have expression patterns consistent with a reproductive function (Tsuchimoto et al. 1993; Angenent et al. 1995; Kater et al. 1998). Ectopic expression of pMADS3 either via 35S promoter in transgenic plants or via gain-offunction alleles led to the homeotic conversion of whorl 1 and whorl 2 generating organs with carpelloid and staminoid features, respectively. However, ectopic expression of FBP6 did not lead to homeotic transformations (Kater et al. 1998).

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The Antirrhinum ortholog of the A class gene AP1 is SQUAMOSA (SQUA). SQUA, like AP1, plays a role both in meristem identity and organ identity; however, its role in organ identity determination is rather limited, as flowers of squa mutants are often nearly wild type (Huijser et al. 1992). The Antirrhinum mutant ovulata exhibits a phenotype similar to that of ap2. However, ovulata is a gain-of-function allele of the C class gene PLE that is caused by a transposon insertion into the second intron of PLE and may have disrupted binding sites for negative regulators (Bradley et al. 1993). Two genes STYLOSA (STY) and FISTULATA (FIS) together control the restriction of the C gene PLE to the inner whorls of the flower. Genetic and expression studies, however, indicated that the effect of STY and FIS on PLE expression is indirect and that STY and FIS are more general regulators of flower development (Motte et al. 1998). In petunia, the blind (bl) mutants exhibit a phenotype similar to A class mutants, which includes homeotic conversion from sepals to carpelloid sepals and petals into antheroid structures (de Vlaming et al. 1984; Angenent et al. 1992; Tsuchimoto et al. 1993). In addition, the petunia C class gene pMADS3 was ectopically expressed in bl mutants (Tsuchimoto et al. 1993), indicating that BL, like AP2, is a negative regulator of C class genes. However, the molecular nature of BL is unknown. Orthologs of Arabidopsis AP2 have not been identified in Antirrhinum, petunia, or other species (Table 2.1). The tomato TM5 gene and its petunia homolog FBP2 are expressed in the inner three whorls, all of which are defective when TM5 or FBP2 are inactivated by antisense constructs or co-suppression (Angenent et al. 1994; Pneuli et al. 1994). Although the phenotypes in tomato and petunia are slightly different, in both cases, petals are transformed into sepals or leaf-like organs, and additional whorls of organs or new flowers can develop in the center of the flower. This phenotype resembles the Arabidopsis sep1 sep2 sep3 triple mutants (Pelaz et al. 2000). Sequence analyses indicate that indeed SEP1, SEP2, and SEP3 are most closely related to TM5 and FBP2 (Purugganan et al. 1995). The presence of three redundant SEP genes in Arabidopsis suggests that tomato and petunia may also possess multiple genes for the same function. The antisense and co-suppression approaches used to knock out TM5 and FBP2 might have simultaneously abolished the activity of other redundant genes of TM5 or FBP2 due to their high levels of sequence similarity. B. Monocotyledonous Species Maize (Zea mays) is a monocot grass species that has been extensively characterized resulting in the development of a host of useful molecular genetic tools. In particular, transposon tagging and other reverse

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genetic approaches are available. A number of maize MADS box genes have been isolated and their functions have been assayed by reverse genetic, as well as forward genetic, approaches. Grass flowers have stamens and carpels, but lack obvious sepals and petals. Instead, grass-specific organs known as glumes, lemma, palea, and lodicules surround the stamens and carpels. Recent studies of a maize mutant silky1 provides compelling developmental evidence for recognizing lodicules as modified petals, and possibly, palea and lemma as modified sepals (Ambrose et al. 2000). SILKY1 encodes an AP3-like gene in maize. Homeotic transformations of stamens to pistils and lodicules to organs resembling lemma/palea are exhibited in silky1 mutants. In situ hybridization indicates SILKY1 is localized to lodicule and stamen primordia. Thus genetic, morphological, sequence, and expression data all support that SILKY1 is a B function gene (Ambrose et al. 2000; Ma and dePamphilis 2000). The maize C class gene ZAG1 was identified first by sequence homology to AG (Schmidt et al. 1993). Subsequently, reverse genetic approaches created a putative null allele of zag1 (Mena et al. 1995). While the ABC model would predict a loss of both reproductive organ development and floral meristem determinacy, only the later phenotype was evident, with supernumerary carpels being reiterated within the zag1 florets. Although ZAG1 is expressed in both stamens and carpels, the zag1 mutation does not affect stamen development. One possible explanation is the existence of partially redundant C class genes in maize. Indeed, ZMM2, a gene closely related to ZAG1, has been isolated (Mena et al. 1995; Schmidt and Ambrose 1998). The expression pattern, sequence, and map position of ZMM2 all suggest that it is a duplicate gene with activities that are non-identical, but partially overlapping with those of ZAG1. Consistent with the genetic analyses, ZAG1 is more highly expressed in carpels and ZMM2 more highly expressed in stamens. Thus, it is highly likely that in maize the AG-like activity is shared by two genes. Several MADS box genes that play important roles in controlling flower development in rice have also been studied. Using antisense experiments, Kang et al. (1998) demonstrated that the rice MADS box genes OsMADS3 (Oryza sativa MADS box gene 3) and OsMADS4 are putative orthologs of AG and PI respectively. OsMADS16 gene has been proposed as a homolog of AP3 based on its amino acid sequence similarity to AP3, its expression pattern, and its interaction with OsMADS4 in yeast (Moon et al. 1999). The leafy hull sterile 1 (lhs1) mutations display mutant phenotypes similar to transgenic plants expressing a dominant-negative mutant form of OsMADS1, suggesting that the lhs1 mutation may be defective in the OsMADS1 gene (Kinoshita et al. 1976; Jeon et al. 2000). The fact that

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wild-type OsMADS1 can rescue lhs1 mutants further indicates that OsMADS1 and LHS1 encode the same gene (Jeon et al. 2000). Strong lhs mutants exhibit leafy palea and lemma, and partial transformation from lodicules to leafy palea and lemma. Additional carpels and a new flower may be generated. This mutant phenotype and the sequence similarity between LHS1 (OsMADS1) and TM5 and FBP2 suggests that LHS1 may encode a functional homolog of TM5, FBP2, and the Arabidopsis SEP genes. However, the phenotype of lhs also suggests that LHS1 has an additional role during the development of palea and lemma during late stages of flower development. In this respect LHS is similar to AP1.

III. POSITIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Meristem Identity Genes: LFY and AP1 At the beginning stages of Arabidopsis development, the primary shoot produces leaves with axillary second order shoots. Later, at the transition to reproductive phase, the primary shoot switches to producing flowers. Two genes LEAFY (LFY) and AP1 are necessary and sufficient for this developmental switch (Irish and Sussex 1990; Huala and Sussex 1992; Weigel et al. 1992; Bowman et al. 1993; Mandel and Yanofsky 1995; Weigel and Nilsson 1995). Loss-of-function mutations in these two genes cause the conversion (to varying degrees) from flowers to second order shoots. Conversely, constitutive expression of either LFY or AP1 cause the conversion from shoots to flowers. Thus LFY and AP1 are referred to as “meristem identity genes.” Specifically, in lfy mutants, flowers are replaced by leaves and second order shoots. In ap1 mutants, (leaf-like) bracts develop in the first whorl and secondary flowers develop in the axils of first whorl floral organs. Most strikingly, ap1 enhances the defects of lfy. In lfy ap1 double mutants, leaf-like organs arise in a spiral fashion (a feature of shoot) rather than whorled fashion (a feature of flowers), and all flowers are replaced by shoot-like structures. Thus, LFY and AP1 have partially redundant roles in floral meristem identity specification, with LFY playing a more prominent role than AP1. For more specific reviews regarding floral inductive pathways leading to the activation of LFY and AP1, see Yanofsky (1995), Koornneef (1997), Weigel (1997 and 1998), Ma (1998), and van Nocker (2001). Although the LFY protein does not share sequence homology with other known families of DNA-binding proteins, LFY can bind DNA in a sequence specific manner and stimulate transcription in yeast cells

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when fused to a heterologous activation domain (Weigel et al. 1992; Parcy et al. 1998). Thus, LFY may activate the floral program by transcriptional activation of target genes involved in flower development. This hypothesis is consistent with LFY RNA and protein expression that precedes the transcriptional activation of ABC genes. Specifically, LFY mRNA is expressed in early floral meristem, and is transiently expressed in sepal, petal, stamen, and carpel primordia (Weigel et al. 1992). The LFY protein is expressed in a similar pattern and localizes to the nucleus, consistent with a role in transcriptional activation (Parcy et al. 1998). To further understand the regulatory relationship between meristem identity genes and ABC genes, Weigel and Meyerowitz (1993) examined ABC gene expression in lfy and ap1 single and double mutants. They found that LFY and AP1 are positive regulators of ABC gene transcription. In lfy mutants, early AG expression is delayed, and the initial expression domain is smaller than in wild-type flowers. The expression of AG mRNA in ap1 mutants is relatively normal, however, AG RNA entirely fails to accumulate in the center of ap1 lfy double mutant flowers. Therefore, AG expression is more strongly affected in the ap1 lfy double mutants than in either single mutant. Similarly, both the amount and the domain of expression of AP3 and PI are severely reduced in strong lfy mutants. The function of AP1 in activating AP3 and PI only becomes obvious when LFY activity is reduced or eliminated as shown in lfy ap1 double mutants. Like B and C class genes, the expression of AP1 is delayed and reduced in lfy mutants (Ruiz-Garcia et al. 1997; Liljegren et al. 1999). Since AP2 RNA is detected in a variety of non-floral tissues, including leaves and stems, AP2 transcription is likely regulated independently of the meristem identity genes (Jofuku et al. 1994). B. LFY, a Direct Activator of AP1 The dual roles of AP1 as a meristem identity gene and an A class organ identity gene correlate well with its two phases of expression. AP1 is initially expressed in the entire floral meristem and later becomes restricted to the first two whorls (Bowman et al. 1993; Mandel et al. 1992b; Gustafson-Brown et al. 1994). This expression pattern correlates well with an early function of AP1 in meristem identity specification and a later function of AP1 in sepal and petal identity specification. LFY is an obvious candidate activator of AP1 expression as AP1 RNA expression in the floral meristem is initiated soon after LFY is first detected and AP1 RNA expression is delayed and reduced in lfy mutants (Ruiz-Garcia et al. 1997; Liljegren et al. 1999).

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Evidence that LFY can directly activate AP1 comes from experiments in which constitutive and ectopic expression of LFY under the 35S promoter led to precocious expression of AP1 in young seedlings (Parcy et al. 1998). Thus the role of LFY in activating AP1 can be separated from its role in floral meristem specification. Consistent with the idea that AP1 expression is directly regulated by LFY, in vitro DNA-binding assays showed that a high affinity binding site for LFY is present in a minimal AP1 promoter. Additionally, LFY, upon fusion to a strong transcriptional activation domain, can activate the expression of a reporter gene in yeast, which is under the control of an AP1 promoter (Parcy et al. 1998). Using a post-translational inducible system, Wagner et al. (1999) demonstrated that AP1 is an immediate target of LFY. Specifically, a steroid hormone-inducible LFY switch was constructed. This construct (35S::LFY-GR) uses the CaMV-35S promoter to express the LFY coding sequence that has been fused to a glucocorticoid receptor (GR) hormone binding domain. In the absence of the steroid hormone dexamethasone, the LFY-GR fusion protein is held in the cytoplasm and is non-functional. In the presence of dexamethasone, the LFY-GR fusion protein moves to the nucleus and is able to perform its function as a transcriptional activator. As the translocation of the LFY-GR protein into the nucleus does not depend on protein synthesis, a direct effect of LFY on its target gene transcription can be evaluated in the presence of cyclohexamide (a protein synthesis inhibitor). The LFY-GR protein was able to rescue defects of AP1 expression at early stages even in the presence of cyclohexamide, indicating that LFY directly activates AP1 at early stages. However, the ability of LFY-GR to rescue defects of AP1 expression during later stages is dependent on protein synthesis. Thus, the two phases of AP1 expression appear to be controlled by different regulatory mechanisms. While LFY directly activates AP1 expression during early stages of flower development, the effect of LFY on AP1 expression is indirect at later stages. C. LFY, a Direct Activator of AG Despite the knowledge that LFY and AP1 are required for AG expression, it has been difficult to determine if the effect of LFY and AP1 on AG expression is direct or indirect. For example, a failure to activate AG might simply result from the fact that the shoot structures formed in lfy or lfy ap1 mutants never acquire any floral identity. Fortunately, a gainof-function LFY protein LFY:VP16 is able to separate the role of LFY in meristem identity specification from the role of LFY in regulating AG

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(Parcy et al. 1998). Specifically, LFY:VP16 is a fusion protein between LFY and a strong transcriptional activation domain from the Herpes Simplex Virus (HSV) VP16 protein, generating a form of LFY that is constitutively active with respect to transcriptional activation. The LFY:VP16 fusion protein was expressed in developing plants from the endogenous LFY promoter, and thus the expression pattern of LFY:VP16 mimics the expression of the wild-type LFY protein. Transgenic plants harboring the LFY:VP16 construct initiate floral meristem formation normally; however, the floral organs display homeotic transformations, where sepals are transformed into carpels in whorl 1 and petals are transformed into stamens in whorl 2. In situ hybridization showed that these LFY:VP16 plants exhibit both ectopic and precocious AG RNA expression. When the LFY:VP16 plants were crossed to the strong loss-of-function ag-1 mutant, the floral organs in whorls 1–2 were largely restored to normal sepals and petals. Hence, ectopic and precocious AG expression is chiefly responsible for the abnormal floral organs formed in these LFY:VP16 plants. When the LFY: VP16 was ectopically expressed in developing seedlings under the control of the 35S promoter, seedlings were growth-arrested and AG was ectopically expressed (Parcy et al. 1998). The induction of ectopic AG expression in pre-flowering seedlings by LFY:VP16 further indicates that LFY:VP16 is able to activate AG in non-floral tissues. Thus, the effect of LFY:VP16 on AG is rather direct and does not require proper floral meristem formation. However, a 35S::LFY construct that drives the ectopic expression of the wild-type LFY protein is not sufficient to generate pre-flowering seedling expression of AG; rather the presence of the strong VP16 activation domain is required (Parcy et al. 1998). Two models were proposed to explain this observation. The first model postulates that there is a repressor that is present in whorls 1–2 of the floral meristem and in the vegetative tissues that normally prevents LFY from activating AG expression. In this model the enhanced transcriptional activity of the LFY:VP16 protein overcomes this repressor activity. Alternatively, in wild-type plants, the LFY protein is assisted by the action of a co-activator only expressed in the center of the floral meristem. Hence, AG is only activated by LFY in the center of a wild-type flower. According to this model, the strong transcriptional activity of LFY:VP16 can activate AG transcription independently of any co-activators. Analysis of the AG cis-regulatory sequences revealed that sequences within the second AG intron are necessary and sufficient for the wildtype AG expression pattern (Sieburth and Meyerowitz 1997; Busch et al. 1999). At least two redundant enhancers within the intronic sequences

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mediate the expression of AG (Busch et al. 1999). These enhancers are LFY responsive, as they respond appropriately to expression of the LFY:VP16 construct or to the loss of LFY activity in the lfy mutant. Using deletion analysis, one functional enhancer was found within a 230 base pair (bp) fragment of the intron. Within this fragment, two LFY binding sequences, that are 31 bp apart, were detected by gel shift analysis. These sequences are similar to each other and to previously defined LFY binding sites in the AP1 promoter (Parcy et al. 1998). Alterations to the binding site sequence that prevent the binding of LFY in vitro also cause the enhancer fragments to lose their responsiveness to both wild type LFY as well as LFY:VP16 proteins in vivo (Busch et al. 1999). These results suggest that LFY-dependent stimulation of AG expression requires the direct binding of LFY to the enhancer sequences of AG. In addition, two CArG boxes have been identified within the 3′ activation cis-element where a LFY binding site is also located (Deyholos and Sieburth 2000). These CArG boxes could serve as binding sites for MADS-domain proteins such as AP1. D. Two Phases of Regulation: Initiation and Maintenance of B Gene Expression The regulation of AP3 and PI can be divided into two phases: the early initiation phase and the later maintenance phase. These two different phases are regulated by distinct sets of genes. The early phase of AP3 and PI expression are positively regulated by meristem identity genes LFY and AP1 (Weigel and Meyerowitz 1993; Goto and Meyerowitz 1994). AP3 and PI expression is significantly reduced in strong lfy mutants, whose flowers lack petals and stamens. Hence, LFY is required to initiate AP3 and PI expression. Although, ap1 mutations alone have little effect on B class gene expression, lfy ap1 double mutants display no detectable AP3 and PI expression (Weigel and Meyerowitz 1993), suggesting that AP1 does play a role in B class gene activation. Activation of AP3 by LFY apparently relies on a mechanism different from those for the activation of AG and AP1. AP1 and AG can be activated in ectopic tissues in response to the ectopic expression of wild type LFY (in the case of AP1) or LFY:VP16 (in the case of AG). However, ectopic and constitutive expression of LFY by 35S::LFY or LFY::VP16 failed to activate AP3 ectopically. Apparently, B class genes require additional factors for their activation. Results from several experiments (see below) indicate that the activation of the B class genes requires the UNUSUAL FLORAL ORGANS (UFO) gene. When LFY and UFO are both constitutively expressed in seedlings (35S::LFY and 35S::UFO), they

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can induce AP3 expression in seedlings (Parcy et al. 1998). Thus, activation of AP3, like that of AP1 and AG, is not dependent on the formation of floral meristems. Similarly, flower-independent activation of PI is observed in seedlings where both LFY and UFO are expressed constitutively (Honma and Goto 2000). LFY-GR experiments that directly test the effect of LFY and UFO on AP3 or PI expression in vivo in the presence of protein synthesis inhibitors will be required to determine if LFY and UFO are immediate upstream activators of AP3 and PI. The maintenance (late) phase of AP3 and PI expression is dependent on the activity of AP3 and PI. The autoregulatory role of AP3 and PI is observed in ap3 and pi mutants where both AP3 and PI late phase expression is reduced while the early phase expression is not affected (Jack et al. 1994; Goto and Meyerowitz 1994). Three CArG boxes were identified between –90 to –180 of the AP3 promoter (Hill et al. 1998; Tilly et al. 1998). These same elements are necessary for maintaining AP3 expression in petals and stamens (Hill et al. 1998). AP3/PI heterodimers can bind to CArG box 1 and 3 in vitro in a sequence specific manner (Hill et al. 1998; Tilly et al. 1998). In addition, AP3-GR, which is activated by steroid hormone, can induce AP3 expression in the absence of de novo protein synthesis (Honma and Goto 2000). Thus, direct interaction between AP3/PI and the CArG boxes in AP3 promoter is responsible for maintaining late phase AP3 transcription. Interestingly, the promoter or intron sequences of PI do not contain any CArG box. This raised the possibility that AP3/PI heterodimers may not directly bind to PI cis-elements. Indeed, electrophoretic mobility shift assays (EMSA) failed to detect binding of AP3/PI to the proximal promoter element of PI. Using a similar AP3-GR system, it was found that the ability of AP3/PI heterodimer to activate PI transcription requires de novo protein synthesis (Honma and Goto 2000). Thus, in contrast to the direct autoregulation of AP3 by AP3/PI, autoregulation of PI transcription by AP3/PI is indirect and requires de novo synthesis of additional factor(s). Using a promoter fusion to the uidA reporter gene encoding bglucuronidase (GUS), the cis-regulatory region of AP3 and PI promoters have been dissected. Minimal promoters of about 727 bp for AP3 (Hill et al. 1998) and 498 bp for PI (Honma and Goto 2000) were identified that can direct the wild-type pattern of respective gene expression. Hence, these fragments contain all necessary cis-regulatory elements. Within 727 bp of the AP3 minimal promoter, multiple cis-acting elements were identified that control temporal and spatial subsets of the AP3 expression. Two elements for initiating early stage AP3 expression were identified; one is located proximally and the other is located

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distally within the promoter of AP3. The three CArG boxes described earlier exist within the proximal early element (Hill et al. 1998). In addition, a petal-specific element and two stamen-elements were identified that direct AP3 expression during maintenance phase (Hill et al. 1998; Tilly et al. 1998). Interestingly, genomic sequences of the AP3 and PI promoters do not show any sequence similarity (Honma and Goto 2000). Within the 498 bp PI promoter, multiple, discrete cis-regulatory elements were identified. The elements located in the distal region of the PI promoter are generally involved in the early initiation phase of PI expression while the element more proximal to the PI promoter is involved in the maintenance phase regulation. Thus, while cis-elements required for initiation and maintenance are located in distinct, separable regions of the PI promoter, the cis-elements required for initiation and maintenance of AP3 expression overlap. E. UFO, a Coregulator of B Gene Expression As described earlier, UFO activity is essential for B gene expression and ufo mutants exhibit floral organ identity defects that are similar to partial loss-of-function lfy and to B class mutants (Wilkinson and Haughn 1995; Levin and Meyerowitz 1995). In ufo mutants, AP3 and PI RNA level is reduced (Wilkinson and Haughn 1995; Levin and Meyerowitz 1995; Hill et al. 1998). Constitutive expression of UFO under the control of 35S promoter results in precocious and ectopic activation of AP3 and PI in flowers, confirming a positive regulatory role of UFO for B gene expression (Lee et al. 1997; Honma and Goto 2000). Consistent with its role as an upstream activator, UFO RNA accumulates in the floral meristem before the onset of AP3 expression. UFO is expressed initially throughout the entire floral meristem, but later is restricted to whorls 2–3 (Ingram et al. 1995; Lee et al. 1997). Thus, the presence of both LFY and UFO in whorls 2–3 might be necessary to initiate B gene expression. What is the relationship between LFY and UFO in activating AP3? Clearly, the simple hierarchical models of UFO acting downstream of LFY or LFY acting downstream of UFO are inconsistent with the following observations. First, LFY and UFO expression is activated independently (Levin and Meyerowitz 1995; Lee et al. 1997). Second, constitutive expression of LFY fails to rescue ufo mutants and, conversely, constitutive expression of UFO fails to rescue lfy mutants (Weigel and Nilsson 1995; Lee et al. 1997). In other words, the ability of the 35S::UFO construct to activate B class gene expression is dependent on wild-type LFY activity (Lee et al. 1997) and vice versa. Based on these

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data, a model has been proposed in which UFO and LFY are coregulators of B genes. LFY is responsible for general floral-specific activation of B gene transcription and UFO confers regional specificity to whorls 2–3 (Lee at al. 1997). The UFO gene encodes a protein with a F-box motif (Ingram et al. 1995). F-box proteins are part of the heteromeric ubiquitin ligase complex known as SCF (Skp1, Cdc53/cullin, F-box), which plays a key role in the degradation of a variety of regulatory proteins (Patton et al. 1998). The F-box protein in each SCF complex probably acts as a receptor to recruit specific protein targets for degradation. UFO has been shown to interact in vitro with Arabidopsis Skp1-like proteins ASK1 and ASK2 (Samach et al. 1999), supporting the idea that UFO may be a component of the SCF complex. In addition, ask1 mutants exhibit mosaic organs in their flowers similar to those seen in ufo mutants (Zhao et al. 1999), suggesting a role of Skp1-like proteins in B class gene regulation. Thus, LFY and UFO might act by distinct mechanisms to coordinately regulate B class gene expression. For example, LFY binds to AP3 promoter and activates AP3 transcription, whereas UFO might act as a member of the heteromeric ubiquitin ligase complex that specifically removes repressors of AP3 transcription via ubiquitin-mediated protein degradation.

IV. NEGATIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES It has become evident that combined activities of both positive regulators, such as LFY, and negative regulators are necessary to specify the proper temporal and spatial domains of ABC gene expression. A number of negative regulators of the ABC genes in Arabidopsis have been identified that play crucial roles in delimiting ABC gene expression to specific domains. Many of these negative regulators have been identified through genetic screens for floral mutants that exhibit partial or complete homeotic transformation from one floral organ type to another. Based on the ABC model, one can interpret the mutant phenotype by predicting an ectopic expression of a particular class or classes of the ABC genes. The predictions can be easily tested with in situ hybridization experiments to examine the specific A, B, or C gene expression in the newly isolated mutant background. Once confirmed, these newly isolated mutations define negative regulators of the ABC genes. Despite the identification and molecular cloning of a large number of the negative regulators of the C class gene AG, the mechanism of negative regulation is still not well understood.

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Work to date suggests that a number of negative regulators work in a coordinated fashion to initiate and maintain AG repression. A. Temporal and Spatial Regulators of AG 1. APETALA2 (AP2). AP2 is both an A class organ identity gene and a repressor of AG, specifically in whorls 1–2. The DNA binding motifs found in AP2 suggests that AP2 could directly interact with DNA and AP2-domain containing proteins have been shown to bind to DNA elements termed GCC boxes (Ohme-Takagi and Shinshi 1995). However, the absence of any GCC box in the cis-regulatory elements of AG (Deyholos and Sieburth 2000) indicates that AP2 may mediate negative regulation indirectly, via other intermediate steps. Thus far, no direct DNA binding of AP2 to the AG cis-elements has been reported. Bomblies et al. (1999) examined the cis-regulatory sequences of AG that mediate the repressive action of AP2. They also tested if the repressive effect of AP2 on AG depends on the activity of LFY. Sequences within the second intron of AG that have been shown to be required for activation of AG expression by LFY (Busch et al. 1999) are also required for the repression of AG expression by AP2 (Sieburth and Meyerowitz 1997; Bomblies et al. 1999). The two independent LFY responsive enhancers identified within the large intron (Busch et al. 1999) are also involved in mediating the repressive effect of AP2 (Bomblies et al. 1999). However, additional experiments suggest that AP2 may regulate AG expression through both LFY-dependent and LFY-independent mechanisms. It is possible that the LFY-independent mechanism might be mediated by AP1 (Deyholos and Sieburth 2000). 2. LEUNIG (LUG). Since AP2 is expressed throughout the developing floral meristem, the spatially restricted activity of AP2 in repressing AG in the first two whorls must depend on additional levels of regulation (Jofuku et al. 1994). This spatially restricted activity of AP2 was initially thought to be conferred by the presence of other co-regulators that are only present in the first two whorls. One candidate is LUG, which was identified in a screen for enhancers of a weak ap2 allele (Liu and Meyerowitz 1995). Plants with mutations in the LUG gene exhibit homeotic transformations similar to, but less severe than, ap2 mutants (Komaki et al. 1988; Liu and Meyerowitz 1995). These lug mutants also display ectopic and precocious expression of AG RNA, suggesting that LUG is required for proper repression of AG. Furthermore, lug ap2 double mutants exhibit more severe homeotic transformations than either single mutants. In situ hybridization experiments indicated that the

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mechanism of this enhancement is through the increased ectopic expression of AG. The enhancement of defects seen in the lug ap2 double mutants suggests that LUG and AP2 share partially redundant functions. In addition, dominant interactions were observed between strong ap2 alleles and lug mutations, suggesting either that these two gene products might interact directly or that an activity composed of both LUG and AP2 is required above a threshold level for proper AG repression. LUG was recently cloned (Conner and Liu 2000) and found to encode a nuclear protein that has an overall domain structure similar to a class of functionally related transcriptional co-repressors including Tup1p of yeast and Groucho of Drosophila (Hartley et al. 1988; Williams and Trumbly 1990). A common structure shared by all members of this class includes Q-rich regions near the N-terminus of the protein and 6–7 WD repeats at the C-terminus. The WD repeats [named so because the repeat often ends with the amino acids tryptophan (W) and aspartic acid (D)] have been shown to mediate protein-protein interactions and are found in proteins with a wide variety of biochemical functions (Neer et al. 1994; Smith et al. 1999). The mechanism of this class of transcriptional co-repressors has been extensively studied in yeast and Drosophila. Several mechanisms were implicated such as interfering with the interaction between activators and the general transcriptional machinery (quenching); interacting with the general transcriptional machinery (direct repression) or by affecting chromatin organization. The Tup1p protein, although it cannot bind to DNA on its own, can interact with a variety of DNA binding transcription factors and mediate transcriptional repression through any of the above mechanisms. In particular, it was shown that Tup1p can organize repressive chromatin structure through direct interaction with the N-terminal region of histones H3 and H4 (Edmondson et al. 1996). LUG may function similarly by interacting with AP2 or other unidentified DNA-binding transcription factors to bring about transcriptional repression of AG. In situ hybridization showed that LUG mRNA is ubiquitously expressed in all floral whorls (Conner and Liu 2000). Hence, like AP2, additional factors or post-transcriptional modifications are needed to limit the activity of LUG to whorls 1–2. Recently, two additional genetic enhancers of lug were identified, SEUSS (SEU) and LARSON (LSN) (R. Franks, X. Bao and Z. Liu, unpublished data). The seu mutants display a phenotype that is very similar to, albeit weaker than, lug mutants. Furthermore, seu lug double mutants display an enhanced phenotype that is characterized by strong precocious and ectopic expression of AG and enhanced homeotic transformations of floral organs, particularly in the first two whorls. lsn mutant

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does not have an obvious phenotype on its own but shows strong enhancement of lug in floral organ identity specification. Both SEU and LSN, together with LUG and AP2, are candidates for members of a transcriptional repression system that regulates AG expression. 3. AINTEGUMENTA (ANT). Mutations in ANT cause narrower floral organ shape and a decrease in floral organ number (Elliott et al. 1996; Klucher et al. 1996). Furthermore, ant mutants are female sterile due to failure of integument initiation and megasporogenesis. Although ant single mutants rarely show homeotic transformation of organ identity, ant dramatically enhances the organ identity defects of ap2 and lug (Elliott et al. 1996; Krizek et al. 2000; Liu et al. 2000). This enhancement correlates with an increased ectopic AG expression in ant ap2 and ant lug double mutants. Hence, ANT is likely another redundant repressor of AG. ANT encodes a member of the AP2 family of DNA-binding transcriptional regulators (Elliott et al. 1996; Klucher et al. 1996); the sequence similarity between ANT and AP2 may underlie their functional redundancy in AG repression. 4. CURLY LEAF (CLF). The clf mutants are characterized by narrow and curled rosette and cauline leaves as well as short stem internodes (Goodrich et al. 1997). The clf flowers display narrow petals, and partial homeotic transformations in whorls 1–2. These phenotypes resemble those reported for plants in which the AG gene was ectopically expressed (Mizukami and Ma 1992). RNA gel blot and in situ analysis indicated that AG was ectopically expressed in leaves and in developing petals of clf mutants at later stages of flower development. Double mutant analyses with ag-3 indicate that the ag mutation is epistatic to clf and thus the clf phenotype in leaves and flowers was dependent upon AG activity. Hence, CLF is another repressor of AG expression with primary roles in leaves, stems and, to a lesser extent, flowers. CLF encodes a protein with extensive sequence similarity to the product of a Drosophila polycomb group gene, ENHANCER OF ZESTE (Goodrich et al. 1997). Drosophila polycomb group genes appear to form multimeric complexes that interact with DNA to bring about heritable maintenance of transcriptional states and there is indirect evidence that they do so by modifying chromatin structure (Carrington and Jones 1996). Like the polycomb group genes, CLF appears to be required for the maintenance of repression and not for the initiation of repression of AG. This interpretation is consistent with the observation that ectopic AG expression was only detected at later stages of flower development. CLF is expressed in 8-day-old seedlings throughout the apical meristem,

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leaf primordia, and leaves (Goodrich et al. 1997). It is also expressed in the infloresence meristem and in all four whorls of the flower. The fact that AG and CLF RNA are co-expressed suggests that CLF RNA expression is not sufficient to repress AG expression. 5. STERILE APETALA (SAP). Flowers of sap mutants resemble those of ap2 or lug mutants with carpelloid sepals and loss of petals (Byzova et al. 1999). In sap mutants, AG RNA is not only detected ectopically in floral whorls 1 and 2, but also in inflorescence meristems. SAP is thus another negative regulator of AG expression. Consistent with a role in AG repression, mutations in SAP enhance the organ identity defects of ap2. To test the regulatory relationship between SAP and AP2, AP2 RNA expression was examined in sap mutants and vice versa. AP2 RNA expression was unaltered in sap mutants and SAP RNA expression was not altered in ap2 mutants. Thus, SAP and AP2 do not appear to regulate each other at the transcriptional level. Like other negative regulators of AG, SAP appears to possess additional functions as revealed by defects of sap mutants in female gametophyte development and by defects of sap ag double mutants in meristem identity determination. SAP encodes a protein with serine-rich and glycine-rich domains that are often found in eukaryotic transcriptional regulators. 6. FILAMENTOUS FLOWER (FIL). The effect of the fil mutation is complex; genetic analyses of fil mutants indicated that FIL is required for the maintenance and growth of inflorescence meristems, floral meristems, and floral organs (Sawa et al. 1999a; Chen et al. 1999). More relevant to this review are the findings that AG is ectopically expressed in fil mutants in floral whorls 1 and 2 and that homeotic transformations in whorls 1–2 are enhanced in ap2 fil and lug fil double mutants (Chen et al. 1999). These data suggest that FIL is yet another member of the AG negative regulators. FIL encodes a nuclear protein that contains a zinc finger and an HMG box-like domain, suggesting a role in transcriptional regulation (Sawa et al. 1999b). Unexpectedly, FIL RNA expression is restricted to the abaxial side of the developing leaves and floral organs, and 35S::FIL plants display an abaxialization of leaves (Sawa et al. 1999b). Thus, FIL controls the identity of the abaxial side of lateral organs. B. Repression of AP1 Expression in Floral Whorls 3–4 In wild type, AP1 is initially expressed in the entire floral primordia, but at later stages AP1 RNA is restricted to whorls 1–2 (Mandel et al. 1992b; Gustafson-Brown et al. 1994). The inhibition of AP1 expression in

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whorls 3–4 results from negative regulation by AG that becomes expressed in whorls 3 and 4 at this stage. In ag loss-of-function mutants, AP1 RNA is expanded to all four whorls (Gustafson-Brown et al. 1994). In the ap2 or lug single mutants, when AG expression is expanded to all four whorls, AP1 RNA is absent from all four whorls (Gustafson-Brown et al. 1994; Liu and Meyerowitz 1995), suggesting that the ectopic AG in whorls 1–2 represses AP1 transcription. However, the mechanism by which AG brings about repression of AP1 transcription is presently unclear. The C class genes HUA1 and HUA2 also participate in the negative regulation of AP1 (Chen and Meyerowitz 1999). In hua1 hua2 double mutants, ectopic AP1 expression was seen in late stages in carpel walls and occasionally in stamens. HUA1 and HUA2 may act in parallel with AG to repress the expression of AP1 in whorls 3–4, possibly as transcriptional co-regulators. C. Restriction of B Gene Expression to Floral Whorls 2–3 1. SUPERMAN (SUP). The sup mutants exhibit supernumerary stamens interior to the third whorl stamens at the expense of carpels (Schultz et al. 1991; Bowman et al. 1992). In situ hybridization experiments indicate that the B class genes AP3 and PI are ectopically expressed in whorl 4 in sup mutants. Additionally, ap3 sup and pi sup double mutants exhibit phenotypes similar to the ap3 or pi single mutants. From these molecular and genetic experiments, SUP was originally thought to function as a negative regulator of B class genes in whorl 4. SUP encodes a nuclear protein with a single zinc finger and a putative basic leucine zipper motif, suggesting a role in transcriptional regulation (Sakai et al. 1995). However, in situ hybridization revealed that SUP is expressed in whorl 3, not in whorl 4. Further, SUP RNA expression is, in fact, dependent on AP3 (Sakai et al. 1995). Initial AP3 expression precedes SUP RNA expression, and ectopic AP3 expression under 35S promoter causes ectopic SUP expression. In addition, SUP RNA is much reduced or absent in ap3 mutant flowers, while the onset of AP3 expression in the sup mutant is normal. These new findings do not support the earlier hypothesis that SUP represses B gene expression in whorl 4. Two alternative models were proposed (Sakai et al. 1995); in one SUP functions to prevent the spread of AP3 activity from whorl 3 to whorl 4, while in the second model SUP functions to limit the extent of cell proliferation in whorl 3. In either model, SUP acts to maintain a boundary between whorls 3 and 4.

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2. LEUNIG (LUG) and CURLY LEAF (CLF). In addition to their roles as negative regulators of AG, LUG and CLF also negatively regulate B class gene expression. Patches of ectopic AP3 and PI expression detected in whorl 1 organs of lug mutant flowers suggest that LUG represses B class gene expression in whorl 1 (Liu and Meyerowitz 1995). Similarly, ectopic AP3 expression was detected in leaves of clf mutants, suggesting a role of CLF in repressing AP3 expression in leaves (Goodrich et al. 1997). Whether these effects reflect a direct action of LUG or CLF on B class genes is unknown. These studies, however, suggest that the regulation of B genes employs at least two mechanisms: a region-specific coactivator such as UFO and negative regulators such as LUG.

V. SUMMARY The initial activation of the ABC genes in a flower specific fashion is dependent upon meristem identity genes such as LFY and AP1. Later, ABC gene expression is spatially refined by a combination of other positive regulators, such as UFO, and negative regulators, such as AP2, LUG, ANT, CLF, and SAP. The combined activity of both positive and negative regulators insures proper spatial and temporal expression of the ABC genes and thus the stereotypical structure of a given flower. Now that many of the key regulatory molecules have been identified and isolated, the challenge for the future is to further clarify the molecular mechanisms underlying ABC gene regulation. Clearly a variety of mechanisms are employed in this process. Evidence to date suggests that both transcriptional and post-transcriptional mechanisms are employed and that generating the proper ABC expression domains likely requires targeted degradation of specific repressors, autoregulatory enforcement mechanisms, and recruitment of transcriptional co-repressors. The identification of an increasingly large number of genes involved in ABC gene regulation suggests that the mechanism of ABC gene regulation is rather complex and many questions remained unanswered. For example, what is the molecular or biochemical basis underlying the genetic enhancement or dominant interaction among these mutants? Do these ABC regulators physically interact directly? Do they regulate each other’s expression? Future experiments involving immunoprecipitation assays and/or yeast two-hybrid assays will allow us to test physical interactions among these ABC regulators. Examination of the expression of these genes by RNA in situ hybridization and immunolocalization in different mutant backgrounds will illuminate the regulatory

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relationship among these genes. For genes that encode DNA-binding domains, gel-shift assays may help to identify their target ABC genes. Further deletion analyses of the promoters of the target ABC genes and testing these deletion constructs using gel-shift and transgenic reporter assays will help to locate sequence motifs that are crucial for ABC gene expression. Similarly, site-directed mutagenesis and domain-swamping —for example, between AP2 and ANT—may help to assess their DNAbinding and transcriptional activation potential and target-site specificity. Thus far, many of the expression studies have only looked at RNA expression. It is now necessary to examine the protein expression of these genes to determine if translation of the RNA molecules is spatially or temporally regulated. Alternatively, regulation of the subcellular localization of regulatory proteins may explain the whorl-specific gene activity. These studies will lead to a more complete picture of the molecular hierarchy responsible for ABC gene regulation. This review has focused on the genetic basis of ABC gene regulation and has treated floral organ identity specification as independent from environmental events. As we further clarify the molecular mechanisms of floral organ identity, we may be able to better predict the effects of environmental influences, such as the effect of photoperiod, hormone, and temperature on floral development. Understanding the interaction between the genetic programs and environmental factors will be critical for controlling traits of agricultural varieties in the field. As our understanding of the genetic mechanisms of organ identity specification grows, so does our ability to engineer new floral variants. One can envision a multitude of applications in the areas of horticulture and agriculture. For example, to engineer environmentally friendly ornamental types (such as pollen-free or fruit-free cultivars), or to facilitate outcrossing and simplify breeding programs, novel methods of generating male-sterile plants will be highly desirable. By specifically repressing the B or C class genes in whorl 3 through transgenic techniques, one may create flowers whose stamens are converted into carpels or petals. Alternatively, our knowledge of organ identity genes could be used to increase the visual diversity of floral variants. Replacing reproductive structures with petals leads to showier flowers, as is seen in a number of presently existing “double flowers” (Acquaah et al. 1992) and in Arabidopsis transgenic plants (Fig. 2.2F). New chimeric organs that result from a partial homeotic transformation may have useful or visually interesting properties: stamenoid petals that are more tubular or sepalloid petals that are more resistant to wilting and thus increase the life of the cut flower. Increasing the proportion of a given organ within the flower may enhance the yield of certain floral-derived products. Yields

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of saffron, derived from stigmatic tissue of the crocus flower, might be improved if ectopic expression of C class genes is employed to generate additional carpels. As we continue to illustrate the “blueprints” of floral development, we hope to enable “agricultural architects” of the future to rationally design floral types to better meet societal needs. In addition to the technical hurdles that lie ahead, we also face the multi-disciplinary challenge of managing these new variants such that environmental and socioeconomic effects are carefully considered.

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3 Lingonberry: Botany and Horticulture Inger Hjalmarsson The Nordic Gene Bank, Smedjevägen 3, P.O. Box 41, S-230 53 Alnarp, Sweden Rodomiro Ortiz The Royal Veterinary and Agricultural University, Department of Agricultural Sciences, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark

I. INTRODUCTION II. HISTORY A. Early B. 18th and 19th Centuries C. 20th Century D. Horticultural Research in the Northern Hemisphere 1. Nordic Region 2. Other Sites in the Northern Hemisphere III. BOTANY A. Taxonomy and Geographic Distribution B. Morphology 1. Vegetative 2. Reproductive C. Ecology D. Other Vaccinium Species in Scandinavia 1. Bilberry 2. Bog Bilberry 3. Small-fruited European Cranberry 4. Dwarf Cranberry IV. MANAGEMENT OF NATURAL STANDS A. Photosynthesis B. Biomass Production C. Seed Ecology and Regeneration D. Berry Production E. Effects of Forestry Management F. Experiments in Natural Habitats Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 79

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I. HJALMARSSON AND R. ORTIZ

V. HORTICULTURE A. Propagation 1. Plants Collected Directly from the Wild 2. Shoot Cuttings and Rhizomes 3. Seedling Plants B. Frost Protection C. Culture 1. Soil 2. Nutrients 3. Mycorrhiza 4. Mulching 5. Irrigation D. Plant Pathology 1. Weed Control 2. Diseases E. Crop Improvement 1. Early Studies of Ecotypes 2. Breeding at Balsgård 3. Description of Swedish Cultivars 4. Description of North American Cultivars VI. SUMMARY AND FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION Lingonberry (Vaccinium vitis-idaea L., Ericaceae) is a perennial, evergreen dwarf shrub that is indigenous to Scandinavia, where the peasized, bright-red fruit is picked from wild stands. Lingonberry is known as puolukka in Finland, as tyttebær in Norway and Denmark, and as lingon in Sweden. Lingonberry jam, with or without sugar, may be eaten with porridge, potatoes, bread, pancakes, cow and reindeer milk, herring, black blood pudding, meatballs, and steak among other foods. The berries have also been used for soups and beverage. Retzius (1806) recommended lingonberry drinks for fever patients. Furthermore, lingonberry has been used as an anti-scorbutic (Nyman 1868), and because of its richness in glycosides (Bandzatiene 1999), as a diarrhea medication (Stodola and Volak 1986). Folk medicine recommends that lingonberry tea, derived from leaves, be used against rheumatism (Henriksson 1923b) and as a remedy for urinary tract infections (Nielsen 1978). Recent reports suggest that lingonberry may have anticancer attributes due to high anthocyanin content (Bomser et al. 1996). Lingonberry jam is a traditional delicacy and, although considered a luxury today, it was once one of the few staples available to poor people. While no longer necessary for survival, berry picking has become a recreational activity for many and jam making has moved from homes

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to processing plants. Industry requires a continuous supply of the berries and shortages of labor have led to the start of domestication of the crop. Lingonberry has also been grown as an ornamental plant or shrub in Scandinavian gardens and landscapes since the 17th century (Adelswärd 1994; Balwoll and Weisaeth 1994; Lundqvist 1995) and is an important component of Christmas decorations. Since the 1960s, cultural studies have been carried out in Sweden, Finland, and Norway but published information is scattered. This review article focuses on Nordic lingonberry and includes botany, management of natural stands, cultural practices, and breeding. Information about lingonberry research in Germany, the former Soviet Union, Poland, and North America has also been included.

II. HISTORY A. Early There is little documented information about berry utilization in Scandinavia before Linnaeus. Remnants of lingonberry wine in Danish graves from the Bronze Age are the first proof of its use in the home (Brøndegaard 1987). The Icelandic law books (Grágrás) of the 13th century stipulated that berry-picking on other people’s land must be limited to what can be eaten on the spot, thereby indicating the importance of lingonberry as human food (Armfelt Hansell 1969). There are a few other published reports of wild lingonberry in the Middle Ages (Eriksson et al. 1979). The Italian diplomat Magalotti (1674) wrote the first thorough description of wild lingonberry in Sweden after his journey through the country in the 17th century. By that time lingonberry was also mentioned in Nordic gardening books. In 1651, André Mollet, the French gardener working for Queen Christina, published Le Jardin de Plaisir and suggested the use of lingonberry for parterres de broderie (hedge gardens) instead of the less adapted box model (Adelswärd 1994; Lundqvist 1995). The first Norwegian gardening book, written in 1694 by Christian Gartner, recommended planting lingonberry for culinary and medical purposes (Balwoll and Weisaeth 1994). B. 18th and 19th Centuries Botanists such as Linnaeus (1748) and Retzius (1806) described lingonberry during the 18th and 19th centuries. The economist Fischerström (1779) also discussed lingonberry in his dictionary about Swedish

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households and natural science. Furthermore, lingonberry recipes were published in the cookbooks of the time. In addition, lingonberry was included in crofters’ (tenants’) contracts and mentioned in local horticultural records. For example, Waern (1834) reported on the cultivation of white lingonberry, Vaccinium Vitis idaea fructu albo, in the garden of Baldersnäs (Sweden). Lingonberry was also considered in ethnology studies by Hyltén-Cavallius (1868) and in diet investigations by Keyland (1919) and Grøn (1942). Although lingonberry has been considered among the most important of the fruit jellies (Retzius 1806), no evidence exists about its importance in the diet of earlier times. Eriksson et al. (1979) indicated that berry harvest was most valuable to poor people, especially in years of bad crops when lingonberry could mean survival. Crofters’ contracts in the 19th century often stated that the crofter’s family should pick a certain quantity of berries for further delivery to the estate (Armfelt Hansell 1969). Women and children did most of this work and they also picked the family supply. The berries were originally picked by hand, but Fischerström (1779) described the earliest picking tool. The use of these tools became more widespread and, at the beginning of the 20th century, a debate started about whether this practice was harmful. However, experiments at the Royal Swedish Academy of Agriculture concluded that the tools were harmless to the plants (Sylvén 1918). Johansson (1983), von Zabeltizt (1989), and Dale et al. (1994) have described the development of other lingonberry harvesting aids. Throughout the history of this region, lingonberry has been important as a supplier of energy and vitamins. Lingonberry fruit differs from most of the other wild berry species owing to its long-term storage potential. Consequently, berries are kept from one year to the next without sugar, a product that was rare in most Scandinavian homes until the 19th century (Kuuse 1982). Traditionally, berries are placed in jars and preserved by pouring clear water over them to produce a dish known as “water-lingon” in Swedish cuisine (Retzius 1806). The storage ability of the berries depends primarily on their benzoic acid content, with up to 65 mg benzoic acid per 100 g of berries (Karlsson and Malmberg 1974). Lingonberry fruit also contains large amounts of aroma compounds and anthocyanins (Anjou and von Sydow 1967; Andersen 1985), negligible amounts of proteins, and only small amounts of minerals, although lingonberry fruit provides 4 mg vitamin C, 0.02 mg carotene, and 67 kcal of energy per 100 g (Statens Livsmedelsverk 1978). According to Fuchs and Wretling (1991), lingonberry fruit has 7 g sugar/100 g and 24 g titrable acids/L fruit juice.

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Lingonberry has a long history of commerce in the Nordic countries. Linnaeus (1732) described how farmers in northern Sweden sent their berries to the markets in Stockholm. The moor farmers of Jylland (Denmark) traded lingonberry with Copenhagen in the 1800s (Brøndegaard 1987). The first reference to export of lingonberry from Norway was dated 1835 (Valset 1976; Graff 1991). However, the “days of glory” for lingonberry began at the turn of the century, in conjunction with the development of the railway. In 1902, a record quantity of 20 million kg of lingonberries was exported from Sweden (Wikmark 1907). The Swedish berries were mainly sold to Germany, where they competed with berries from Norway and Finland. C. 20th Century Taking care of wild lingonberries captured people’s interest in Finland, Norway, and Sweden at the beginning of the 20th century. For example, Norwegian committees were established to make berry picking more efficient (Valset 1976; Graff 1991), whereas Swedes started an industry with dried lingonberry and blueberry (Lind 1916). According to official statistics, the exports remained high throughout the 1930s (Eriksson et al. 1979; Graff 1991). Finnish exports during this decade varied between 2.4 and 6.7 million kg yearly (Anon 1983). The demand for lingonberry continued to be high during World War II, when people were encouraged by their governments to create contingency stocks in case there were shortages of other fruits. Lingonberry exports never surpassed earlier levels after World War II (Eriksson et al. 1979). But commercial lingonberry harvest has been reported from Alaska, Nova Scotia, and Newfoundland in North America (Holloway 1984). The commercial lingonberry harvest in Finland from 1977 to 1985 was estimated to vary between 1.7 and 10.2 million kg annually, of which 35 to 80% was exported (Hiirsalmi and Lehmushovi 1993). Swedish exports during the same period fluctuated between 1.5 and 4.2 million kg (Holmberg 1987). In 1985 the highest economic return (85 million Swedish Krone) was realized. Finland and Sweden import some wild berries but remain net exporters of lingonberries. In Norway, however, more fruit are imported than exported. The Norwegian food industry uses about 1 million kg of lingonberry yearly and an additional 0.2 to 0.4 million kg are sold on the fresh market. The majority of these berries are imported (Nes 1994). Foreign trade in wild lingonberry remains important in Scandinavia (Statistics Sweden 1994). Per capita consumption of wild small fruits (mainly lingonberry and blueberry) in Sweden was estimated to be 0.6 kg in 1990 (Statens Jordbruksverk 1994).

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D. Horticultural Research in the Northern Hemisphere 1. Nordic Region. Lingonberry is a healthy component of the Scandinavian diet and is considered an exotic fruit by many people outside the region, which suggests that this crop will always have a market. However, the question persists whether the crop should be picked solely from native stands or through commercial production, or both. Interest in commercial harvest from wild stands has decreased but the industry demands a reliable annual supply. Sweden. AB Bjäre Industrier, the Swedish producer of lingonberry jam, initiated the first lingonberry plantings at Vången (Skepparslöv) in 1962 and another 5 ha were planted at the Ottarp farm (Ryssby) from 1966 to 1968 (Teär 1972). Additional small plantations were started later in both middle and northern Sweden, and supplementary experiments were carried out in the Department of Pomology at the former Agricultural College of Sweden in Alnarp (Fernqvist 1977). A Nordic symposium about domestication of wild berries was held in 1974 at Karlstad (Fernqvist 1974). Most of this early research was published in Swedish, but English abstracts were printed for the ISHS International Symposium on Vaccinium Culture (Fernqvist 1977; Hjalmarsson 1993). Since the late 1970s, most lingonberry research has been carried out at the Department of Horticultural Breeding in the Swedish University of Agricultural Sciences (SLU) in Balsgård. Pilot plantations are run parallel to the plant breeding experiments and provide continuous experience on practical cultivation. The first plantations were planted with micropropagated plants of ‘Sussi’ and ‘Sanna’. Plant spacing was 40 cm in a zig-zag pattern that aimed to create a dense carpet of lingonberry vegetation (Eckerbom 1990). Currently a row system with plant spacing of 25–30 cm × 80 × 100 cm is recommended (Nilsson and Rumpunen 1997). Finland. The first cultivation experiments began in 1968 at the Institute of Horticulture in Piikkiö (southwest Finland). The broad research program involved experiments in natural habitats, field studies on plant material, soil, liming, fertilization, and shading among others. Research results were reported in Finnish journals, and review articles were also written (Lehmushovi 1977b; Hiirslami 1989). Current research is carried out by the Department of Plant Production of the University of Helsinki and focuses on weed control (Saario 1998). Norway. The first commercial lingonberry field in Norway was planted in the mid-1960s. In 1974 public research began at Kise Research Station, where the experiments focused on plant material, plant establish-

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ment, water requirement, and weed control (Nes 1994). In 1983, a group of farmers in southeastern Norway started a lingonberry project; however, it did not lead to any extensive production (Vestrheim et al. 1994). Seven years later, a private farmer imported Dutch and German cultivars for field planting. At the same time, the Agricultural University of Norway (NLH) started cultivar trials, which are still going on (Vestrheim et al. 1994). 2. Other Sites in the Northern Hemisphere Eastern Europe. Former Soviet researchers started their research on lingonberry domestication in the mid 1960s (Paal 1992) owing to a rising demand and low productivity of wild lingonberry. Propagation methods, seed germination, factors influencing root development in cuttings, mulching, phenology, somatic embryogenesis, and micropropagation were included in their research agenda (Butkus et al. 1989; Labokas and Budriuniene 1989; Audrina 1996; Banner 1996, 1998; Bandzaitiene 1998; Kutas 1998; Kutas and Sidorovitch 1998). In Poland, mulching was also investigated (Pliszka and Scibisz 1985). Plant growth was enhanced in mulched plots but it did not improve fruit yield. Testing foreign lingonberry germplasm to identify new high-yielding cultivars has also been an important activity in Eastern Europe. A high second harvest was reported for Dutch and German cultivars in Byelorussia (Pavlovsky and Ruban 1998). However, this second harvest of foreign cultivars was low in Latvia (Audrina 1996) and Russia (Tiak and Cherkasov 1998). Four promising selections were made from the local material in Latvia (Audrina 1996) and two Russian cultivars (‘Kostromskaya Rozovaya’ and ‘Kostromichka’) were released in the mid1990s (Tiak and Cherkasov 1998). It seems that seedlings from northern regions have earlier growth and faster development than those from southern sites (Reier and Paal 1998). Plant breeding through chromosome doubling (CD) with colchicine was unsuccessful in Byelorussia. The CD-derived plants did not improve fruit yield because of few flowers and pollen sterility (Morozov 1998). Wild tetraploid lingonberry accessions collected in Magadan (Russia) were crossed with other Vaccinium species, but the hybrid seeds did not always germinate. Germany. Lingonberry research was started in 1973 by Prof. G. Liebster at the Institute of Fruit in Weinhenstephan (Liebster 1977, 1984; Müller 1982). Morphology and physiology research led to an efficient method for vegetative propagation of the Dutch cultivar ‘Koralle’ and the German

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cultivars (‘Erntedank,’ ‘Erntekrone,’ and ‘Erntesegen’). The German cultivars are selections from wild lingonberry stands (Zillmer 1985). Further investigations at Wilhelm Dierking Beerenobst included soils for lingonberry cultivation, mulching, and fertilizers (Dierking and Krüger 1984; Dierking 1985). The University of Hannover started research on lingonberry nutrition in the 1980s to determine the correlation between yield and growth with nutrient levels using the leaf analysis (Krüger and Naumann 1984a,b,c; Krüger 1985). There were some attempts by German scientists to obtain hybrids between lingonberry and cranberry (Vaccinium macrocarpon Ait.) (Christ 1977), as well as in vitro propagation of lingonberry clones (Gebhardt and Friedrich 1986). Tissue culture has been confirmed as a means for rapid mass propagation of lingonberry (Riechers and Bünemann 1989) and to facilitate fruit picking, a harvest machine was developed by von Zabelitz (1989). Nowadays, about 35 ha are grown commercially in Germany (Dierking and Dierking 1993). North America. Wild lingonberry fruit are collected commercially in Alaska, Nova Scotia, and Newfoundland (Holloway 1984; Hendrickson 1997). Since 1965 researchers at Fairbanks (Alaska) have been working in lingonberry improvement. Early investigations included assessment of substrates for lingonberry cultivation, influence of light intensity on growth, gibberellic acid effect on fruit set, chilling temperature requirements, factors affecting rooting of stem cuttings, and seed propagation (Hall and Bell 1970; Holloway et al. 1982a,b; Holloway et al. 1982; Holloway et al. 1983; Holloway 1985). In Newfoundland, lingonberry researchers are studying crop establishment, maturity dates, pests affecting the crop, and the best cultural practices to enhance earliness and fruit yield (Penney et al. 1997). Researchers working in the Department of Horticulture at the University of Wisconsin–Madison have investigated physiology (photoperiod response) and cultural practices (weed control, humus application) needed to introduce lingonberry as a new fruit crop in the northern United States (Stang et al. 1993a,b, 1994; Stang 1994). They have established small-scale demonstration plots and determined fruit processing requirements. Cuddy (1998) has reviewed advances in lingonberry cultivation at Wisconsin. North American scientists have tested cultivars and selections from the wild (Penney et al. 1977; Estabrooks 1997). In the mid-1990s, the University of Wisconsin–Madison released two lingonberry cultivars for commercial production (Stang et al. 1994), ‘Splendor’ and ‘Regal’, both

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selections from Finnish exotic wild germplasm. Vaccinium reticulatum has been crossed with lingonberry because this diploid evergreen wild species, native to Hawaii, has a large fruit size among other interesting characteristics (Zeldin and McCown 1997). Some North American researchers (Hosier et al. 1985; Serres et al. 1994) have investigated micropropagation methods including basal media, plant growth regulators, and culture conditions. III. BOTANY A. Taxonomy and Geographic Distribution The genus Vaccinium includes approximately 400 species (Galletta and Ballington 1996) dispersed from the arctic to subtropical regions and mountainous tropics (Hutchinson 1969). Five Vaccinium species occur in Scandinavia: V. vitis idaea (2n = 24), V. myrtillus L. (2n = 24), V. uliginosum L. (2n = 24, 48, 72), V. oxycoccus L. (2n = 24, 48, 72), and V. microcarpum (Turcz.) Hook. (2n = 24) (Hylander 1955) (Plate 3.1A). The chromosome numbers in brackets are those reported by Luby et al. (1990). The rare hybrid Vaccinium × intermedia Ruthe was described by Ritchii (1955a) as an intermediate between V. vitis-idaea L. and V. myrtillus L. Although this hybrid has been seldom reported in the Nordic Region, it has been seen sometimes in Sweden (Scania and Stockholm) and in two locations at Jylland, Denmark (Lagerberg 1948). Triploid forms of Vaccinium vitis-idaea have been reported in Sweden (Ising 1950) and in Finland (Ahokas 1971). Hultén and Fries (1986) have mapped the distribution of the circumpolar lingonberry (Fig. 3.1), and Hultén (1971) provided details about the abundance of lingonberry in the forests of Finland, Norway, and Sweden and its sparse occurrence on the calcareous soils of southern Sweden and the Danish islands. Lingonberry is also common on the moors of Jylland (Brøndegaard 1987; Hultén 1971) and grows at 1800 m above sea level in Jotunheimen, Norway (Hultén 1958; Lagerberg 1948). Lingonberry seems to be a European crop (Plate 3.1B), although the Pacific Northwest in the United States has observed a significant planting of lingonberry in recent years (Finn 1999). Vaccinium vitis-idaea is the only species in the section Vitis-idaea (Moench) Koch (Galetta and Ballington 1996). Hultén (1949) divided lingonberry into two subspecies: subsp. vitis-idaea L. and subsp. minus (G. Lodd.) Hultén. Both subspecies are found in the arctic mountains of Norway. Subspecies vitis-idaea predominates in Eurasia, while subsp.

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Fig. 3.1. Lingonberry in the Northern Hemisphere (From: Hultén and Fries 1986, with kind authorization from Koeltz Scientific Books).

minus prevails in the mountains of North America. Apparently, the two subspecies hybridize in Scandinavia (Hultén and Fries 1986). B. Morphology 1. Vegetative. Lingonberry is as an evergreen, small shrub with subterranean rhizomes, and aerial shoots varying from 5 to 30 cm tall (Hultén 1958) (Fig. 3.2). The subsp. minus is shorter and has smaller leaves and berries than subsp. vitis-idaea. The leaves of subsp. vitis-idaea possess conspicuous venation, while the venation is inconspicuous in subsp. minus (Hultén 1949). The leathery leaves of lingonberry are ovate with a thick, glossy upper surface and a pale, glandular lower surface. These characteristics allow the plant to survive the cold and windy winters of the North without desiccation. The lingonberry plants typically have leaves ranging in length from 4 to 29 mm and width from 2 to 16 mm (Teär 1972).

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Fig. 3.2.

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Lingonberry plants collected from the Nordic forest.

2. Reproductive. Floral initiation in Vaccinium species starts in late spring or early summer (Bell and Burchill 1955; Eck 1966b). Flowering occurs for one month between May and June in the southern part of Scandinavia, and four weeks later in Lapland (Hultén 1971) (Fig 3.3). In these northern latitudes, secondary flowering on current year shoots is rare, as noted in middle Europe by Hegi (1927) and Ritchii (1955b). At Wisconsin, flower initiation occurs at 8 to 12 h day length and a minimum of 8 weeks seems to be needed for maximum flower induction (Stang et al. 1993a). It is possible to distinguish between floral and vegetative buds of lingonberry in August at northern latitudes. The vegetative buds are larger (2–3 mm) and wider (1 mm) and they have a tendency to bend downward. The inflorescence is a slightly pendulous raceme of 4 to 6 flowers (Teär 1972). The lingonberry flowers (Plate 3.1C) are white to pinkishred, 4 to 6 mm in length, urceolate, and possess 4 to 5 petals (Knuth 1899). These flowers are hermaphroditic and epigynous with 4 to 5 locules per ovary and 15 to 20 ovules per carpel. The flower has 8 stamens that open by pores (also known as porandrous). The form of their sepals, hairiness of their filaments, and length of their style (Hultén

Fig. 3.3.

Lingonberry plants in blossom.

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1949) differentiate the flowers of the two subspecies. The dark red, globular berry of the subsp. vitis-idaea ripens in September and has, on an average, a diameter of 6 to 8 mm (Teär 1972). The style is closely surrounded by anthers; the stigma and anthers mature nearly simultaneously but the anthers may mature later. The stigma rises outside the corolla, which suggests that wind might be important for pollination (Hagerup 1954). However, most authors (Haslerud 1974; Lehmushovi 1977a) consider entomogamy essential for fruit set. Bumblebees (Bombus terrestis L. and B. pratorum L.) were the principal pollinating species at Ottarp (Eriksson 1975). However, some years the bumblebee workers were not ready until the last half of the flowering period (Ängeby 1978). Nectaries, hidden beneath the stamens, produce a large amount of nectar that attracts insects. Cross-pollination is effected as the insect passes the stigma, depositing pollen, and in the process of getting to the nectaries, pollen from the anthers is transferred to the insect. The anthers (Plate 3.1D) are touched afterwards, thus releasing pollen grains to the surrounding environment. Pollen grains of lingonberry are arranged in tetrads, and the rate of germination exceeds 80% (Eriksson 1975; Lehmushovi 1977a). Pollen tubes required five days to grow through the style. Different botanical varieties of lingonberry are mentioned in the literature. Among them, variety ovata J. Henriksson, exhibiting oblong-ovate berries, occurs in the county of Dalsland (Sweden) and in northern Norway (Henriksson 1923a; Jørstad 1960). Swedish botanists have also noted lingonberry stands with white berries (Nyman 1868; Lagerberg 1948). Temperatures between 15 and 20°C enhance pollination, while temperatures above 25°C lower fruit set (Eriksson 1975; Hjalmarsson 1997). Fruit set was greatest following artificial cross-pollination (64%) and open-pollination (58%), whereas fruit set after self-pollination and in isolated flowers were only 28% and 2%, respectively, in wild stands. Seed set ranged from 10.9 per berry in open-pollinated plants to 4.1 in isolated plants. There were 8.6 seeds per berry after cross-pollination, while 3.8 seeds were obtained after self-pollination. Similar results were obtained with the cultivars ‘Sanna’ and ‘Sussi’ (Hjalmarsson 1997). C. Ecology Teär (1972) was the first Nordic scientist to thoroughly investigate vegetative and reproductive growth of wild lingonberry. His research was aimed at obtaining information about plant ecology to facilitate a predictable process of domestication. Since then botanists and foresters

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have further investigated native lingonberry populations together with other common understory species in the forests of northern Scandinavia. The effect of light on the annual growth differed according to species (Kellomäki 1977). In lingonberry the relationship between the amount of photosynthesis and productivity was nonlinear. Maximum growth was reached at low photosynthetic light flux. Bilberry and lingonberry were abundant after the closure of the canopy at the expense of more light-dependent species. Kellomäki argued that lingonberry possesses phenotypic and reproductive plasticity, which is common among vascular plants in ground cover communities. Branching occurred on one-year-old shoots in young ramets as well as from buds on older shoots after rejuvenation in deciduous bilberry and evergreen lingonberry in habitats of northern Finland (Tolvanen 1995). Lingonberry has a predominantly monopodial growth habit, but shoot growth stopped in terminal inflorescences after a few growing seasons. This indicates a sympodial branching system, i.e., older ramets grew more horizontally than younger ramets. Terminal buds mainly developed into vegetative shoots in the northern Finnish forest understory, whereas a great number of lateral buds were activated in open habitats. This, however, did not lead to any change in the total number of new shoots. Instead, flower production was greatest in open habitats. Differences in growth habits between the two sites indicated high morphological plasticity, allowing the species to respond rapidly to changing environments. D. Other Vaccinium Species in Scandinavia Berry crops have always been important components of human diets, although some of the berry species have remained economically important only locally. Lingonberry has been considered among the major new berry crops (Finn 1999), but other Vaccinium species indicated below have been locally harvested and may become economically important new crops in the Nordic region. Most of their fruit continues to be harvested from wild stands, although owing to their attributes some of these species are attracting the attention of the industry either for processing (e.g., for juices or jams) or for pharmaceutical purposes. As expected in this process, lingonberry and other Vaccinium species are being domesticated to shift the harvest from wild or native stands to their commercial cultivation in farmers’ fields, which will ensure a stable fruit supply for the industry (Finn 1999). Hence, potential markets for these new crops are playing an important role in the process of domestication of lingonberry and other Nordic Vaccinium species.

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1. Bilberry (V. myrtillus). This species is a perennial low bush whose leaves fall off in autumn (Mossberg et al. 1992). The angular twigs remain green during the winter. The leaves are not leathery as with lingonberry, but are thin and pointed with denting edges. Bilberry flowers between May and June, and the flowers grow one by one. The flower is almost round and pale green with a reddish tone. Berries ripen in July to August and most commonly they are blue and covered with dew. However, they may also be black and shiny and are called “shoemaker’s berry.” Bilberry is common in most of Scandinavia, is harvested from the wild and eaten fresh, as jam, cream, or soup. Bilberries, which have a long history in European folk medicine (Morazzoni and Bombardelli 1996), are still used as a medical treatment against diarrhea. 2. Bog Bilberry (V. uliginosum). This small bush (10–75 cm tall), which flowers between May and June in Scandinavia, stretches across circumboreal Northern Hemisphere regions (Finn 1999). It may be seen as a common under shrub, especially where heath-lands are turning to swamp and along lakeside forest. The berries are blue, oval, covered with dew and harvested from the wild plants (Mossberg et al. 1992). Some accessions have been crossed with V. corymbosum to improve winter hardiness and obtain early harvest in highbush blueberry. The cultivar ‘Aron’ was developed following this breeding approach (Hiirsalmi 1989; Hiirsalmi and Lehmushovi 1993). The fruit juice has no color (opposite to bilberry), and the taste is often described as flat and stale. 3. Small-fruited European Cranberry (V. oxycoccus). This species is similar to the American cranberry, but is much smaller (4–8 cm tall). The leaves (6-8 mm long) are evergreen (Mossberg et al. 1992). The plant flowers between June and July, and each flower cluster has 2 to 4 flowers. The round-shaped berry diameters are 8 to 10 mm, and the berry ripens late. Hence, berry picking is preferred after the first winter frost. This small-fruited European cranberry is common in Scandinavia, although not in the very northern mountain area. The berries are used for jam or alcoholic beverage. 4. Dwarf Cranberry (V. microcarpum). This Vaccinium species is even smaller than the small-fruited European cranberry and extends to the very north of Scandinavia (Mossberg et al. 1992). The leaves are 3 to 8 mm long, and the plant flowers between June and July. The flowers grow one by one or two together, and the berries (5–6 mm) are more oval than those of the small-fruited European cranberry.

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IV. MANAGEMENT OF NATURAL STANDS A. Photosynthesis Seasonal carbon dioxide assimilation per unit leaf mass was three times higher in the deciduous bog bilberry (V. uliginosum) than in evergreen lingonberry in sub-arctic environments (Karlsson 1982). However, lingonberry was light saturated for a longer period, i.e., 80 to 86% of the time in July and 60 to 70% in early August. Lingonberry leaves are largest on the mid-position of each yearly shoot segment, and have axes with uniform and horizontal angles. Old lingonberry leaves are important for a rapid CO2 assimilation in the spring (Karlsson 1982). Old leaves need two weeks to build up to full photosynthetic capacity. Similarly, lingonberry plants are able to extend their growing season late in autumn. Increased leaf age, however, affects maximal photosynthetic capacity. Leaves will only retain 2/3 of their original capacity during the second growing season, and in the following years an additional 10% will be lost annually (Karlsson 1982). Hence, lingonberry leaves need four growing seasons to assimilate the same amount of carbon dioxide as bog bilberry leaves assimilate during one season. Current year shoot growth in lingonberry was mainly supported during early summer by photosynthetic products of older leaves (Karlsson 1982). Photosynthesis in bog bilberry and lingonberry was similar. In response to drought, lingonberry has a great ability to survive in dry environments. The two species respond differently to light utilization and water economy, and occupy distinct niches in natural subarctic environments. B. Biomass Production There were, on average, 101 to 231 m rhizomes per m2 in a large number of selected lingonberry plots in natural habitats in Sweden (Teär 1972). Annual production was calculated at 13 to 55 new rhizomes per m2. New rhizomes typically grew 15 to 20 cm, and never had a growth rate exceeding 40 cm. Rhizomes had buds, which may develop new shoots under or above ground. A total of 150 dormant buds were found per m of rhizome. New plants were usually developed in groups near where the rhizomes originated, and only about 15% of these originated from buds at the terminal ends. Every year 10 to 25% of the old plants were replaced by new plants through rejuvenation. The average plant age was 4 years, but plants as old as 11 years were found. The total amount of dry biomass varied between 153 and 579 g per m2 (Teär 1972). About

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half the biomass was underground and rhizomes accounted for 10 to 20%. The above ground biomass was equally divided between shoots and leaves, with the latter constituting 78% of the annual production of biomass. A lingonberry plant had on average 3.3 shoots, and on average produced 1.7 new shoots per plant every year, but more than half the plants had only one new shoot. Most of the shoots were produced from vegetative one-year-old shoots. Large plants produced more shoots than small plants, though the ratio (new shoots/total number of shoots) was lower. Furthermore, flowering shoots were shorter than vegetative shoots, and produced new shoots from stems that flowered late in the growing season. On average there were 8 to 9 leaves per shoot. Flowering shoots had more leaves than those that were strictly vegetative. At the beginning of the summer, current-year leaves accounted for 70% of the total leaf number (Teär 1972). Leaves growing on shoot tips were more pointed than others growing below the shoot tips. The number of plants with flower buds increased with the age of the plants. Plants with 5 to 6 older shoots had mostly new flowering shoots (2.8–3.1). However, the share of new fertile shoots within a plant decreased as it grew in size. Most fertile shoots were in clear felled areas, while the opposite occurred in forests with Norway spruce. On average, there was one flower bud per flowering shoot. The number of flowers per cluster varied from 4.1 to 5.9, while there were, on average, 3 to 4 berries and a maximum of 14 berries per cluster. Increased shoot growth and higher levels of nitrogen, phosphorous, and potassium were noted after irrigation and fertilization, while the photosynthetic rate was the same in controls and treatments (Karlsson 1985). These changes were more conspicuous in lingonberry than in bog bilberry. Lingonberry had fewer old leaves per shoot after the treatment. These results suggested that lingonberry, as previously reported in other evergreen species, has decreased leaf longevity when nutrient levels are increased in the substrate. C. Seed Ecology and Regeneration Fruit set was lower after self-pollination than after open-pollination (Fröborg 1996). A few lingonberry seeds were recorded in the seed bank of a sub-arctic pine-birch forest in Lapland, Finland (Vieno et al. 1993). Vaccinium species lack developed seed banks even though they are known to have high seed production (Eriksson and Fröborg 1996). The work of Eriksson and Fröborg focused on “windows of opportunity,” i.e., spatially and temporally unpredictable conditions in which seedling

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recruitment was possible. Lingonberry seedlings favor moist soil with high organic content. Likewise, sudden optimal conditions at a specific micro-site may lead to establishment of new seedlings within stands of adults. Extensive production of well dispersed seeds but no extended dormancy will take advantage of such favorable situations. Furthermore, recruitment through “windows of opportunity” could explain unexpected high genetic variation within populations of persistent clonal plants. Regeneration of understory species after fire was investigated in Northern Sweden (Schimmel 1989, Schimmel and Granström 1996). To survive or escape a forest fire, plant material must either be able to withstand high temperatures or be deeply buried. Rhizomes of lingonberry were killed after 10 minutes at 55° to 59°C. The main part of the subterranean runners grew in the middle or a little below the middle of the humus layer known as mor, which occurs by decomposition in the superficial soil layers instead of its surface. A small proportion of rhizomes in the mineral soil occurred in thick humus layer. The total bud bank ranged from 470 to 950 shoots per m2. Slightly burned plots produced more sprouts than clipped plots that weren’t burned. Sprouting from rhizomes in the upper horizon increased after burning. Improved nutrient status in the soil could explain this phenomenon. If fires were limited to the moss layer, pre-fire coverages were reached within 2 to 4 years. The most severe fires, however, eliminated lingonberry as well as other Vaccinium species. Tolvanen et al. (1995) mapped the recovery of lingonberry after removal of annual branches or ramets in a boreal forest. Recovery proceeded unexpectedly high even after the most severe treatment (100% removal of ramets). When whole ramets were removed the percentage of new growth emerging from basal buds increased. About 42 to 112% of the above ground biomass and 60 to 70% of the coverage were regained after three growing seasons. D. Berry Production The National Forestry Survey did the first research project regarding wild berry production in Swedish forests (Eriksson et al. 1979; Kardell 1980). Production of bilberry, raspberry, and lingonberry were analyzed on 44,000 sites during three years (1974 to 1977), and berries were counted and weighed. Lingonberry occurred on 1.2 million ha out of 23.5 million ha of productive Swedish forests. The occurrence of lingonberry varied little with the density of the forest. Coverage was about 5% on clear-cut areas and in young forests, while about 7% were

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recorded for old established forests. In northern Sweden, lingonberry was more common than bilberry and raspberry. In this region, coverage was highest (8–9%) in stands of Scots pine, while it was 4% for Norwegian spruce. Productivity was relatively high on clear-cut areas, in young and very old forests, whereas productivity was low on 80 to 90% of the forests. Most of the berries (70–80%) were produced in the forests of northern and central Sweden. Between 70 and 80% of the total production was considered available for harvest. The National Forest Survey for 1978 through 1980 investigated the occurrence of cloudberry, small-fruited cranberry, and lingonberry (Kardell and Carlsson 1982; Kardell 1986). The inventories involved both forests and bogs. The latter covered 5.1 million ha with average lingonberry coverage of 1.1%. The results indicated that lingonberry preferred mineral soil, where the coverage increased from south to north, while the opposite occurred in bogs. A negative relationship was reported between lingonberry coverage and site elevation. The total mean berry production during this investigation was 209 million kg yearly. Only 4% of this yield was from bogs. The yield as a whole was widely distributed over the country. The highest yields (13.8 and 17.3 kg /ha) were noted from forests and bogs in central Sweden. Graff (1991) calculated the total Norwegian lingonberry production, which varied between 44 and 115 million kg per year. Raatikainen (1988) and Raatikainen et al. (1984) noted that variation in berry production depended on different factors, such as tree-canopy density and lingonberry coverage in Finland. The total annual production was estimated at 180 to 200 million kg, with an average yield of 8 kg/ha in the forests. About 80% of these berries were considered harvestable. However, lingonberry yield varies from one year to another due to night frost in June (Kardell and Carlsson 1982). About 50% of the flower buds, flowers, and green fruits are killed at temperatures below –1.5°, –3.1°, or –3.5°C, respectively (Teär 1972). There was also an association between snow cover and yield. Lingonberry buds were unable to withstand minimum temperatures, which varied between –25° and –32°C in January (Raatikainen and Vänninen 1988). After the exceptionally cold winter of 1985, highest yields were harvested from plants that had overwintered below a thick snow cover. The annual production of lingonberry in the Nordic countries can be estimated at 500 million kg, of which 80% is considered available for harvest. However, only 2 to 11% of the total production is collected. Through a mail survey, Hultman (1983) found that 38% of the Swedish population picked 15 L of lingonberry per person annually. People in northern Sweden gathered more berries than those in the south. The per-

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centage of berries picked was 28% in the south and 7% in the north, i.e., 11% of the production was harvested in Sweden. Only 3 to 5% of the total lingonberry production was picked in Norway (Graff 1991), and 2 to 7% of lingonberry production was harvested in Finland (Saastamoinen 1981). During the 1970s the average picker gathered 14 L per year (Hultman 1983). Saastamoinen and Lohiniva (1989) studied lingonberry picking in the Rovaniemi region of Lapland. About 70% of the households participated in the harvest. The total quantity of wild berries collected was 2 to 4 times larger than the national average. This is considered typical for rural communities in northern, eastern, and central Finland. Approximately 86% of the families in five communities in central Finland picked lingonberry and 9 to 44% of the total production in this area was gathered (Rossi et al. 1984). E. Effects of Forestry Management As lingonberry picking is a popular family pastime, there is an apprehension that modern forestry could threaten berry production. Consequently, the section of Environmental Forestry of the Swedish University of Agricultural Sciences (SLU) started a series of experiments at 27 testing sites across Sweden in 1976 with the aim of studying the relationship between silviculture and the development of grand cover vegetation. The original status of the sites was mapped during the first year. Different treatments that included thinning, clear cutting, fertilization, and soil disruption were carried out. Results from these experiments were reported after 5, 10, and 15 years (Kardell and Eriksson 1983, 1990, 1995). Lingonberry biomass was reduced by 15 to 20% directly after thinning of the overstory trees. Recovery was slow over time but thinning was positive for vegetative lingonberry development and, after 15 years, the coverage was slightly higher than in the control plots. Additionally, thinning resulted in a 3- to 4-fold yield increase. Clear cutting also led to decreased lingonberry biomass in the beginning, but after 15 years the plants had completely regained their positions. Lingonberry thus has good competing capacity on clear cuts and takes advantage of extra light. Clear cuts may be exposed to spring frosts, but in general these are good areas for berry production. On average a 4-fold yield gain was recorded. Development after fertilization with 150 kg N/ha was negative, as lingonberry coverage and yield decreased by 12 and 45%, respectively, following fertilization. Soil disruption caused a 20% biomass reduction after clear cutting and reduced berry production over 14 years by 39%.

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During a six-year period, removal of branches in clear-cut areas doubled yield, while stump removal had the opposite effect (Kardell and Wärne 1981; Kardell 1992). Pinus contorta forests allow less light penetration and lingonberry yields were 46% lower in those forests than those growing with P. silvestris (Kardell and Eriksson 1989, 1990). Kardell and Eriksson (1990) predicted that lingonberry production in Sweden could decrease by 10%, owing to soil fertility, mechanical soil disruption, and the widespread introduction of Pinus contorta. In contrast, minimal use of fertilizers and machinery, thinning, and clear cutting followed by dead branch removal appears to benefit lingonberry growth and production. Flavor of berries harvested from plants grown in fertilized plots was judged by a test panel to be slightly inferior to those plants grown in unfertilized plots (Kardell et al. 1981; Åkerstrand et al. 1988). The berries from fertilized plots had higher N levels during the year of treatment. Furthermore, molds and yeast were common in the samples from fertilized plots, resulting in reduced storage life. However, the effects of fertilization were considered small in comparison with those associated with environment and post-harvest handling. F. Experiments in Natural Habitats A prerequisite for successful field planting is the knowledge of the conditions prevailing in natural habitats (Teär 1972; Lehmushovi 1977a). This point was considered for ecological research by Teär as well as by Finnish researchers, who focused their investigations on wild stands from 1968 to 1976. Soil characteristics, temperature, light, and moisture were investigated to elucidate the factors promoting vegetative and reproductive development. The effects of fertilizer applications were also studied. One of the most important factors influencing yield is the weather during flowering. Frost, severe drought, or abundant rain may lead to 60 to 100% loss of buds, flowers, and unripe berries (Lehmushovi 1977a). In the years with favorable weather, this loss ranged from 30 to 60%. Crosspollination by insects and good light conditions are also essential for fruit set. The results of fertilizer trials in natural populations in forests are contradictory. A 2- to 3-fold gain in fruit yield after fertilization has been reported (Lehmushovi and Hiirslami 1972; Lehmushovi 1977a), but if the natural habitat included competing grasses and broad-leaved herbs, there was no benefit. Teär (1972) found a lower percentage of flowering shoots in fertilized plots than in unfertilized plots. The fertilizers did not

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affect the number of flower buds per shoot; however, their use resulted in slight increase in the production of rhizomes, new shoots, and leaves.

V. HORTICULTURE A. Propagation 1. Plants Collected Directly from the Wild. The cheapest way to propagate lingonberry is by digging up plants from natural habitats. Plants may be dug early in the spring, before growth starts, or late in the autumn when it has ceased. If homogeneous clumps of lingonberry were chosen and subdivided before planting, 73% survived when there were four plants per division, while only 18% survived when there were 15 plants per division (Teär 1972). Material collected from native stands should be planted with only the shoots above ground, because the lingonberry plants develop roots in the uppermost soil layer. These plants were susceptible to drought and frost heaving due to their fine, fibrous, and shallow root system. Plant survival ranged from 30 to 90% (Öster 1974). Plants could be established more reliably if the plants were first grown in humid peat nursery beds. After three growing seasons, plants that were first grown in nursery beds had better coverage (80%) than those directly planted from the forest (50%), though these coverages were 80% and 90%, respectively, the following year (I. Hjalmarsson, unpublished). After 3 to 5 years with an initial density of 80 to 100 plants per m2, an even cover was achieved with 400 to 600 plants per m2 (Teär 1972). Plant materials from sunny locations were more difficult to establish than those from shaded locations (Öster 1974). Selected plants had stronger sympodial growth, shorter shoots, greater number of leaves, twice as many flower buds per flowering shoot, and 3 to 4 times more shoots per plant than those from native stands (Teär 1972). In a 5- to 6-year-old lingonberry field, more than 1000 flower buds per m2 were observed, while there were only 300 to 500 flower buds per m2 in the forest (Lehmushovi 1975). The major drawbacks with wild plantlets are that they have a poor rate of establishment, are heterogeneous, and are difficult to handle. 2. Shoot Cuttings and Rhizomes. Propagation by shoot cuttings has been successful (Öster 1974; Lehmushovi 1975). The best results were obtained with mature spring and autumn shoots, while soft wood shoots taken during the summer were the most difficult to root (I. Hjalmarsson, unpublished). The shoots required about eight weeks for rooting and the

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most favorable substrate was peat. An average rooting percentage of 85% was achieved when mist and peat beds were used (Lehmushovi 1975). Pre-treatments with auxins also promoted rooting and almost 100% rooting was obtained if cuttings were over-wintered in non-heated greenhouses. The shoots were taken from September to October, placed in moistened peat, and wrapped with transparent plastic. By March or April the plants were ready to be transplanted in the field. The best shoot cuttings of ‘Sanna’ were collected in spring and late summer (Gustavsson 1998). However, cuttings from the current year growth were more uniform than mature shoots. Thus, softwood cuttings taken in late summer were recommended. Rooting was more successful in outdoor plastic tunnels than in the greenhouse for ‘Sanna’ (Gustavsson 1998). Plants from cuttings generally grow well in the field and tend to crop early. However, their rhizomes develop slowly. Plants raised from micro propagation produce rhizomes more easily. Rhizomes can propagate lingonberry, therefore shallowly planting 5 to 10 cm rhizome pieces in boxes with moist peat has been recommended (Öster 1974). One or two shoots normally develop at the terminal end, while roots are produced basically. Best results were achieved during spring and late summer, when 60 to 80% of the material was rooted. Similar results were reported by Lehmushovi (1975), who, nevertheless, concluded that rhizomes were difficult to procure and use for propagation. Lack of strong rhizomes with well-developed buds may result in weak plants. Also, rhizomes are sensitive to drought and therefore impossible to plant directly in the field without first growing in a nursery. 3. Seedling Plants. Lingonberry is easily propagated by seeds. The berry contains many seeds that can be cleaned by pulverizing the berry with water in a blender. Well-developed seeds will sink to the bottom and the rest of the mix is then decanted. Seeds germinate well directly after harvest but can also be cold-stored. About 72% seed germination (in dry seeds that were kept in a refrigerator for six months) has been observed after two weeks in damp sand (Lehmushovi 1975). Results from experiments with whole berries, pulverized berries, or direct seed sowing in the field at Ottarp were not encouraging. The acids in the fruit flesh inhibited seed germination (Karlsson and Malmberg 1974), and seedling plants were very susceptible to drought immediately after sprouting. The most preferred sowing appears to be in boxes with fertilizer-free peat and a top layer of sand during winter (Hjalmarsson 1993). The seeds are not covered because light is necessary

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for germination, which begins in about three weeks at 20° to 25°C. Seedlings are watered once a week with a complete nutrient solution, given additional light, and then transplanted as soon as they can be handled. After hardening-off at the beginning of July, they are transplanted to the fields. In greenhouse experiments, the number of leaves increased from 5 to 23 after three months, and shoot length from 1 to 7 cm (Hjalmarsson 1977). The development of leaves and shoots was simultaneous. However, the burst of leaves at the terminal end occurred in flushes and 3 to 4 leaves were born at a time, with 10 days between each flush. When the plants had reached 4 cm in height (11 leaves), the first lateral shoots became visible just above the cotyledons. Seedlings grown outdoors produced dense stands; after two years, for each m2 there were 2755 current year shoots, 330 flowering shoots, 430 flower clusters, and 28 m of rhizomes, which accounted for 9.4% of the total biomass dry weight (Hjalmarsson 1977). Lingonberry seedlings also develop rhizomes when they are very young. In addition, seedlings offer a quick way of producing a large quantity of plants, but fruit production could be delayed by 2 to 3 years due to juvenility and seedlings are genetically diverse. B. Frost Protection An experimental field at Ottarp was consistently exposed to spring frosts. Consequently a frost protection experiment was established in 1976 (in 4 blocks of 12 m2), which consisted of gravel mulch, covering with transparent plastic film, and spruce twigs along with a control. The transparent plastic was laid on wooden frames and, as with the spruce twigs, was removed during warm days. In the sandy soil at Ottarp the gravel mulch increased the temperature near the surface by 0.5°C, whereas the temperatures for plastic film and spruce twigs were 1.4° and 1.6°C greater than ambient temperatures. In 1978, a year with four frosts during spring, all plots were harvested and it was observed that all treatments increased yield. The best result was obtained with plastic film, where a 4-fold yield gain was obtained compared to the control. In mulched plots lower temperatures were observed than in control plots at Balsgård (Gustavsson 1993). This was especially noted in sawdust plots, where the difference was 1.8°C. Transparent plastic covers during the night raised the temperature in the control 1.4°C higher, as in the Ottarp experiment. The positive response to transparent plastic film covering was less pronounced with peat and sawdust.

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C. Culture 1. Soil. In nature, lingonberry grows most abundantly in conifer forests, where the environment is characterized by leached soils with low pH, low base saturation, and low calcium content. Nutrient turnover and nutrient availability is low. Rhizomes and roots grow shallowly, mainly in the humus horizon, which has a porous structure and is considered the principal source for nutrients (Teär 1972). The effects of different soil types were investigated in a replicated trial that included eight different types of soils varying in the amount of clay, humus, and loamy sand plus two organic soils with a high percentage of humus and pure peat at an outdoor frame-yard. The pH ranged from 5.8 in the silt loam to 4.2 in the peat substrate. Within each frame an area was photographed every autumn and spring to record plant development and rhizome spreading. With the aid of these photos the surface coverage of lingonberry shoots and leaves was assessed. The best and fastest plant development was recorded in the pure peat and loamy sands with moderate humus content. These results, supported by Finnish researchers, documented superior growth and coverage in peat (Lehmushovi and Hiirsalmi 1973; Lehmushovi and Säkö 1975). Plants grown in peat, in which coverage was 95%, had greater biomass, of which 24% was accounted for by rhizomes and roots. One quarter of the total shoots from plots in peat were fertile, whereas in the silty loam only 2% were fertile. The highest fruit yields were recorded in plots with the best vegetative development. However, length of the juvenile phase and the beginning of fertility varied among different seedlings. In another experiment the effects of soil types, nitrogen fertilizer rates, and peat mulch were investigated (Hjalmarsson 1980). Fertilizer was added at four nitrogen levels. The organic soil provided the best environment for vegetative growth and the silt loam the poorest. Plant analyses at the end of the experiment indicated that nutrient uptake was similar in the different soil types. However, the percentage of calcium was significantly higher in plants grown in the calcium-rich silt loam. The first-year fruit yield was only 10 g per plot, except for plots with sand, where the yield was 40 g. The following two years the sandy soils were superior for yield, whereas those plants grown in the silt loam had the lowest yield. It was speculated that vigorous plants grown in the organic soil showed delayed flower bud initiation but would have higher productivity at an older age.

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2. Nutrients. Nutrition research in lingonberry has concentrated on nitrogen fertilization; however, the effects of phosphorous, potassium, and lime have also been investigated. Nitrogen fertilization is beneficial to vegetative growth and up to 5 g N per m2 has enhanced growth without a decrease in berry quality. At rates greater than 5 g N per m2, berry production was negatively affected. In these studies nitrogen was applied in a solution that contained 100 g N (NH4–N/NO3–N 40/60), 13 g P, 65 g K, 9 g S plus trace elements, but no calcium. Leaf analysis showed that nitrogen and potassium concentration or content increased and calcium content decreased concomitantly to increased fertilizer rates. Studies of the effect of dolomitic lime (6 and 12 kg per m2) and a balanced fertilizer (NPK 11-11-22 at 5 or 10 g/m2) showed that liming slightly decreased spreading, shoot height, and significantly lowered berry production (Lehmushovi and Hiirsalmi 1973; Lehmushovi and Säkö 1975). In contrast, the greatest ground coverage, tallest shoots, and the highest yields were recorded in plots with fertilizer, although berry size was small in these plots. Joint application of lime and fertilizer negatively affected all characteristics. In another experiment, nitrogen, potassium, and phosphorus were applied singly or in mixtures with two or three elements, and with or without trace elements on a mineral soil (Lehmushovi 1977a). Yields were low because the plants did not grow well in the mineral soil with clay. Nevertheless, fertilizers in small amounts increased fruit yield, while larger application rates did not increase yields. In the investigations with a range of nitrogen fertilizer rates and saltpeter plus sulfate of potassium-magnesium, the best growth was in the control and at the lowest nitrogen application (Sakshaug 1974; Hjalmarsson 1980). Soil analyses in autumn showed that the higher the fertilizer rates, the lower the soil pH and the higher total salt concentration. Nitrate also increased, while ammonium, phosphorous, and potassium were more stable in the soil. Yield responses could not be evaluated due to annual frost, although berry size was consistent over years and across treatments. Slow release sources of nitrogen on sandy soils have not proven to increase growth or yield of cultivars when compared to standard fertilizers. In solution, ammonium uptake was faster than nitrate uptake and lethal levels were reached at 400 mg/L (Ingestad 1973, 1974). Based on growth studies, a 40 ammonium/60 nitrate ratio was recommended. The nutrient requirements to grow lingonberry seedlings were 100 N, 50 K, 13 P, 7 Ca, and 8.5 Mg mg/L (Ingestad 1973, 1974). With the above

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combination of nutrients, maximal growth with lingonberry seedlings was achieved at pH 4.5 to 5.0 and conductance 0.4 to 0.5 mS, which are typical for calcifuges. Potassium regulates water movement in plants and may be less critical in xeromorphic lingonberry. Lingonberry seedlings grown in nutrient solution have the same mechanism regulating potassium uptake as spring wheat, cucumber, birch, pine, and spruce (Jensén and Pettersson 1978). However, in contrast to these species, lingonberry did not appear to be able to accumulate potassium ions. Uptake was limited to what was needed for current growth. A remarkable characteristic of lingonberry was its rapid absorption of calcium (Ingestad 1973, 1974). Such a phenomenon is also typical of species that are adapted to acid soils, but still have a physiological calcium requirement like the other species. Calcium was taken up together with nitrate. Lower growth rates and yellowing leaves were noted in the treatments with high nitrate concentrations, indicating that inhibitory levels of calcium had been reached. The chlorosis was also ascribed to low mobility and activity of iron within the plant tissues due to internal increase of pH. Ingestad (1973) pointed out the practical consequences of his findings. First, the potassium/calcium ratio may have a bearing on water economy, especially if lingonberry is grown in soils with high calcium availability. Low potassium level may lead to poor water-holding capacity, and leaves with lime-chlorosis are more drought-sensitive than normal green leaves. Second, sensitivity to high total salt concentration indicates the importance of small but frequent fertilizer applications and the need to avoid drought. 3. Mycorrhiza. All the members of Ericales are characterized by ericoid mycorrhizae and lack of root hairs (Lihnell 1974; Önner 1977). The mycorrhizae are endotrophic and the fungal hyphae penetrate the epidermal cells of the roots, resulting in a root system in which a high proportion of the biomass is composed of fungal material. Young active roots tend to be infected by mycorrhizae, while they are not found in older roots that have lost their epidermis. Some American scientists such as Goulart et al. (1993) ascribed the ericoid mycorrhizae as most important in their role as nitrogen supplier, though they may also enhance phosphorous uptake. Interactions with heavy metals and soil-borne root diseases were also discussed by these researchers. The fungal species Hymenoscyphus ericae (Read) Korf and Kernan (formerly called Peziella ericae Read) was found on lingonberry (Önner 1977).

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Experimentally it has been shown that lingonberry seeds can germinate and develop normally under sterile conditions (Lihnell 1974), consequently the symbiosis is not obligatory. Mycorrhizae were observed in all living epidermal root cells from all samples collected in wild stands (Önner 1977). There were no differences in degree of infection between samples from wild stands or from those of experimental fields. Weak growing plants tended to have fewer epidermal cells and therefore less mycorrhizae; nevertheless, this did not explain the weak growth of lingonberry plants. Önner (1977) claimed that mycorrhizae are very important for lingonberry in wild stands characterized by low fertility, especially in dry environments. The symbiosis appears to be less important when water and nutrient levels are not the limiting factors. The fact that most of the hyphae are found inside the root could be ascribed to the low pH in the soil, which inhibits the growth of mycorrhizae. It has, therefore, been suggested (Goulart et al. 1993) that the mycorrhizae can assist the host to adapt to a slightly higher soil pH. 4. Mulching. Mulches have been established for frost protection and for other purposes. During the 1970s, the aim was to imitate the humus horizon in natural habitats to obtain a better soil substrate. Later experiments focused on weed control. The growth of wild plants dug from the forest was studied following addition of different amounts of peat mulch (Sakshaug 1974; Hjalmarsson 1980). In addition, different levels of nitrogen in combination with the peat mulch were tested. The nitrogen fertilizer (3 g N/m2) was split into six applications of 0.5 g N/m2 per year—the first three times as ammonium sulfate, thereafter as calcium ammonium nitrate. In addition sulfate of potash-magnesium was included for the first three times. The plots were mulched with wet peat at the end of May 1972. The peat was limed with 1 kg of dolomitic lime/m2 and the peat mulching was repeated in 1974. Weeding was performed by hand in the first year. The following years herbicides, which had been tested at Ottarp previously, were used. Furthermore, fungicides were applied against “leaf falling disease.” In the first year after application of peat, if the peat layer was 4 cm greater, shoot growth was damaged. However, upon application of the mulch two years later, there was no negative effect. Plants mulched with 4 cm of mulch produced twice as many new shoots as the unmulched control. When nitrogen was added in the peat, the fertilized plots with 2 cm peat had a similar positive effect as the treatment with 4 cm peat. Generally the treatments without peat mulch showed the

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weakest growth, and there was a trend that mulched plots with fertilizer had more shoots than their unfertilized counterparts. No effects of mulch or fertilizer on survival of frost at bloom were noted, but differences between individual years were observed and associated with frost damage. Peat mulch caused a decrease in salt concentration, nitrate, phosphorous, and potassium contents, which may explain the more vigorous growth in those plots. Other organic (peat, sawdust, pine needle) and non-organic mulches (gravel, plastic) were also tested (Gustavsson 1998, 1999). Vegetative growth and fruiting were consistently superior with peat mulch. Winter frost damage was highest on the organic mulch treatments, while the plots with plastic mulch and gravel were unaffected. Growth in sawdust mulch was poor and the experimental plots were scored for fungal disease. Fungal disease symptoms were most severe with gravel mulch and control, and least severe with pine needle mulch. In summary, genetic growth habit affects the outcome of soil surface treatments. Low-growing and rapidly spreading cultivars like ‘Sussi’ grow well without mulch, while bush-like cultivars like ‘Sanna’ tend to suffer from broken branches on open soil (Saario and Voipio 1997). Covering the soil with plastic film mulch is not recommended for rhizomeforming cultivars like ‘Sussi’. 5. Irrigation. Irrigation provides a means to achieve consistently high yields and high fruit quality in most small fruit crops. Lingonberry grows best with a combination of irrigation and peat mulch (Hjalmarsson 1980). More shoots are observed where extra water has been provided. Increasing irrigation from 40% of field capacity to 100% field capacity of loamy sand soil increased rhizome number by 37% and shoot number per plant by 39% (Stang et al. 1993a). Peat mulch favors shoots developing from rhizomes. D. Plant Pathology 1. Weed Control. Weeds were a major problem in the early attempts to grow lingonberry. Young plants are unable to compete against weeds and the rhizomes close to the surface are easily damaged by mechanical cultivation. Herbicides (Andersson 1974, 1976; Fernqvist 1977; Hjalmarsson 1980), mulching (Gustavsson 1993, 1996; Saario and Voipio 1997), and allelopathy (Saario 1998) have been studied as possible ways to suppress weeds. A number of herbicide trials were performed at Ottarp early in the 1970s (Andersson 1974, 1976; Fernqvist 1977; Hjalmarsson 1980).

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Promising results were obtained with lenacil and linuron for annual weeds and with dichlobenil for perennial weeds in the Nordic Region of Europe. However, berries from untreated plots had the best taste (Ingelög et al. 1977). None of the herbicides or dosages in various experiments were harmful to lingonberry unless summer applications were used; therefore, they should be avoided. In Wisconsin, terbacil, oryzalin, and simazine herbicides provided effective weed control at application rates recommended for other perennial crops (Stang et al. 1993a). At present there are no herbicides registered by official authorities for use in cultivation of lingonberry in Scandinavia or in the United States. In studies comparing mulching materials (peat, wood chips, and sawdust) and herbicide treatments (glyphosate and propyzamide), the latter eradicated perennial weeds during winter, but propyzamide was no longer inhibiting weed germination in spring and summer. By summer the applied herbicides were no longer effective and the plots had many weeds. Sawdust mulch has consistently and reliably given the best weed control. Mulching with peat and wood chips can also be effective. Herbicides can reduce berry numbers, even though the plants look healthy. The general recommendation for successful weed control, safe plant development, and clean berries involves a combination of herbicides and mulching (Gustavsson 1999). 2. Diseases. Disease attacks have not yet caused damage of economic importance in lingonberry plantings (Gustavsson 1997). Nilsson (1974) described the pests and types of damage found in domesticated lingonberry and divided them into four classes: deformed plants, leaf spots, berry symptoms, and dead shoots. In the first group is lingonberry tumor caused by the fungi Exobasidium vaccinii Fuckel (Woronin), which causes leaves to form thick reddish knobs. However, tumors are more common in the forest than in the field, probably due to humidity. A more serious pest is the little leaf disease, which was found in the field at Ottarp. This disease is spread by cicada and caused by a phytoplasma (Tomenius and Åhman 1983). The risk of infection, which may result in stunted plants and dwarf leaves, can be reduced by always using healthy plant material and eliminating suspected plants in the surroundings. There are two main foliar diseases; one of them, mostly seen in forests, is thought to be caused by the fungus Mycosphaerella stemmatea (Fr.: Fr.) Romell (Magnusson 1976). The infection is characterized by dark spots (4–5 mm in diameter) surrounded by red-brown edges. “Leaf falling disease” is another condition that causes spots that appear in the autumn. These spots have diffused edges and cover large areas, and the

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infected leaves fall off during autumn and spring. In addition, infected plants are thought to be less winter hardy. Magnusson (1976) tried unsuccessfully to isolate the causal fungus. Nilsson (1974) tested fungicides, commonly used in orchards, to control “leaf falling disease” and good results were obtained when fungicide applications started in June (suggesting that the fungi disperse spore early). Monilinia urnula (Weinm.) Whetzel, whose symptoms are grey and hard berries, may have a negative economic impact in the future (Magnusson 1976). The same is true of Godronia cassandrae Peck, which has been found in plantations of highbush blueberry (V. corymbosum) (Magnusson 1976). It has also been reported on wild lingonberry in Sweden, Finland (Eriksson 1970), and Norway (Gjaerum 1969). E. Crop Improvement 1. Early Studies of Ecotypes. During the first attempts to domesticate lingonberry, wild germplasm was collected for comparative studies. A collection of 500 accessions from all over Sweden were planted from 1962 to 1965 and studied by Teär (1972), who observed variation in fertility, number of clusters per shoot, and number of flowers per cluster. About 10 promising ecotypes were propagated for further studies and sent to Öjebyn Experimental Station in northern Sweden. The same year (1969) the station also received a large collection of unselected accessions from southern Sweden. Unfortunately, the ecotypes were collected as sods and it was therefore uncertain whether they consisted of one or more clones (Öster 1974). The material varied considerably in growth behavior and yield. The Finnish wild material has also been surveyed at Piikkiö. A field experiment was established with 88 clones from all over the country in 1969–1970 and evaluated in 1973–1974. The original clones were divided into ten geographic areas to facilitate statistical analysis (Lehmushovi 1986). Data on shoot characteristics, flowering, fruit set, yield, and berry weight were collected. The comparison indicated a trend of increasing shoot height from south to north. The flowering period was found to be rather long, on average, 30 and 33 days. Bloom was shortest (23–26 days) in the northern clones and longest (40–45 days) in the southern clones. Coastal clones were more fertile than the inland clones. The total number of flowers was highest in the southern material and lowest in the northern. Fruit set varied between 23 and 75%. The lowest percentage was noted on plants from Åland and the highest on plants from southeastern locations. In addition the accessions from Åland proved to be least productive, while plants from a southeast

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area yielded most. Berry size increased towards the north. Lehmushovi (1986) suggested that tall shoots, large berries, and late but short bloom are important characteristics for new commercial cultivars. 2. Breeding at Balsgård. The first two Nordic cultivars, ‘Sussi’ and ‘Sanna’, were introduced at Balsgård in 1987 and 1988, respectively (Trajkovski and Sjöstedt 1986; Eckerbom 1988). Both cultivars originated from open-pollinated seed samples collected in Småland (Sweden) forest from large stands of high-yielding genotypes by the late Professor Dr. Sven Dalbro of the Royal Veterinary and Agricultural University (KVL), Denmark. Seedlings were selected for vegetative growth, fruiting habit, and plant health in Balsgård. Four populations originating from the above-mentioned material were studied in a growth chamber at Alnarp (Hjalmarsson and Ortiz 1998). The results suggested that in wild lingonberry spreading ability (i.e., number of rhizomes), growth, plant height, and number of vegetative shoots and flowering shoots are genetically controlled. In Balsgård, lingonberry breeding was intensified in 1990 (Gustavsson 1992, 1993, 1996, 1997). Breeders at that time had 50 genotypes that had been selected for high yield, large berry size, concentrated maturity, and resistance against “little leaf disease” from seedling plants originating in Småland and northern Sweden. Outstanding clones were propagated for comparative studies together with other known cultivars as well as some Latvian and Lithuanian selections. To further broaden the genetic base for breeding, seedlings from Fennoscandia, the Baltic States, Russia, and Japan were raised in 1992 and 1993. Three years later there were 84 lingonberry accessions from different natural populations in Balsgård, from which second and third generation seedlings were planted out for assessment (Gustavsson 1997). Plant height, size of leaves and berries, precocity, plant vigor, and amount of rhizomatous growth, productivity, fruit ripening time, tendency towards off-season flowering, winter hardiness, and disease resistance were evaluated. Crosses began in 1993 to determine the best breeding method. A modification of the method described for blueberry (Galletta 1975) was identified as the best. In 1994 and 1995 several crosses were made using ‘Sussi’ and ‘Sanna’ as well as two American cultivars, ‘Splendor’ and ‘Regal’ that were derived from a Finnish seed lot and released by the University of Wisconsin at Madison (Stang et al. 1994). Gustavsson (1997) found large variation in fruit set and seeds per berry between crosses and years. In some crosses very few berries were obtained, and these berries sometimes contained only non-viable seeds.

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Gustavsson postulated that this phenomenon might be caused by sterility barriers existing between certain cultivars. The first lingonberry cultivars on the market were selections from wild stands in Germany and The Netherlands (Zillmer 1984). The German cultivars ‘Erntedank,’ ‘Erntekrone,’ and ‘Erntesegen’ were developed by A. Zillmer and released in 1975, 1978, and 1981, respectively (Zillmer 1984, 1985). These cultivars and the Dutch ‘Koralle’ were the first plants whose fruit were harvested commercially in Europe (Dierking and Dierking 1993). Today there are about a dozen lingonberry cultivars worldwide selected from wild material. The crosses undertaken at Balsgård appear to be the first in which several known cultivars were combined to obtain improved germplasm (Gustavsson 1997). 3. Description of Swedish Cultivars. The descriptions are based on information included in the catalogue of the Swedish Elitplantstation (Nilsson and Rumpunen 1997) as well as other publications (Gustavsson and Trajkovski 1999). Lingonberry growers in Germany are interested in early Scandinavian cultivars and their ability to shorten the harvest time (Dierking and Dierking 1993). ‘Ida’. Released in 1997. The plant is average in size (0.1–0.2 m) and rather dense. Production is about 140 g per plant (three-year-old stands). This cultivar may be harvested twice in southern Sweden (middle of August and October). The berries are large (0.6 g per berry) and it is popular as an ornamental plant because of its beautiful leaves and abundant and repeated flowering periods. ‘Linnea’. Released in 1997. The plant is upright (0.15–0.25 m) and rather dense with a few rhizomes. Production is about 150 g per plant (threeyear-old stands) and the very good quality, medium-sized berries (0.4 g) ripen in the late season. ‘Sanna’. Released in 1988. The plant is upright and its height ranges from 0.2 to 0.3 m. Production varies between 200 and 600 g per plant or 5 to 10 metric t ha–1 (four-year-old plants). Fruit ripen in midseason, i.e., from the middle of August to the beginning of September. The bright red berries are larger than the average size of wild accessions (see Plate 3.1B), and are excellent for processing, particularly jam making. ‘Sussi’. Released in 1986. The plant habit is low growing (0.15–0.25 m) and the rhizomes spread rapidly. Production ranges from 200 to 300 g

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berries per plant (four-year-old stands). Fruit mature in mid-season (around August 20), and are dark red berries that are larger than the average wild berries. ‘Sussi’ is excellent for processing, especially for jam production. 4. Description of North American Cultivars. The University of Wisconsin–Madison named and released two new cultivars (‘Splendor’ and ‘Regal’) derived from open-pollinated seed collected in southwest Finland in the mid-1990s (Stang et al. 1994). ‘Splendor’. Released in 1994. The plants are precocious and show moderate spreading and plant height (0.15–0.19 m) at maturity. Fruit yield is about 25 g per plant. The brilliant carmine red fruits are medium (0.41 g) and ripen in mid to late September. ‘Regal’. Released in 1994. The plants are precocious and show moderate spreading and plant height (0.18–0.22 m) at maturity. Fruit yield is about 29 g per plant and the fruit are small (0.33 g), bright red, and the best harvest time is in late September.

VI. SUMMARY AND FUTURE PROSPECTS The Vaccinium genus comprises many interesting berry species. Among them are the American blueberry and cranberry, which were successfully introduced as commercial crops during the last century. There is reason to believe that the European lingonberry has a similar potential. Today large quantities are harvested from the wild and there is an important worldwide trade. Other Vaccinium species might be considered for cultivation as well. Vaccinium plants in general have certain highly appreciated qualities. The plants are easy to grow and can be grown on marginal land with a minimum of fertilizers and pesticides. The berries, consumed either as fresh or processed, possess an attractive taste and healthful properties. In addition they are suitable for machine harvest and have good keeping qualities. Through breeding, it is also possible to combine unique and valuable properties from different species. For example, one challenge for breeders would be to combine the high levels of antioxidantia in the European bilberry with the high productivity of its North American counterparts. Most certainly continued research in Vaccinium will lead to an

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increased number of species and hybrids in cultivation, an expansion of the cultivated area both in acreage and geographic distribution, and new functional food products on the market. This review has emphasized Nordic research on lingonberry and has provided information on the design and outcome of Swedish attempts for domestication of this species. Additional research has been reported in Germany (Liebster 1984; Dierking and Krüger 1984) and in the former Soviet Union (Paal 1992). More recently researchers in Alaska (Holloway 1984), Wisconsin (Stang et al. 1990, 1993b, 1994), and Canada (Estabrooks 1997) have also focused on the domestication of lingonberry as a new crop. According to Galletta and Ballington (1996), lingonberry may extend Vaccinium culture further north (in North America and Europe) and south (in South America). However, weed control and the high cost of planting material remain the two major constraints for lingonberry cultivation (Zillmer 1998). As with the highbush blueberry in the United States (Eck 1966a), the first Nordic experiments with lingonberry were aimed at determining soil and environment requirements for its cultivation, followed by an emphasis on breeding. Both organic soils and sandy soils with moderate to high content of humus were preferred by lingonberry, while soils with high clay content inhibited plant development. A well-drained acid substrate is essential for the cultivation of this species. Application of peat mulch increases vegetative growth and berry yield. In addition field experiments indicated the importance of irrigation in dry periods. Furthermore, although contradictory results were sometimes obtained, the experiments suggested that application of nitrogen fertilizer at low levels (5–10 g/m2) positively affected the vegetative lingonberry growth. Experiments with hydroponics confirmed the sensitivity of this species to high salt concentrations. The plasticity of lingonberry was noted in a number of different investigations. There is improved adaptation to open and sunny fields through sympodial growth, and an increased number of flowers as compared to cultivation in shaded areas. Increased yields were also noted in open habitats unless the plants were affected by spring frost. The source of plant material affected performance and clonally propagated wild plants, dug from the forest, cropped early and had good rhizome development compared to seedling propagules. Seedlings were adequate producers of daughter plants, but were not as precocious. Currently propagation by cuttings is the method used for commercial production of lingonberry, whereas plants dug from native stands and derived from seedlings remain important for genetic enhancement.

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The ability to spread through rhizomes has been considered a problem rather than an asset in row cultivation. However, the early establishment of a dense carpet of vegetation offers some advantages. A mixture of genotypes in the field through the use of seedlings may insure cross-pollination, which will increase fruit set and yield. It is also likely that such a plantation system would be a long-term investment because rhizome systems of Vaccinium species have a long life, e.g., up to 100 years (Sjörs 1989). According to Galletta and Ballington (1996) there has been interest in establishing lowbush blueberry fields by using seedling progenies from elite clones. This idea may also be applied to lingonberry cultivation. Pioneering work was carried out by Lehmushovi (1986), who investigated wild plant material collected from across Finland in an experimental field in Piikkiö. Bloom length was one of the characteristics that changed according to origin and genetic background. However, all the Finnish clones exhibited one distinct flowering period, while lingonberry originating in central Europe tended to flower twice. Germplasm exchange between regions may therefore significantly affect plant biorhythms (Paal 1992). Collection, characterization, and evaluation of clones as well as the development of broad base germplasm are essential to achieve success in lingonberry breeding. Lingonberry, being a new crop, offers a unique challenge to breeders and genebank curators. Germplasm collections must be enriched to meet the need for the genetic enhancement of this species. Luby et al. (1990) recommended comprehensive seed collections of native forms as well as field genebanks comprised of elite wild clones and cultivars for the conservation of Vaccinium genetic resources. Based on the results reported in this review, we recommend that further research also focus on the uptake and utilization of nutrients by lingonberry. Xeromorphic leaves, symbiosis with mycorrhiza, and enhanced adaptability to survive in distinct environments create a complex system that is not fully understood. The results of such research may affect breeding strategy and crop husbandry of lingonberry. In this process, highest priority should be given to develop an ecologically friendly cultivation system for lingonberry. The development of enhanced methods for pollination, weeding, and rejuvenation of lingonberry should be emphasized. Pests are another area that needs further study. The fungus causing “leaf falling disease” has not yet been identified and its potential interaction with decreased leaf longevity at high nutrient levels has not been investigated.

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Despite 40 years of lingonberry research and production, the industry associated with this crop is still in its infancy. One factor that has hindered its expansion is the economic return, which tends to fluctuate according to the size of the wild lingonberry harvest rather than market demand. Thus aggressive marketing of domesticated lingonberry appears to be crucial. Demand for lingonberry products will not increase unless the unique taste and health attributes of this species are promoted. The industry should consider products other than the traditional jam. In Finland the aroma of lingonberry is already appreciated in baby food, yogurt, ice cream, and liquor (Hiirsalmi and Lehmushovi 1993). Cranberry juice has become a popular drink in the United States, owing (partially) to its ability to prevent infections in the urinary tract (Avorn et al. 1994). Folk medicine suggests that the lingonberry juice may have similar qualities. In addition, recent research indicates that several Vaccinium species, among them lingonberry, contain anti-cancer compounds (Bomser et al. 1996). Lingonberry may also have an expanded future as a new berry crop in home gardens and as an ornamental plant. LITERATURE CITED Adelswärd, G. 1994. Vad menade André Mollet? Ljung eller lingon? Lustgården 74:47–60. Ahokas, H. 1971. Notes on polyploidy and hybridity in Vaccinium species. Ann. Bot. Fennica 8:254–256. Andersen, M. Ø. 1985. Chromatographic separation of anthocyanins in cowberry (lingonberry) Vaccinium vitis-idaea. L. J. Food Sci. 50:1230–1232. Andersson, C.-R. 1974. Kemisk ogräsbekämpning i lingon odling. Swedish Univ. of Agr. Sci. Konsulentavdelningen. Trädgård 71:33–36. Andersson, C.-R. 1976. Herbicide trials in lingonberries (Vaccinium vitis-idaea) during 1971–75. Unpublished report. Agr. College of Sweden, Dept. Pomology, Alnarp. Anjou, K., and E. von Sydow. 1967. The aroma of cranberries. Acta Chem. Scand. 21:945–952. Anonymous. 1983. Finland, the encyclopedia. Holger Schildts förlag, Helsingfors. Armfelt Hansell, Ö. 1969. Bärboken. P.A. Nordstedt och Söners Förlag, Stockholm. Audrina, B. 1996. The first results of cowberry breeding in Latvia. p. 48–56. In: K. Buivids (ed.), The Baltic Botanical Gardens in 1994–1995. National Botanic Gardens of Latvia, Salaspils. Avorn, J. M., J. H. Gurwitz, R. J. Glynn, I. Choodnovskiy, and L. A. Lipsitz. 1994. Reduction of bacteria and pyuria after ingestion of cranberry juice. J. Am. Med. Assoc. 271:751–754. Balwoll, G., and G. Weisæth. 1994. Horticultura. Norsk hagebok frå 1694 av Christian Gartner. Landbruksforlaget, Otta, Norway.

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Statens jordbruksverk. 1994. Livsmedelskonsumtionen 1990–1993. Jordbruksverkets rapport 6. Jönköping, Sweden. Statens livsmedelsverk. 1978. Livsmedelstabeller. Energi och vissa näringsämnen. Uppsala, Sweden. Statistics Sweden. 1994. Foreign trade 1993: Imports and exports. Commodities according to harmonized system. Official Statistics Sweden. SCB Tryck, Örebro. Stodola, J., and J. Volak. 1986. Tidens stora bok om Läkeväxter. (B. Wahlin, trans.) Tidens förlag, Stockholm. (Original work published 1984). Sylvén, N. 1918. Jämförande försök med maskin och handplockning av skogsbär. Kungl. Lantbruksakademiens handlingar och tidskrift 57:62–76. Teär, J. 1972. Vegetativ och fruktifikativ utveckling hos vildväxande och odlade lingon. Ph.D. diss. Alfa-Lavals Offsettryckeri, Tumba, Sweden. Tiak, G., and A. F. Cherkasov. 1998. Experiences in growing Vaccinium vitis-idaea L. in European Russian central parts. In: T. Paal (ed.), Wild berry culture: an exchange of western and eastern experiences. Estonian Agr. Univ., Forest Research Inst., Tartu. Forest Studies 30:198–200. Tomenius, K., and G. Åhman. 1983. Mycoplasma-like organisms in the phloem of little leaf diseased plants of Vaccinium vitis-idaea and V. myrtillus in Sweden. Swedish J. Agric. Res. 13:205–209. Tolvanen, A. 1995. Aboveground growth habits of two Vaccinium species in relation to habitat. Can. J. Bot. 73:465–473. Tolvanen, A., K. Laine, T. Pakonen, and P. Havas. 1995. Recovery of evergreen clonal dwarf shrub Vaccinium vitis-idea after simulated microtine herbivory in a boreal forest. Vegetatio 116:1–5. Trajkovski, V., and B. Sjöstedt. 1986. Breeding of lingonberries and lowbush blueberries. Swedish Univ. Agr. Sci. Div. Fruit Breed.—Balsgård. Rep. 1984–1985:49–51. Valset, K. 1976. Ville vekster—nyttige frukter. Det norske hageselskap og Grøndahl and Søns Forlag, Oslo. Vestrheim, S., K. Haffner, and L. Øyre. 1994. Dyrking av tyttebær. Norsk Landbruk 113:9. Vieno, M., M. Komulainen, and S. Neuvonen, S. 1993. Seed bank composition in a subarctic pine-birch forest in Finnish Lapland: Natural variation and the effect of simulated acid rain. Can. J. Bot. 71:379–384. von Zabeltitz, C. 1989. Entwicklung einer Erntemaschine fur Kulturpreiselbeeren. Erwerbsobstbau 31:165–168. Waern, C. F. 1834. Underrättelser för år 1833, om Baldersnäs Trädgård, i Norra Dalsland. Svenska Trädgårdsföreningens Årsskrift 1:66–74. Wikmark, E. 1907. Om en svensk framtid och om ett fosterländskt företag: Några ord. Göteborg, Sweden. Zeldin, E. L., and B. H. McCown. 1997. Intersectional hybrids of lingonberry (Vaccinium vitis-idaea, section vitis-idaea) and cranberry (V. macrocarpon, section oxycoccus) to Vaccinium reticulatum (section macropelma). Acta Hort. 446:235–238. Zillmer, A. 1984. Beschreibung von fünf Preiselbeersorten. Erwerbsobstbau 26:282–283. Zillmer, A. 1985. Account of my three types of Vaccinium vitis-idaea—‘Erntedank’, ‘Erntekrone’, ‘Erntesegen’. Acta Hort. 165:295–297. Zillmer, A. 1998. Some thoughts about the cultivation of lingonberries Vaccinium vitisidaea L. In: T. Paal (ed.), Wild berry culture: an exchange of western and eastern experiences. Estonian Agr. Univ., Forest Research Inst., Tartu. Forest Studies 30:198–200. Åkerstrand, K., L. Kardell, and T. Möller. 1988. Undersökning av blåbär och lingon från kvävegödslade provytor. Vår Föda 40:259–270.

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Ängeby, O. 1978. Apis mellifera as pollinator of Vaccinium myrtillus and Vaccinium vitis idaea. Proc. 4th Int. Symp. on Pollination. Maryland Agr. Expt. Sta. Spec. Misc. Publ. 1:165–170. Önner, B. 1977. Studier av mycorrhizaförekomsten hos vildväxande och odlade lingon. M.Sc. thesis. Swedish Univ. Agr. Sci. Dept. Pomology, Alnarp. Öster, H.-E. 1974. Försök med odling av lingon. Swedish Univ. Agric. Sci. Konsulentavdelningen. Trädgård 64.

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4 Caper Bush: Botany and Horticulture Gabriel O. Sozzi* Departamento de Biología Aplicada y Alimentos Facultad de Agronomía, Universidad de Buenos Aires Avda. San Martín 4453. C 1417 DSE—Buenos Aires, Argentina.

I. INTRODUCTION A. History B. World Production II. BOTANY A. Taxonomy and Distribution B. Morphology and Anatomy C. Floral Biology and Seed Dispersal III. ECOPHYSIOLOGY A. Environmental Requirements B. Growth and Flowering C. Adaptations to Water Stress and Poor Soils IV. HORTICULTURE A. Biotypes B. Propagation 1. Seed 2. Vegetative C. Cultural Practices 1. Plant Establishment 2. Intercropping 3. Pruning 4. Plant Nutrition 5. Irrigation 6. Weed Control 7. Pests and Diseases 8. Harvest and Yield

*I am grateful to Prof. John M. Labavitch for a critical review of the manuscript, to Prof. Jules Janick for his helpful comments, and to María M. Quiroga for her support during the preparation of this review. Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 125

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V. POSTHARVEST TECHNOLOGY A. Capers 1. Handling and Curing 2. Industrial Treatment and Packaging B. Caperberries VI. COMPOSITION AND UTILIZATION A. Composition B. Utilization 1. Food Use 2. Ornamental Use 3. Medicinal and Cosmetic Value VII. INTERNATIONAL TRADE VIII. CONCLUDING REMARKS LITERATURE CITED

I. INTRODUCTION The caper bush (Capparis spinosa L., Capparidaceae) has been introduced as a specialized culture in some European countries during the last three decades. The economic importance of caper plant (young flower buds, known as capers, are greatly favored for seasoning and different parts of the plant are used in the manufacture of medicines and cosmetics) led to a significant increase in both the area under cultivation and production levels during the late 1980s. The main production areas are in harsh environments found in Morocco, the southeastern Iberian peninsula, Turkey, and the Italian islands of Pantelleria and Salina. This species has developed special mechanisms in order to survive in Mediterranean conditions, and introduction in semiarid lands may help to prevent the disruption of the equilibrium of those fragile ecosystems. Little information on this species is available despite the increasing worldwide demand for capers and the socioeconomic influence of the caper crop. In the context of the potential use of this species as an alternative for marginal lands, caper bush deserves further research and diffusion. A. History Capers (flower buds) and caperberries (caper fruits) have a long history of use by humans. A fragment of thick fruit skin of the caper type was obtained by archaeological excavations from an Old World Paleolithic site (Wadi Kubbaniya, west of Nile Valley, Upper Egypt) and provides direct evidence of the consumption of Capparis spp. from 18,000 to 17,000 years ago (Hillman 1989). Prehistoric remains of wild caperber-

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ries were also found in southwest Iran and in Iraq (Tigris) and dated to 6000 BCE (Renfrew 1973). Caper seeds were found in quantity at Tell esSawwan (Iraq) and dated to 5800 BCE (H. Helback, cited by Renfrew 1973). The earliest known mention of capers is in a Sumerian legend of 2000 or 3000 BCE (Stromme 1988; Trager 1995). The Greek term Capparis (καππαρις) has no convincing etymology (Carnoy 1959). Nevertheless, in ancient Greece, capers were used as a condiment and for other purposes. Hippocrates first wrote about the medicinal properties of different caper plant tissues, including use as a treatment for pneumonia (Diseases III, 7, 142, 7), pleurisy (Diseases III, 7, 150, 11), and fistulas (Fist. VI, 460, 3, 6). Theophrastus suggested that wild caper plants could have greater pungency and that caper bush could not grow well on cultivated lands (De Causis Plantarum III, 1, 4). Capers were also used in cosmetics. Phryne (4th century BCE), who was said to have modeled for both her lover the sculptor Praxiteles and the painter Apelles, used them regularly (Cerio 1983). A caper cream was utilized to help one stay young and to keep the skin free of wrinkles and with a healthy color (Castro Ramos and Nosti Vega 1987). Capers were known as an appetizer by ancient Hebrews (Duke 1983). They are mentioned in a nostalgic poem of the Hebrew Bible (Ecclesiastes 12, 5) probably written in the 3rd century BCE. Moreover, the Babylonian Talmud cites caper bush several times (Shabbath 30b and 150b; Bezah 25b; Baba Bathra 28b), as well as caper flower (Abodah Zarah 38b) and capers (Demai I, 1, 6; Abodah Zarah 40b). The Midrash Rabbah describes how a caper bush grew up and fenced a breach in the vineyard of a pious man (Leviticus -Behar- 34, 16). Dioscorides indicated the therapeutic properties of capers in his De Medica Materia (II, 192). In Latin literature, Plinius Secundus included several passages about the caper bush in his Historia Naturalis. He recommended that capers coming from foreign countries not be used, as they could be a health risk (XIII, 44, 1), but considered that the direct consumption of Italian capers or extracts from different parts of the plant could be beneficial for the organism (XX, 59, 1). He claimed that caper bushes should be sown in dry localities and sandy soils, “. . . the plot being hollowed out and surrounded with an embankment of stones erected around it: if this precaution is not taken, it will spread all over the adjoining land” (XIX, 48, 2). Apicius classified capers as a condiment in De Re Coquinaria (IV, 1) and the poet Marcus Martialis mentioned them in one of his famous Epigrams (III, 77). Claudius Galenus cited the caper bush among medicinal plants and Columella gave a description of this perennial bush in his agricultural treatise De Re Rustica (XI, 3).

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Athenaeus mentioned capers several times in his cookery book Deipnosophistae and cited comedians who included them in their plays: Aristophon (II, 63a), Nicostratus (IV, 133c), Alexis (IV, 170a), Timocles (XIII, 567e), and Antiphanes (II, 68a; IV, 161e). According to Plutarch’s Moralia (Quaestiones Convivales), pickled capers were used either alone to restore the appetite (VI, 2, 687d), or as an ingredient of a delicious meal (IV, 4, 668a–b). Remains of capers have been recovered from Mons Claudianus, a Roman quarry settlement (1st–2nd centuries; van der Veen 1999) and from the Roman port of Berenik (1st–4th centuries; Cappers 1999). During the Middle Ages, the School of Salerno documented caper bush as a medicinal plant in the Regimen Sanitatis (II -Materia Medica-, II, 24; VIII -Therapeutica-, VI). Platina (1475) included capers in the first dated cookery book, De Honesta Voluptate et Valetudine (IV, 15) and added new medical advice. Mattioli described caper bush and its qualities (De Plantis Epitome Utilissima) and pointed out some therapeutic properties of capers (Commentari al Dioscoride II, 169). During the Renaissance, Spanish writers and poets such as Lope de Vega, Juan de Zabaleta, and Alfonso Ortiz de Ovalle cited them. In France, Olivier de Serres reported different preparation methods in his Théâtre d’ Agriculture (XI, 6), and the famous surgeon Ambroise Paré wrote: “Capers are good, in that they sharpen the appetite and relieve bile” (ToussaintSamat 1992). Capers were used not only by common people but also at the tables of the higher classes; e.g., in the famous dinner to honor the Holy Roman Emperor Ferdinand III’s ambassador in Rome in 1638 (Trager 1995). Some documents reveal caper consumption in England during the 17th century (Allen 1994), which is corroborated by the cookery books of that time (Hackwood 1911). Pierre Belon du Mans (16th century) described caper plants growing in the regions of Alexandria, Suez, and Sinai (Darby et al. 1977). He found those bushes different from other European types, as they were thornless and kept their leaves in winter. Coles (1657) distinguished five kinds of caper plants without clear morphological differences and indicated places and seasons in which they grew. At the beginning of the 19th century, the Spanish poet Gaspar de Jovellanos mentioned the caper plant in his description of the castle of Bellver, Palma of Majorca (Font Quer 1962), where it grows on the ramparts. Alfred Kaiser found C. spinosa var. aegyptia in Sinai; it was used as fodder, and its buds and seeds were utilized as condiments (Darby et al. 1977). Alexandre Dumas (1873) pointed out the Asiatic origin of caper bush and marked caper qualities as an aperitif in his Le Grand Dictionnaire de Cuisine.

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The caper bush was grown in Provence during the 18th and 19th centuries. At the end of the 19th century French capers were replaced by the Spanish and Italian product. B. World Production Caper production and trade have become highly competitive. Spain, Morocco, Turkey, and Italy are the four major producers. Beginning in 1977/78, Spain had a rapid increase in caper growing areas and its production rose to first place in just a few years. Spain had the most important caper-producing area from 1978 through 1991, with an average production of 3,550 t/year and a maximum of 4,685 t in 1985 (Fig. 4.1). The ready availability of seeds and seedlings, the favorable soil and climate, the possibility of using low yielding soils, its ready sale and attractive profits, low initial investment, low production costs, and the crisis of traditional cultures such as that of the olive tree in Andalusia

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contributed to the success of caper bush growing in Spain. Different government research services, such as the Centro Regional de Levante, developed and transferred new technologies and helped to remove technical barriers (e.g., propagation) for caper bush culture (Luna Lorente and Massa Moreno 1979; Luna Lorente and Pérez Vicente 1979). During the 1980s, caper culture extended to different provinces: Almería, Murcia, Balearic Islands, Jaén, Granada, and Córdoba and, to a lesser extent, Ciudad Real, Cadiz, Málaga, Alicante, Barcelona, and Valencia. Most of the caper plantings (90%) had less than 5 ha, but fields with more than 5 ha represented 49% of the total area (Millan Campos 1987). The unchecked expansion of production, the competition of Morocco and Turkey, and the lack of promotion in Spain (Ruíz Avilés 1987; Villena 1988) brought about an oversupply and a 40% drop in the price between 1985/86 and 1994. This caused both the removal of many caper plantings and the decline in the average yield (Fig. 4.1), because the remaining plantings are located in marginal lands. Morocco is a major caper-exporting country. In Morocco, production is mainly based on prickly wild plants of different closely related species. Local consumption is negligible, as capers are not usually utilized in the traditional Moroccan cuisine. Most of the produce is exported to European countries (Anon. 1982). During 1986, exports exceeded 3,000 t. One third of the produce was sold to Italy but substantial amounts were also exported to France, the United States, Germany, and Switzerland (Barbera 1991). Three-fourths of the Moroccan production comes from the region of Fez, Taounate, and Boulemane; other areas of minor production are located in the surroundings of Safi and southwest of Marrakech. Traditionally, no cultural practices were carried out. Harvest began around mid June and lasted two months. The Moroccan product quality was not considered outstanding. It generally consisted of a mix of capers from different species that were not adequately processed. The creation of regional associations and the promotion of caper plantings during the past 15 years has resulted in the stabilization of profits and the improvement of caper production and quality. Turkey has come to be the leading caper-exporting country, although local consumption is trivial. Turkey began to export capers in 1982. The average annual production is estimated to be around 3,500–4,000 t per year. Turkey exported 1,095 t of capers in 1989, 5,072 t in 1994, and 3,500 t in 1998 (Turkey Embassy in Argentina, pers. commun.). In 1999, 3,226 t were exported in large packs and 1,025 t in small packages for retail sale (Aegean Exporters’ Unions, Izmir, Turkey, pers. commun.) but

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these figures may be overestimated, as they are based on gross weights that include the packaging materials. Most of Turkish production of capers comes from the provinces on the Aegean and Mediterranean coast (Çanakkale, Balikesir, Izmir, Aydin, Mugla, Antalya, Içel, Adana, Hatay). The remainder of production is obtained from warm places in the provinces of Us¸ak, Denizli, Konya, Ankara, Zonguldak, Tokat, Malatya, and from others in the northeast and southeast of the country (Rize, Artvin, Erzurum, Kars, Igdir, Van, Mardin, and Sanliurfa). Caper plants grow naturally in these areas. However, agricultural production in private arable lands has started in recent years. Italy is not only a traditional caper producer but also the country where the caper bush has been grown intensively for a long period. Nevertheless, the caper culture still shows an important territorial concentration in two Sicilian insular areas that produce 95% of the Italian capers: Pantelleria Island (36° 47′ N, 12° 00′ E), and the Aeolian Archipelago, especially in Salina Island (38° 34′ N, 14° 51′ E) and, to a lesser extent, in the islands of Lipari and Filicudi. The remaining plantings are located in Apulia, Sicily, and in other Italian islands (Ventotene, Ustica, Egadas) (Barbera 1991). Between 1960 and 1985, there was an increase in caper production and areas under cultivation in Pantelleria and Salina due to: (1) the decline of traditional cultures such as grape vine and olive tree; (2) the increasing internal demand for capers, which provided a better profit margin; (3) improvement in cultural techniques and early preservation procedures; (4) the creation of mutual associations; and (5) improvement in the socioeconomic levels (Caccetta 1985; Barbera and Butera 1992). Between 1973 and 1983, the area under cultivation increased by 67% and production levels by 90%. Caper production in 1983 was 1,900 t, 1,360 in Pantelleria and 440 in Salina; and the cultivated area was 1,000 ha, 770 in Pantelleria and 180 in Salina (Caccetta 1985), but these figures may have been overestimated (Barbera and Di Lorenzo 1984; Barbera 1991). After 1983, Italian caper production reached the historically lowest levels: 600 t in Pantelleria and 250 t in Salina (Barbera 1991), due to the open competition with Spain and Morocco. In the early 1990s, Italian production was supported by a better organized and dynamic marketing, and the improvement of Italian quality standards. Tunisia produces 300 t/year obtained from wild plants with or without stipular spines. Most of these wild specimens grow in the mountainous region situated at the north and northeast of Tunisia. Most of the spineless genotypes are situated around Beja (Barbera 1991). The main problem is the high cost of harvesting capers, which could be

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significantly reduced by increasing the density of spontaneous plants and initiating specialized plantings. Studies performed in the kibbutz Maagan Michael (south of Haifa) in 1973 discarded caper bush as a profitable intensive culture due to the spiny populations available in Israel and the high cost of harvesting (Putievsky 1977). In France, it is still grown in the departments of the Maritime Alps, Var, and Bouches-du-Rhône. In other countries around the Mediterranean basin such as Greece and Cyprus occasional harvests from wild plants are carried out. In the United States, Stromme (1988) considered caper bush to be invaluable for hillside planting in California but growers could only recently propagate it. The first caper farm was established in Gilroy, California, but it supplied only local restaurants. The United States imported more than $5 million worth of processed capers annually during the late 1980s and almost $10 million during the late 1990s (USDA Foreign Agricultural Service 1999). In Argentina, genus Capparis is represented by several species (Gómez 1953); some of them are an important supplement to the cattle diet during the dry season (Van den Bosch et al. 1997). Caper bush plantings were first established in the province of San Juan (Dimitri 1959). Nowadays, there are small (0.5–5 ha) plantings in the provinces of San Juan (Rawson) and Mendoza (San Martín, Maipú, Junín); some of them are nurseries of young plants. In 1990, the Instituto Nacional de Tecnología Agropecuaria (INTA) initiated a research program to determine the feasibility of caper production in the provinces of Catamarca (Andalgalá) and La Rioja (Arauco) (Paunero et al. 1996).

II. BOTANY A. Taxonomy and Distribution The caper bush is known as alcaparro, alcaparra, tapanera, tapenera, tapena, tápano, alcaparrera, or caparro in Spanish; câprier in French; cappero in Italian; Kappernstrauch in German; alcaparreira in Portuguese; ahveeyonah in Hebrew; al-kabbar in Arabic; kapari or kebere in Turkish; and dàmá in Chinese. Alkire (1998) has compiled the name of caper bush, capers, and caperberries in 16 different languages. The caper plant is a member of the Capparidaceae, which comprises 40 to 50 tropical, subtropical, and temperate genera with 700–930 species of trees, shrubs, and herbs (Zohary 1966; Heywood 1985; Zomlefer 1994), from

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which only Capparis, Cleome, Crateva, and Polanisia are cultivated (Bond 1990). Most of the members of this family are distributed in the low-altitude regions of the tropics and subtropics in both hemispheres, as well as in temperate climates of the Mediterranean basin (Heywood 1985). In Europe, only Cleome and Capparis have been found (Heywood 1964). Lately, Capparis and relatives have been proposed to form a basal paraphyletic complex within Brassicaceae (Zomlefer 1994; Judd et al. 1999) on the basis of molecular (Rodman et al. 1993) and morphological (Judd et al. 1994) cladistic analyses. In fact, taxonomists have long agreed that the caper family is very closely related to Cruciferae, based on some major shared characters, particularly the original bicarpellate ovary with parietal placentae, the vacuolar and utricular cysternae of the endoplasmic reticulum, the presence of myrosin cells and glucosinolate production (Rodman 1991a,b; Zomlefer 1994). Capparis is the largest genus of this family but has been taxonomically neglected (Jacobs 1965). The number of species in this genus varies according to different authors: 150 (Luna Lorente and Pérez Vicente 1985), 250 (Jacobs 1965), 300 (Bond 1990), or over 350 (Stocker 1974; Barbera et al. 1991; Judd et al. 1999). Only a few are economic plants: 9 (Uphof 1968) to 15 (Bond 1990). Species identification in this highly variable genus is very difficult and there are different opinions concerning the rank assigned to the different taxa and to their subordination (Zohary 1960; Jacobs 1965; Higton and Akeroyd 1991). The limits of the species are not always clear, because intraspecific crosses are relatively common in the overlapping areas. Capparis spinosa may be considered a species complex (Rao and Das 1978) and continuous flux of genes (Jiménez 1987) throughout its evolution has made relationship determination hard. The existence of morphological variations with many intergrading forms has led to the recognition of many varieties, resulting in an unsatisfactory working classification (Rao and Das 1978). According to Jacobs (1965), Capparis spinosa L. represents one polymorphic and variable species that embraces C. antanossarum Baill., C. cartilaginea Decne., C. cordiflora Lam., C. elliptica Hausskn. & Bornm., C. galeata Fres., C. hereroensis Schinz, C. himalayensis Jafri, C. leucophylla DC., C. mariana Jacq., C. mucronifolia Boiss., C. murrayana Grah., C. napaulensis DC., C. nummularia DC., C. obovata Royle, C. ovata Desf., C. sandwichiana DC., and C. uncinata Edgew. Thus, this polymorphic species is widespread in Europe (Mediterranean region), Africa (northern, northeastern and southwestern part, as well as the eastern coast and Madagascar), Asia (Turkey, the Caucasus, the Near East, Arabia,

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Iran, Afghanistan, West Pakistan to Kashmir and Nepal, India, East Malaysia, and the Pacific) and Australia. On the other hand, some of the taxa included in C. spinosa by Jacobs (1965) have been considered as independent species by other authors (Zohary 1960; Bokhari and Hedge 1975; Rao and Das 1978). Higton and Akeroyd (1991) recognized three entities in Europe, mainly based on vegetative characters: (1) C. spinosa subsp. rupestris (Sibth. & Sm.) Nyman, distributed in the coastal rocks and cliffs of the Mediterranean basin; (2) C. spinosa subsp. spinosa var. spinosa, predominantly found in Spain, France, and Italy and thought to be related to the cultivated caper bush; (3) C. spinosa subsp. spinosa var. canescens Cosson, widespread in Southern Europe and Anatolia and proposed to be the native variant of the cultivated plant. Nevertheless, Fici and Gianguzzi (1997) suggested that C. spinosa subsp. spinosa var. spinosa and var. canescens could represent two edaphic variants of the same entity. Both C. spinosa subsp. spinosa var. canescens and C. spinosa subsp. rupestris are present in Pantelleria Island. The first one is widespread on regosols, lithosols, and sedimentary rocks such as clay and marl; the second one is present along the coastal limestone and volcanic cliffs (Fici and Gianguzzi 1997). Caper bush is present in almost all the circum-Mediterranean countries (Greuter et al. 1984) and is included in the floristic composition of most of them (e.g., Willkomm and Lange 1880; Halácsy 1901; Post 1932; Rechinger 1943a,b; Colom Casanovas 1957; Garnier et al. 1961; Heywood 1964; Davis 1965; Maire 1965; Zohary 1966; Blatter 1978; Danin 1983; Barclay 1986), but whether it is indigenous to this region is uncertain (Zohary 1960; Pugnaire 1989). Although the flora of the Mediterranean region has considerable endemism, the caper bush could have a tropical origin and only been naturalized in the Mediterranean basin (Pugnaire 1989). B. Morphology and Anatomy The caper bush is a shrub 30–50 cm tall but old specimens can achieve 80 cm in height and occupy an area of 15 m2. It is a perennial deciduous plant that becomes woody at maturity. Its roots are deep. Plants have been reported with 6–10 m long roots (Reche Mármol 1967; González Soler 1973; Luna Lorente and Pérez Vicente 1985; Bounous and Barone 1989). The root system may account for 65% of the total biomass (Singh et al. 1992). Caper canopy is made up of 4–6 radial decumbent branches from which many secondary stems grow. In wild bushes, Singh et al.

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Fig. 4.2.

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Stem of caper bush showing flowers and fruit.

(1992) observed up to 47 branches per plant. Branches (Fig. 4.2) are 2 to 3 m long. Stipular pale yellowish spines are often recurved and divaricate but sometimes weakly developed and setaceous. Leaves are alternate, 2–5 cm long, simple, glabrous to densely pubescent, chartaceous or coriaceous, obovate to elliptic or ovate to orbicular, with a rounded base and a mucronate, obtuse, or emarginate apex. Flowers are 5–7 cm across, axillary and solitary, with a sweet scent, scattered along the twigs on a sturdy pedicel. Their calyx, with 4 purplish sepals, is from almost symmetrical to strongly zygomorphic. The 4 petals, slightly exceeding the sepals, are white and imbricate. Stamens are numerous

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(50–190), slightly exceeding the petals, glabrous, with purplish filaments and small subbasifixed introrse anthers. The gynophore is approximately as long as the stamens. The ovary is superior, 1-locular, with 5–10 placentas. The fruit (caperberry) is ellipsoid, ovoid, or obovoid, with a thin pericarp. The fruit bursts when ripe, exposing seeds in a pale crimson flesh. Seeds are 3–4 mm across, reniform, with a circinnate embryo. Germination is epigaeal. A thousand seeds weigh 6–8 g (Gorini 1981; Akgül and Özcan 1999; Li Vigni and Melati 1999). Few studies have been performed on this plant’s internal structure. Ponzi et al. (1978) examined caper bush ovular tissues, while Jørgensen (1981a,b) investigated the presence of myrosin cells; some characteristics of caper endoplasmic reticulum were also found in the sister group Cruciferae. Bokhari and Hedge (1975) studied different anatomical characters and Rao and Das (1978) researched the idioblast typology, in an attempt to shed some light on the taxonomic status of this complex species. Seidemann (1970) described the anatomical characteristics of sepals and petals, while Leins and Metzenauer (1979) examined the ontogenetic sequence of flower buds. Investigations on ultrastructure of caper anther and pollen have been carried out (Gori and Lorito 1988a,b). Recently, Ronse Decraene and Smets (1997a,b) discussed taxonomic affinities and possible trends in floral evolution on the basis of the androecium configuration and the increase in carpel numbers from an original bicarpellate condition. Doaigey et al. (1989) and Psaras et al. (1996) analyzed various anatomical and histological features of caper leaf and stem, which will be reexamined in the next section in relation to the ecophysiology of this species. C. Floral Biology and Seed Dispersal The caper bush is known to be noctiflorous (Jacobs 1965). It blossoms for approximately 16 h, from ca. 18:00 h to ca. 10:00 h the next morning (Ivri 1985; Petanidou et al. 1996). Flowers of Capparis attract different insects, most of which have low pollination efficiency (Eisikowitch et al. 1986). In Israel, the main pollinators are hawkmoths and large bees (Kislev et al. 1972; Eisikowitch et al. 1986; Dafni et al. 1987; Dafni and Shmida 1996). In Greece, flowers are to a major extent pollinated by bees (Petanidou 1991). Flower rewards in genus Capparis vary according to location and year (Petanidou et al. 1996) and differ significantly among taxa. C. spinosa var. aegyptia has a higher pollen grain weight and its nectar is richer in total amino acids (Eisikowitch et al. 1986). On the other hand, higher

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nectar concentration and volume are found in C. ovata (Eisikowitch et al. 1986; Dafni et al. 1987). Most nectar secretion in C. spinosa is nocturnal. Amino acid content and concentration, as well as hexose concentration, increase with flower age, while sucrose concentration decreases (Petanidou et al. 1996). Although alternative nectariferous species are available in the Mediterranean environment, the genus Capparis is almost the sole nectar source for pollinators under desert conditions (Eisikowitch et al. 1986). The juicy fruits of caper bush are consumed by animals. Seeds surrounded by sweet pulp are eaten by birds (Seidemann 1970; Danin 1983) like Sylvia conspicillata, Oenanthe leucura (Hódar 1994), and Chlamydotis [undulata] macqueenii (van Heezik and Seddon 1999), which transport the undigested seeds. Caper plant is also considered a myrmecochorous and saurochorus species; harvester ants (Luna Lorente and Pérez Vicente 1985; Li Vigni and Melati 1999) and lizards like Lacerta lepida (Hódar et al. 1996) feed on the fruit and carry off fragments together with the hard-coated seeds, which are left to germinate afterwards. Wasps are attracted by mature caperberry scent and also act as dispersal agents (Li Vigni and Melati 1999).

III. ECOPHYSIOLOGY A. Environmental Requirements The caper bush requires a semiarid climate. Mean annual temperatures in areas under cultivation are over 14°C and rainfall varies from 200 mm/year in Spain to 460 in Pantelleria and 680 in Salina. In Pantelleria, it rains only 35 mm from May through August, and 84 mm in Salina. A rainy spring and a hot dry summer are considered advantageous (Barbera 1991). A harvest duration of at least 3 months is necessary for profitability. Intense daylight and a long growing period are necessary to secure high yields. The caper bush can withstand temperatures over 40°C in summer but it is sensitive to frost during its vegetative period. The potential exposure of caper hydraulic architecture to cavitations has recently been proposed as an explanation for its susceptibility to freezing conditions (Psaras and Sofroniou 1999). On the other side, caper bush seems to be able to survive low temperatures in the form of stump, as happens in the foothills of the Alps. Caper plants have been found even 1,000 m above sea level though they are usually grown at lower altitudes (Barbera et al.

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1991). Some Italian and Argentine plantings can withstand strong winds without problems, due to caper bush decumbent architecture and the coriaceous consistency of the leaves in some populations. The caper bush is a rupiculous species. It is widespread on rocky areas (Heywood 1964) and is grown on different soil associations, including alfisols (Fernández Pozo et al. 1993), regosols, and lithosols (Barbera 1991; Fici and Gianguzzi 1997). In different Himalayan locations, C. spinosa tolerates both silty clay and sandy, rocky or gravelly surface soils, with less than 1% organic matter (Ahmed 1986). It grows on bare rocks, crevices, cracks, and sand dunes in Pakistan (Ahmed and Qadir 1976), in dry calcareous escarpments of the Adriatic region (Lovric 1993), in dry coastal ecosystems of Egypt, Libya, and Tunisia (Ayyad and Ghabbour 1993), in transitional zones between the littoral salt marsh and the coastal deserts of the Asian Red Sea coast (Zahran 1993), in the rocky arid bottoms of the Jordan valley (Turrill 1953), in calcareous sandstone cliffs at Ramat Aviv, Israel (Randall 1993), and in central west and northwest coastal dunes of Australia (Specht 1993). It grows spontaneously in wall joints of antique Roman fortresses, on the Wailing Wall, and on the ramparts of the castle of Santa Bárbara (Alicante, Spain). Moreover, this bush happens to grow in the foothills of the southern Alps (Verona, Italy) and is a common species on city walls (Baccaro 1978) in Tuscany (Italy) and on bastions of Mdina and Valletta (Malta) (Brandes 1992a,b). Hruska (1982) included caper as part of the floristic diversity on the walls of Castiglione del Lago (Italy). Clinging caper plants are dominant on the medieval limestone-made ramparts of Alcudia and the bastions of Palma (Majorca, Spain) (Brandes 1992b). This aggressive pioneering has brought about serious problems for the protection of monuments (Fairushina 1974; Brandes 1992b). Deep and well-drained soils with sandy to sandy-loam textures are favorable for caper bush (Barbera and Di Lorenzo 1982, 1984; Ahmed 1986; Özdemir and Öztürk 1996), though this shrub adapts perfectly to calcareous accumulations or moderate percentages of clay (González Soler 1973; Fernández Pozo et al. 1993). It also shows a good response to volcanic (Barbera and Di Lorenzo 1982) or even gypseous soils (Font Quer 1962) but is sensitive to poorly drained soils. Soil pH between 7.5 and 8 are optimum (Gorini 1981), though pH values from 6.1 to 8.5 can be tolerated (Duke and Terrel 1974; Duke and Hurst 1975; Ahmed 1986). Caper bush is usually not considered to be a halophyte but Abbas and El-Oqlah (1992) found this plant in the loamy solonchacks of Bahrain coastal lowlands, where the conductivity may reach 54 mS/cm. On the other hand, aerosols from sea-water-fed cooling towers proved to pro-

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duce leaf chlorosis or necrosis, probably due to chloride toxicity (Polizzi et al. 1995). C. spinosa withstands chronic levels of some toxic gaseous pollutants. Krishnamurthy et al. (1994) reported an unusual 93% retention of leaves when caper bush was exposed to a mixture of sulfur dioxide, oxides of nitrogen, ammonia, and suspended particulate matter, although the photosynthetic area per leaf was reduced by 61% and the fresh weight by 67%. The simultaneous increase in the free amino acid pool was attributed to the production of sulfur-containing amino acids and the hydrolysis of proteins due to pollution. B. Growth and Flowering In winter, most of the biotypes remain in the form of stumps; when spring comes, new stems appear, elongate and bear flower buds. The total growing cycle lasts 5–6 months depending on temperature and early frosts. There is a positive correlation between temperature and productivity (Luna Lorente and Pérez Vicente 1985). Fertility of the nodes is maximum (close to 100%) during the hottest periods and lower at the beginning and end of the season (Barbera et al. 1991). Flower bud appearance is continuous so that all transitional stages of development, from buds to fruit, can be observed simultaneously on the plant throughout most of its ontogeny whenever buds are not harvested. The first ten nodes from the base are usually sterile and the following ten only partially fertile; the subsequent nodes have a caper each, almost to the tip of the stem (Barbera et al. 1991). C. Adaptation to Water Stress and Poor Soils Genus Capparis has a C3 photosynthesis (Alkire 1998). As caper bush grows in semiarid environments, it routinely encounters high radiation levels, high daily temperature, and insufficient soil water during its growing period. The caper bush has developed a series of mechanisms that reduce the impact of said conditions and ensure its survival. It is not only capable of resisting periods of drought but also of making active use of these periods, e.g., by bloom at that time. Some caper anatomical features are typical of xerophytes: small cell size (including guard cells), thick outer epidermal cell walls both in leaves and stems, strongly developed palisade in the mesophyll, and dense vein network (Stocker 1974; Doaigey et al. 1989; Fici et al. 1995).

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C. spinosa subsp. spinosa var. canescens seems to be better adapted to semiarid conditions than C. spinosa var. rupestris due to some characteristics of the cuticle, epidermal cells, and stomata (Fici et al. 1995). Caper photosynthesis is favored by its amphistomatic leaves (Psaras et al. 1996). Mesophyll cells are densely packed and chloroplasts are arranged adjacent to the intercellular air spaces (Psaras et al. 1996). This feature may be of ecological importance in intense daylight regions where CO2 could be the photosynthetic limiting factor. Rhizopoulou (1990) analyzed different caper bush physiological responses to drought. Plasticity of young expanding tissues increases under water deficit with a simultaneous lowering of water potential and the production of a large canopy within a short period. Under water deficit conditions, new leaves reach full size more rapidly. Response to drought also includes regulated wall properties and stomatal opening, as well as increased root density. The radical growth is not inhibited under water deficit but roots change their distribution in the different soil layers and make metabolic adjustments. As its root system also spreads horizontally in the soil, it makes use of rainfall however light it may be. The caper bush is a stenohydric plant (Rhizopoulou et al. 1997) with high photosynthetic rates. This characteristic is supported by a high hydraulic conductivity due to the presence of wide vessels with non-vestured intervessel pits, both in roots and stems (Psaras and Sofroniou 1999). The caper bush also shows characteristics of a plant adapted to poor soils. This shrub has a high root/shoot ratio and the presence of mycorrhizae serves to maximize the uptake of minerals in poor soils (Pugnaire and Esteban 1991). Four different N2-fixing bacterial strains have been isolated from the caper bush rhizosphere (Andrade et al. 1997) and may, at least partially, explain its capacity to acquire and maintain high reserves of that growth-limiting nutrient (Pugnaire and Esteban 1991). Fertilization of cultivated bushes probably leads to a luxury consumption of some nutrients, a typical response of wild plants from infertile environments (Chapin 1980).

IV. HORTICULTURE A. Biotypes The chromosome number (sporophytic count) of Capparis spinosa is 38 (Taylor 1925; Kuhn 1928; Darlington and Wylie 1955; Murín and Chaudhri 1970; Fici et al. 1995). Few, if any, breeding programs have

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been undertaken. Considering the existence of extensive variation (whether one species or several), the possibility of intercrossing and the absence of strict selection of progeny, it is still difficult to define the genetic material available in all the world (Barbera 1991). The main germplasm collections are located in Italy and Spain (Alkire 1998). In any case, there are biotypes that have been chosen by growers according to some advantageous characteristics. Features of interest that should represent the current scope in caper bush improvement programs are: (1) high productivity (long stems, short internodes, and high node fertility); (2) deep green spherical flower buds, with close non-pubescent bracts (to ensure a better commercial quality) and late opening; (3) absence of stipular spines and easy stalk separation to simplify harvest and postharvest operations; (4) processed product with an agreeable appearance; (5) capacity for agamic reproduction; (6) resistance to water stress, cold, and pests; (7) stem tip thick and tender for food use; (8) oval fruit with light green pericarp and few seeds for food use. The most attractive Italian commercial biotypes are ‘Nocellara’ and ‘Nocella’ (Barbera et al. 1991). Both are highly productive and yield high-quality capers (almost spherical shape, conserved integrity after brining). ‘Nocellara’ does not bear spines, and ‘Nocella’ has very small harmless ones. Seedlings are used for the propagation of ‘Nocellara’ in Pantelleria; ‘Nocella’ is propagated in Salina using cuttings. On the other hand, ‘Nocella’ does not resist drought. Also in Italy, the biotypes ‘Ciavulara’, ‘Testa di lucertola’, ‘Spinoso of Pantelleria’, and ‘Spinoso of Salina’ have different disadvantages. ‘Ciavulara’ is less productive and its buds tend to open precociously; capers are flatter and flake easily during postharvest treatments, giving a poor aspect to the final product. ‘Testa di lucertola’ (‘Lizard’s head’) produces capers with a lengthened pyramid shape. ‘Spinoso of Pantelleria’ and ‘Spinoso of Salina’ have conspicuous axillary spines. In ‘Spinoso of Pantelleria’, the leaf tips also bear a small thorn. ‘Spinoso of Salina’ is less productive; its capers are flattened pyramidal and tend to flake during postharvest curing. Other Italian biotypes are ‘Tondino’, grown in Pantelleria, and ‘Dolce di Filicudi-Alicudi’, cultivated in the Aeolian Archipelago. The most important Spanish biotypes are ‘Común’ or ‘del País’ and ‘Mallorquina’ (Luna Lorente and Pérez Vicente 1985). ‘del País’ is a heterogeneous population with spiny stems. This biotype branches frequently. Its capers are covered with a dense indumentum at the beginning of their growth. Stems dry out completely in winter. This biotype is difficult to propagate by cuttings (Jiménez Viudez and Guillamón Garrido 1986).

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‘Mallorquina’ is widely cultivated in Murcia and Almería. It has long spiny stems with bright green leaves. Its fruit is small and seedy. ‘Mallorquina’ is highly productive and shows a very important vegetative growth. In addition, it can be easily propagated by cuttings. This biotype has extraordinary yields under irrigation. ‘Italiana’ is a biotype with a lower vegetative and productive potential. When scions of ‘Italiana’ were bark-grafted using ‘del País’ as rootstock, yields increased significantly. ‘Italiana’ has the advantage of being easily harvested due to the absence of spines. Other biotypes are cultivated to a lesser extent in the Balearic Islands. ‘Rosa’, ‘Redona’, and ‘Fina’ are spiny but highly productive biotypes. ‘Redona’ and ‘Fina’ yield high-quality capers. On the other hand, ‘Fulla Redona’ and ‘Cavall’ are biotypes without spines. ‘Cavall’ has a low productivity but ‘Fulla Redona’ can be considered a promising biotype due to the quality and quantity of its produce. ‘Boscana’ is another spiny biotype, with low productivity and low product quality. B. Propagation 1. Seed. Caper seed germination performance is poor. Caper bush produces a high number of seeds per generative shoot, but low germination percentages under semidesert conditions create a great gap between seed yield and germination (Ziroyan 1980). However, caper bush propagation is usually carried out by seed owing to the serious rooting problems associated with cuttings. In Pantelleria, 5% germination was obtained within 2–3 months of seeding (Barbera and Di Lorenzo 1982, 1984). Cappelletti (1946) in Italy, and Luna Lorente and Pérez Vicente (1985) in Spain, also observed low germination percentages after direct sowing under field conditions. In the United States, using fresh seeds kept in pots above 18°C, Bond (1990) obtained germination percentages of about 10% within 10 days, and another 5–10% over the following month or two. Stromme (1988) described her difficulties when germinating caper seeds in California, although caper bush is fully adapted to the Mediterranean climate. Different pretreatments had been performed in order to improve the germination percentage, including scarification, stratification, soaking in 0.2% K2MnO4, concentrated H2SO4, H2O2, 0.2% KNO3, or gibberellins (GA4+7) and manipulation of the environmental conditions (light/dark, temperature) (Reche Mármol 1967; Ministerio de Agricultura 1980; Orphanos, 1983; Singh et al. 1992; Macchia and Casano 1993; Sozzi and Chiesa 1995; Yildirim 1998; Söyler and Arslan 1999; Tansi 1999). The best results were attained by partially removing

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the seed coats from non-germinated seeds (Sozzi and Chiesa 1995). With this procedure, the germination rate was greatly enhanced and seed dormancy was completely broken: 100% viable embryos germinated within 3–4 days. Caper seed germination shows a complete dependence on the covering structures. The seed of the genus Capparis is bitegmic (Corner 1976). The testa is 0.2–0.3 mm thick, with all its cell walls somewhat lignified, some of them with distinct thickening; its tegmen consists of an outer fibrous, lignified layer 4–10 cells thick, with a lignified endotegmen composed of contiguous cuboid cells, with strongly thickened radial walls. Only the mesophyll between exo- and endotegmen is unlignified (Guignard 1893a; Corner 1976). Since the integrity of the seed coating is very important to the persistence of dormancy in caper seeds, it is likely that the seed coats, with their successive lignified structures, are the most important cause for the problematic germination of this species (Sozzi and Chiesa 1995). The seed coats and the mucilage surrounding the seeds may be ecological adaptations to avoid the loss of water and germination during the dry season (Scialabba et al. 1995). Longitudinally opened mature fruits, with a violaceous dark green pericarp and dark red pulp, are adequate sources of dull brown mature seeds. Seeds obtained from small not-yet-opened fruit are generally light brown and immature. Seeds lie without order in the pericarp mass, each of them surrounded by an adherent layer of pulp. They can be obtained by rubbing and washing, followed by drying in the shade for a couple of days. Seeds are over 90% viable (Orphanos 1983; Sozzi and Chiesa 1995; Tansi 1999) for two years if held at 4°C and low relative humidity. In Spain, many methods to obtain young plants have been tested (Ministerio de Agricultura 1980; Luna Lorente and Pérez Vicente 1985). Commercial lots of seed are usually pre-germinated in February or March. Pre-germination consists of packing the seed in moist river sand, though other materials may also be used, such as compost of two parts turfy loam and one part leaf-mold and sand (Foster and Louden 1980) or mixtures with vermiculite or perlite (Kontaxis 1989). Small lots can be pregerminated in boxes; moderate- to large-sized lots are usually pregerminated in bins located in a greenhouse or another protected place. Two to four layers of seed are packed in each bin and the top one is covered with a 6 cm sand layer. Seeds are sprinkled with water and treated with captan or captafol. The moisture content requires a careful control; use of well-drained containers is essential to ensure thorough wetting as well as aeration (Luna Lorente and Pérez Vicente 1985). Seeds are usually

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submitted to these conditions for 25 to 50 days (Ministerio de Agricultura 1980) depending on temperature. Only sprouted seeds are planted. In Spain, nursery preparation begins in February using calcareous soils with loam to clay-loam textures and available irrigation. Proper cultivating and fumigation under a plastic tarpaulin kills perennial weeds, weed seeds, nematodes, soil insects, and fungi. After removal of the tarp, seeds (1.5–2 g/m) are planted about 1.5 cm deep, in rows 30 or 40 cm apart. Irrigation is needed on a 2-week or even more frequent basis. Most caper nurseries use furrow irrigation (Luna Lorente and Pérez Vicente 1985). Yields of 45–50 seedlings/m are obtained after 30 days. In Pantelleria, planting is performed in a rudimentary way, with few defined cultural practices (Barbera 1991). Transplants may also be produced under protected conditions using floating row covers. Some nurseries seed directly into pots, containers, or polyethylene bags where plants remain until outdoor transplanting. 2. Vegetative Cuttings. Caper bushes grown from cuttings have an advantage over seed-propagated bushes: they are genetically identical with their source. This practice avoids high variability in terms of production and quality. Nevertheless, plants grown from cuttings are more susceptible to drought during the first years after planting. Caper is a difficult-to-root woody species. Successful propagation requires careful consideration of biotypes as well as seasonal and environmental parameters. Propagation from cuttings is the standard method for growing ‘Mallorquina’ and ‘Italiana’ in Spain, and ‘Nocella’ in Salina. In Pantelleria, utilization of rooted cuttings did not yield good results (Barbera and Di Lorenzo 1984). Hardwood cuttings are generally used to propagate ‘Mallorquina’. Workers collect 1-year-old wood, 1.0 to 2.5 cm in diameter, when the plants are still dormant, and cut it into sections 20 to 30 cm long (15 cm minimum, 40 to 50 cm maximum). When cuttings are at least 1.5 cm in diameter, rooting percentages of 55% are possible (Istituto di Coltivazioni Arboree, Università degli Studi di Palermo, unpublished). The shoots are planted in individual rooting plugs or lined out in a nursery row with a humid environment. Rooting percentages depend on cutting harvest time and substrate utilized (Pilone 1990a). Another possibility is to collect stems during February through the beginning of March, pretreat them with captan or captafol, and stratify them outdoors or in a refrigerator at 3–4°C, covered with sand or plastic. Moisture content and drainage should be carefully monitored and maintained until planting (Luna Lorente and Pérez Vicente

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1985). Using semihard cuttings, collected and planted during August and September, low survival rates (under 30%) have been achieved. Softwood cuttings are prepared in April from 25- to 30-day shoots. Each cutting should contain at least 2 nodes and be 6 to 10 cm long. Basal or subterminal cuttings are more successful than terminal ones. Then, cuttings are planted in a greenhouse under a mist system with bottom heat; 150 to 200 cuttings m–2 may be planted. Dipping the cutting basal end into 1500–3000 ppm auxin solution may enhance rooting (Pilone 1990b) but results depend on the type of cutting. Hardwood cuttings do not seem to respond to indole-3-butyric acid (IBA) or a-naphthaleneacetic acid (NAA) pre-treatments. On the other hand, dipping the herbaceous cutting base in a 2000 ppm NAA yielded rooting percentages of 83% (Luna Lorente and Pérez Vicente 1985). Micropropagation. Successful micropropagation was achieved from nodal shoot segments. Four µM 6-benzylaminopurine stimulated proliferation and shoot development; when combined with 0.3 µM indoleacetic acid (IAA) and 0.3 µM GA3, formation of proliferating clusters was enhanced (Rodríguez et al. 1990). High rooting response was obtained by using 30 µM IAA (Rodríguez et al. 1990). The presence of abnormal vitrified shoots was observed in some cases and could be prevented by means of alternate culture in cytokinin-enriched and hormone-free media, or normalized by using sucrose-enriched medium (Safrazbekyan et al. 1990). Because of the difficulties of caper bush conventional propagation, in vitro culture may be a promising alternative technique. Grafting. Grafting is a less common method of propagation for caper bush. In Spain, acceptable results (60% scion take) were obtained using bark grafting in plantings. Nurseries generally whip graft with survival rates of 70–75% (Luna Lorente and Pérez Vicente 1985). C. Cultural Practices 1. Plant Establishment. Caper is a long-lived bush and plantings over 25–30 years old are still productive. Thus, site selection for the planting is an important step. Soil, water availability, and climate are the main aspects to be considered. Physical properties of the soil (texture and depth) are particularly important. Caper bush can develop an extensive root system. Because of that, it grows best on deep, non-stratified, medium-textured, loamy soils. Moldboard plowing and harrowing are usual practices prior to caper plant establishment (Luna Lorente and

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Pérez Vicente 1985). Soil-profile modification practices, such as slip plowing operating 0.6 to 1 m deep, can alleviate restrictions (Massa Moreno 1987). In Pantelleria, digging backhoe pits for each shrub was found to be the most effective means of cultivating caper in small plantings with rocky soils (Barbera 1991). Most caper bush plantings are planted in a square or hedgerow design, as these patterns are very easy to lay out and plant. Subsequently, cultivation and harvest are simplified. Spacing is determined by the vigor of the biotype, the fertility of the soil, the equipment to be used, and the irrigation method, if any. Bush spacing of 2.5 × 2.5 m (Barbera and Di Lorenzo 1982) or 2.5 × 2 m (Bounous and Barone 1989) is common in Pantelleria. In Salina, 3 × 3 m is satisfactory for ‘Nocella’. Even wider spacing is common in Spain: 4 × 4 or 5 × 5 m may be entirely satisfactory for planting on fertile loam soils. ‘Mallorquina’ may be crowded at 3 × 3 or 3 × 4 m in a 5-year planting (Centro de Capacitación Agraria de Lorca—Murcia, unpublished results). If caper bush is used to control soil erosion, especially on slopes, 2- to 2.5-m spacing is appropriate. Fertilization should begin 20–30 days before planting. At that time, 100 kg/ha ammonium sulfate, 400 kg/ha single superphosphate, and 150 kg/ha potassium chloride have been suggested in Spain (Massa Moreno 1987). Fertilizers may be broadcast on the surface and incorporated by tilling or cultivating, or surface band applied. In Pantelleria, plots are enriched with organic or inorganic fertilizers applied to the backhoe pits (Barbera 1991). Nursery plants, propagated as seedlings or rooted cuttings are dug in the nursery row during the dormant season. In the Aeolian Archipelago, transplanting is performed during January through February, but in zones of the Iberian Peninsula with prolonged winter, it takes place during February through early March, after the last frosts. In Argentina, transplanting is generally made in July through August. Transplanting is carried out by hand and caper shrubs may be transplanted either bare-root or containerized. Most plants are handled bareroot and replanted immediately in their permanent location or heeled-in in a convenient place with the roots well covered. Field beds should be well prepared and watered. Afterwards, a good amount of irrigation should be applied. Containerized plants are only used where irrigation is the chief factor limiting transplanting success. Direct field seeding or cutting planting may be alternative practices. Although costs and labor requirements are lower than for transplanting, a major difficulty is the very heavy losses of young plants resulting from drying. Eight to 10 pregerminated seeds or 3 to 4 cuttings are placed by hand into each 30-cm hole after carefully pulverizing the soil. On rocky

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slopes, caper bush can be directly seeded into a natural setting for landscape purposes. 2. Intercropping. Intercropping caper bush with other perennial crops is a practice commonly used by farmers both in Italy and Spain, as it provides a variety of returns from land and labor, increases resource efficiency (e.g., reduces labor peaks; Millan Campos 1987) and attenuates the risk of dependence upon a single crop. In Italy, the intercropping pattern varies from strip cropping to row intercropping. In the strip cropping pattern used in Pantelleria, there are one or two rows of caper bush (usually along the borders of the fields) to several rows of grape vine. In the row intercropping system used in the Aeolian Archipelago (especially in Salina), caper bush is grown in association with olive (Barbera 1991). In Almería, Spain, intercropping of caper bush and almond tree was devolved during the 1970s and 1980s (Lozano Puche 1977). Three different planting patterns were examined, providing more favorable conditions for this intercropping arrangement (Lozano Puche 1977): alternate-row, alternate-plant, and same-hole intercropping systems. No significant differences were detected between systems, and almond tree productivity was not affected significantly. Nevertheless, the alternate-row system is the more appropriate (Cano García 1987) to avoid competition between both species and to prevent erosion. Tillage of orchard middles or soil cultivation for weed control may be performed when caper plant is in the form of stump. 3. Pruning. Caper bush is usually dormant pruned. Where winter temperatures are very low, the pruning operation is delayed until the severest weather is over. After the removal of dead or dying tissue, caper bush must be pruned of weak, non-productive wood and water sprouts. Caper bush benefits from a short and heavy spur pruning that reduces branches to a length of 1 cm (Barbera and Di Lorenzo 1984) to 3 cm (Luna Lorente and Pérez Vicente 1985) or 5–10 cm when the plant is young and vigorous (Barbera and Di Lorenzo 1982). It is important to leave several buds on the spur, as only the 1-year-old stems will bear flower buds for the current season. Early summer pruning involves thinning out weak stems when caper bush is in active shoot growth, 30–40 days after budding. The number of stems to leave upon a vine should be based on its general vigor. A strong plant may have as many as six, strategically distributed to obtain an open canopy with uniform light penetration throughout. Summer pruning also involves heading back a few of the new shoots, to stimulate the formation of new flower buds.

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Table 4.1. Nutrient levels on a fresh weight basis of caper bush flower buds, stems, and leaves with no apparent visual deficiency symptoms. From Centro de Edafología y Biología Aplicada del Segura, Spain, cited by Luna Lorente and Pérez Vicente (1985). Nitrogen Phosphorus (%) (mg/100 g)

Plant tissue Flower buds Stems + Leaves

0.87 1.01

Potassium Calcium Magnesium (mg/100 g) (mg/100 g) (mg/100 g)

86 55

135 173

32 18

55 116

Water (%) 81.1 70.1

4. Plant Nutrition. In Spain, different analyses were performed to make an adequate diagnosis of nutrient deficiencies (Centro de Edafología y Biología Aplicada del Segura, cited by Luna Lorente and Pérez Vicente 1985). The types of fertilizer used and application rates should be related to plant age (Table 4.2) and soil nutrient content. Measurements of the total concentration of a nutrient in the plant (fresh weight basis, Table 4.1) and the extraction of different elements from soil (Table 4.2) can be used for diagnosing mineral deficiencies. Phosphate and potassium fertilizers are generally applied every two to three years (Table 4.3), as caper P-K requirements are lower than that for N. Ammonium fertilizers are incorporated into the soil at the end of the winter, before sprouting. In Pantelleria and Salina, N-P-K fertilizers (15-6.6-12.5, 10-4.4-8.3, 20-4.4-8.3, or 11-9.6-13.3) are applied during winter (December and January) at a rate of 200–300 g/plant (Barbera and Di Lorenzo 1982; Barbera 1991). Bounous and Barone (1989) suggested that fertilizations with

Table 4.2. Extraction of mineral elements during the first seven years after implantation. From Centro de Edafología y Biología Aplicada del Segura, Spain; cited by Luna Lorente and Pérez Vicente (1985). Flower buds, stems, and leaves are on a fresh weight basis. Planting age (years) 2 3 4 5 6 7

Flower buds (kg/ha)

Stems and leaves (kg/ha)

N (kg/ha)

P (kg/ha)

K (kg/ha)

Ca (kg/ha)

Mg (kg/ha)

125 600 1100 1250 1350 1350

500 1500 2000 2250 2500 2500

5.6 2.4 29.8 33.6 37.0 37.0

0.17 0.59 0.87 1.01 1.11 1.11

0.86 2.82 4.07 4.65 5.10 5.10

0.13 0.45 0.70 0.80 0.87 0.87

0.65 2.07 2.92 3.30 3.64 3.64

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Table 4.3. Fertilizer application program for caper bush. From Centro de Edafología y Biología Aplicada del Segura, Spain; cited by Luna Lorente and Pérez Vicente (1985). Planting age (years)

Ammonium sulfate (kg/ha)

Single superphosphate (kg/ha)

Potassium sulfate (kg/ha)

2 3 4 5 6 7

50 150 200 200 250 250

— 25 — 50 — 50

— 25 — 50 — 50

150–200 kg/ha of ammonium sulfate and additional P-K applications would be appropriate for mature plantings. 5. Irrigation. Caper bush is cultivated mostly in non-irrigated areas represented by poor lands generally receiving little attention from farmers. Though the caper plant typically grows in semiarid regions and tolerates water stress well, water is the most limiting production factor. During the first year, caper bush is particularly sensitive to water stress. In Pantelleria and Salina, irrigation is impossible due to the lack of hydrous resources (Barbera and Di Lorenzo 1984). Nevertheless, a type of mulching—which may include placing stones around the young plants—is utilized to protect them from the wind action and thus reduce evaporation. In Spain and Argentina, additional water is usually provided during the first year. The caper bush shows its productive potential under irrigation (longer vegetative cycle, larger bud production that begins earlier, and shorter intervals between harvests), though the plant tends to be more prone to diseases (Jiménez Viudez 1987). In Spain, irrigation begins in January when caper bush is grown with almond trees, but in February or March when grown alone, and it ends in August in either case (Jiménez Viudez 1987). Yields were doubled and even tripled when irrigation was used in Almería (it rains 96 mm from February through August), Jaén (284 mm), and Murcia (156 mm). In 1984, the average yield in Spain was 1365 kg/ha in irrigated plantings and 650 kg/ha in non-irrigated plantings (Ministerio de Agricultura, Pesca y Alimentación 1989). In 1988, 837 ha had been irrigated in Almería, Murcia, and Jaén (Ministerio de Agricultura, Pesca y Alimentación 1988). In 1995, only 41 ha (mainly in Murcia, Córdoba, and Valencia) were still under irrigation due to the

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increasing competition from caper grown in Turkey and Morocco (Ministerio de Agricultura, Pesca y Alimentación 1997). A point source sprinkler system may be utilized. Total volumes of 12–140 L/plantweek, depending on the climatic conditions, are supplied under irrigation (Jiménez Viudez 1987). 6. Weed Control. Competition of weeds may be particularly serious during the seedling establishment. During the first and second years of the planting, preemergence herbicide treatments in combination with mechanical weed removal (4–5 supplementary well-timed operations using discs or rotary tillers) yield satisfactory results whenever direct pulverization on caper stump or stems is avoided. In Pantelleria, paraquat and simazine are used (Barbera 1991). After establishment, most of the ground is rapidly covered by the caper bush canopy and weed development is almost suppressed. In Spain, different preemergence and postemergence herbicides were tested in a 5-year planting prior to outgrowth of the new caper shoots (Lozano Puche 1984). Linuron (50%) at 2 kg/ha, prometryne (50%) at 4 kg/ha, methabenzthiazuron (70%) at 3 kg/ha, and metribuzin (30%) at 3 kg/ha were compared. Prometrine and metribuzin gave the most satisfactory results under such conditions. On the other hand, simazine and prometrine were found to be effective in controlling the “weed” caper bushes (Fairushina 1974). 7. Pests and Diseases Pests. The caper bush is not very sensitive to pest damage when growing without cultivation and insects do not appear to be the limiting problem under field conditions. Nevertheless, some phytophagous species that attack caper in its main production areas have been detected. Insecticide treatments are restricted by the short interval between harvests (7–10 days); only low-persistence active principles can be used, so as to avoid the presence of toxic residues at harvest. Research should also be undertaken to determine the possible influence of caper processing on persistence of different types of residues. In Pantelleria, the caper moth (Capparimyia savastanoi Mart.; Trypetidae) is considered to be the most important pest (Longo and Siscaro 1989; Longo 1996). Its control relies on the removal of infested leaves, combined with the use of poisoned hydrolyzed protein baits in summer when populations are high. Another major pest is the caper bug (Bagrada hilaris Bm.; Pentatomidae), a polyphagous insect that feeds on species of many plant families

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such as Brassicaceae, Fabaceae, Poaceae, Amaranthaceae, Apiaceae, Solanaceae, Rutaceae, Malvaceae, and Asteraceae (Gunn 1913). In Pantelleria, it was first found on wild plants (Carapezza 1981) and, later on, attacking cultivated caper plantings (Genduso 1990). The pale creamy oval eggs, which turn to orange as the insect develops (Mineo and Lo Verde 1991), are laid singly on the ground, in the cracks of the bordering field walls and, more rarely, on the leaves. At the beginning of spring it attacks different wild plants, among them caper bush, which grows weak and rapidly yellows. Pyrethroid formulations are used to control this insect. The chemicals are applied either to the walls or to the plants after harvest is finished (Barbera 1991). The painted bug (Bagrada picta Fabr.; Pentatomidae) is a pest of cruciferous oilseed crops and has been reported to thrive on caper bush at Tandojam during summer (Mahar 1973). Also in Pantelleria, the larval form of a type of weevil, Acalles barbarus Lucas (Curculionidae), causes damage to the root system (Liotta 1977). In general, its targets are weak adult plants previously affected by other insects. The only effective control is the removal of the attacked plants. Other insect pests in Italy are Phyllotreta latevittata Kutsch (Chrysomelidae), which causes oval to round erosions in leaves, leaf yellowing and stem decay, and Asphondylia spp. (Cecidomyiidae) and Cydia capparidana Zeller (Tortricidae), which alter the morphology of buds (Harris 1975; Orphanides 1975, 1976). The braconid Chelonus elaeaphilus Silv., a promising parasite of Prays oleae (an olive pest), was also recovered from C. capparidana infesting caper bush (Fimiani 1978). Rapisarda (1984–85) reported the occurrence of Aleurolobus niloticus Priesner & Hosny (Aleyrodidae), a polyphagous species that feeds only on caper bush leaves in Sicily. In Southern Spain, caper bush is the only larval host plant available during the dry season for various Pieridae: cabbage small white (Pieris rapae L.) and large white (Pieris brassicae L.) butterflies, and desert orange tip (Colotis evagore Klug.). P. rapae has also been found feeding on caper bush in the Badkhyzskii Reserve, Turkmenistan (Murzin 1986), and in California (Kontaxis 1990). The larvae of P. rapae and P. brassicae usually use cruciferous plants in the rainy season and caper bush in summer when Cruciferae are dry (Fernández García 1988). Oviposition takes place preferentially on the ground or on dried material around the food plant. On the other hand, C. evagore larvae are unable to survive on alternative cruciferous hosts (Jordano Barbudo and Retamosa 1988; Jordano et al. 1991) but they complete their life cycle successfully in certain coastal enclaves where caper bush provides sufficient resources throughout the year. The adult lays red eggs singly, on young

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leaves, stems, and inert supports next to the food plant (Fernández et al. 1986; Fernández Haeger and Jordano Barbudo 1986). Caper bush and other related species are also the commonest food plants of other Pieridae in Saudi Arabia, such as Anaphaeis aurota F., Colotis fausta fausta Olivier, and Colotis liagore Klug. (Pittaway 1979, 1980, 1981, 1985). These species deposit the ova on isolated bushes in rocky scarps and cliffs. Eventually, caper plants may be completely stripped of foliage, the resulting bare branches carrying pupae and larvae. Pyrethroids and trichlorfon can be used to control all of these Pieridae pests (Massa Moreno and Luna Lorente 1985). Larvae of Lampides boeticus L. (Lycaenidae), which have anthophagous and carpophagous habits, have also been found to feed on the buds of caper bush (Jordano Barbudo et al. 1988). In Spain, the pentatomid bug Eurydema ornata L. (Fernández et al. 1986) attacks caper bush leaves and may cause serious damage to plants. Other pentatomid bugs (Holcostetus punctatus and Carpocoris lunula) are less frequent. The green stink bug Nezara viridula L. has caused some minor damage in the Iberian Peninsula and Argentina. All these Hemiptera can be controlled by using trichlorfon, endosulphan, dimethoate, or chlorpyriphos. Other insect pests detected in caper include Ceuthorhynchus sp. (Curculionidae) and Heliothis-Helicoverpa (Noctuidae). Many ant species (Camponotus spp., Plagiolepis pygmaea, Crematogaster auberti, Crematogaster sordidula, Formica subrufa, Tetramonium hispanica, Cataglyphis viaticoides) have been found feeding on caper plant (Fernández et al. 1986). In California, caper plants can be damaged by cabbageworm, black vine weevil, and flea beetle, as well as gophers, snails, and slugs (Kontaxis 1998). Mosquitoes (Culex pipiens molestus Forskal; Culicidae) and sandflies (Phlebotomus papatasi Scopoli; Psychodidae), both of which are bloodsucking insects, also feed on caper plants (Schlein and Muller 1995). Photosynthate levels in caper bush may affect sandfly feeding (Schlein and Jacobson 2000). Sandflies are known to be vectors of human pathogens, Leishmania tropica and L. major (Schlein and Warburg 1986; Schlein et al. 1986; Schlein and Yuval 1987), but Leishmania-infected sandflies showed a significant reduction in the number of parasites and 55% of impaired infections when feeding on caper bush. This suggests a role for the plant in the epidemiology of leishmaniasis (Schlein and Jacobson 1994a,b). In fact, extracts of C. spinosa caused extensive parasite agglutination, apparently due to caper plant lectins (Jacobson and Schlein 1999). Screening tests were performed in Spain (Servicio de Sanidad Vegetal, Murcia, data not published) to determine the efficiency of insecti-

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cides (parathion, malathion, chlorpyrifos, deltamethrin, trichlorfon, tetradifon plus dicofol, and dithiocarbamates). These pesticides were found not to be phytotoxic for caper bush. Biologically integrated pest management approaches through the use of biopesticides have not yet been tested. Diseases. Damping-off diseases may be severe. Frequently, caper seedlings are completely destroyed either when they are placed in seedbeds or after being transplanted. Seedlings are usually attacked at the roots or in the stems at or below the soil line, and the invaded areas soon collapse. Damping-off in caper bush is caused by several fungi (Pythium spp., Fusarium spp. Verticillium spp., etc.), which often cause quite similar symptoms. These diseases can be controlled through the use of sterilized soil and chemically treated seeds. A list of fungi that affect caper bush was given by Ciferri (1949). The most important is probably the white rust disease (Albugo capparidis De By.) which affects aboveground plant parts, particularly the leaves and flowers. Neoramularia capparidis spec. nov. produces small grayishwhite leaf spots with a narrow brown margin in India (Bagyanarayana et al. 1994). Besides, caper bush is a host of Leviellula taurica (Lev.) G. Arnaud, causal agent of the powdery mildew (Gupta and Bhardwaj 1998). In California, Botrytis spp. and Pythium spp. attack caper plants (Kontaxis 1990). A caper vein banding virus (CapVbV) was reported in Sicily and was tentatively assigned to the carlavirus group (Majorana 1970). Gallitelli and Di Franco (1987) isolated the same virus and showed that it infects caper plant symptomlessly. Therefore, they suggested the alternative name caper latent virus (CapLV). The real causal agent of vein banding may be a rhabdovirus, the caper vein yellowing virus (CapVYV) that may infect caper bush simultaneously with the CapLV (Di Franco and Gallitelli 1985). New serological tests have shown that CapVYV is indistinguishable from the Pittosporum vein yellowing nucleorhabdovirus (PVYV) (Nuzzaci et al. 1993). 8. Harvest and Yield. Harvest is the costliest operation of caper production. It may represent 2/3 of the total labor in the crop management process (Caccetta 1985; Martínez Capel 1987) because it is done manually. Harvest is difficult and time-consuming due to: (1) the decumbent character of the branches; (2) the presence of stipular spines in some biotypes; (3) high temperatures and insolation during summer in caperproducing areas; and (4) the small diameter of flower buds. Besides, harvest has to be performed several times. Flower buds are arranged along

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twigs that have an indeterminate growth habit. Since bud production is continuous, twigs should not be cut and mechanical harvesting is not a viable option at present. A harvester can collect around 12 kg of capers per day when working in a regularly designed planting (Lozano Puche 1983). Successive harvests are designed to maximize the yield of smaller (more valuable) capers. However, buds that are missed in one harvest will continue to develop and will be collected as caperberries in subsequent harvests. Temperature is the main environmental factor affecting caper harvest dates. In Italy, harvesting takes place from late May through late August. In Spain, caper production continues through September, but there are not always enough buds to justify collection. In Argentina, harvest operations last 75–90 (Mendoza) to 120 days (Catamarca) or even more, depending on the latitude. The genetic nature of the biotype also affects harvesting operations: in Spain, the production peak takes place in June–July when the biotype is ‘del País’, but in July–August if the biotype is ‘Mallorquina’. Harvest frequency has a direct bearing on the final size and quality of the product (Table 4.4). However, determining the optimum time interval is a difficult decision because there are different conflicting factors to consider. Shorter time intervals between successive harvests result in a high-quality product (Fig. 4.3); yet, on the other hand, the number of buds per kg increases when caper size decreases (Table 4.4) and harvesting costs increase when intervals between harvest dates are shorter.

Table 4.4.

Caper international grading system. Number of flower buds/kg

Diameter (mm)

International commercial denomination

14

Non Pareil Surfine Capucine Capote Capote Fine Fine Grosse Hors Calibre

According to Barbera (1991)

According to Luna Lorente and Pérez Vicente (1985)

5,500 4,000 3,250 2,600 2,200 1,900 1,600 — —

7,000 4,000 4,000 2,000 2,000 1,300 1,300 800 —

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30 10-12 days

25

Distribution (%)

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7 days

20

15

10

5

0 6

7

8

9

10 11 12 13 14 15 16

Diameter (mm) Fig. 4.3. Distribution of caper diameters in relation to time interval between successive harvests. Prepared with data from Barbera (1991).

In general, 7- to 10-day intervals between successive harvests are appropriate, but they are usually shortened to 3–5 days during the peakproduction periods, as happens in Spain. Usually, 8 to 10 recollections per year take place in Pantelleria and Salina, and 12–14 in the Iberian peninsula. Caper bush yields are highly variable depending on the growing environment, cultural practices, and biotype, but a maximum yield is expected in the 4th year. Bounous and Barone (1989) indicated average annual yields of 1–1.5 kg/plant and yields as high as 4 kg/plant in the 3rd and 4th years of cultivated growth. Barbera and Di Lorenzo (1982) reported average annual yields of 1–1.5 kg/plant in Pantelleria (maximum yields of 4–5 kg/plant) and 2–3 kg/plant in Salina in 3-year plantings (average annual yields of 3–4 t/ha). According to Barbera (1991), a mature caper plant may produce 4–5 kg/year. On the other hand, Caccetta (1985) estimated annual yields of 1.2–2.5 t/ha in Pantelleria and 1.8–2.6 in Salina. According to Lozano Puche (1977), a wild growing plant yields 2–3 kg/year in Spain, but the same caper bush has the potential to produce

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8 Mallorquina del País

7

Average Yield (kg plant -1)

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6 5 4 3 2 1 0 1

3

5

7

9

Planting Age (years) Fig. 4.4. Caper bush yields in the first years after implantation. Open symbols mark drought years. Prepared with data from Ezequiel Sánchez García (Centro de Capacitación Agraria de Lorca, Murcia), unpublished.

6–9 kg/year when cultivated in irrigated fertile soils (Jiménez Viudez 1987). Great differences in yield are attributed to genetic variations. A 3-year-old ‘del País’ planting yields 1–1.5 t/ha-year, but this production may be doubled and even tripled by using ‘Mallorquina’ (Fig. 4.4).

V. POSTHARVEST TECHNOLOGY A. Capers 1. Handling and Curing. Immediately after harvest, capers are placed in shallow vats. In Spain, postharvest conditioning is generally performed by local traders, cooperatives, or producer associations, and consists of a series of steps. After cleaning away the rest of the leaves and pedicels, a first selection of capers takes place and blemished and open buds are discarded. Then, capers are subjected to a first sieving, which generally

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size-grades them into two size groups, with diameters lower or higher than 8–9 mm. This first classification provides an incentive for recollection of smaller capers and makes the subsequent industrial steps easier. After aeration in a well-ventilated place, capers are packed in wooden or PVC barrels, fiberglass tanks, or large casks and pretreated with high salt brine (ca 16% NaCl w/v at the equilibrium, increasing to 20% after changing the first brine). After filling, the casks are hermetically closed and placed in the sun. In order to reach the equilibrium in salt concentration, barrels are rolled during the early stage of brining. Periodical checks should be performed, to ensure that the brine completely covers the material. The completion of this “wet” curing process lasts 20–30 days (Luna Lorente and Pérez Vicente 1985), but capers may be stored under such conditions for several months, until final industrial conditioning takes place. Thus, capers may be classified as fully brined vegetables (Ranken 1988) that may be regarded as a stable product during storage. Fresh capers have an intensely bitter flavor and one of the purposes of the pickling process, besides that of preservation, is to remove this unpleasant characteristic. This is due to the presence of the glucoside glucocapparin, which is readily hydrolyzed to by-products completely lacking the bitter taste. Although Spanish regulations still accept the use of brine concentrations up to 25°Baumé (Dirección de Comercio Exterior 1984), high salt brines are increasingly being objected to (Alvarruiz et al. 1990; Rodrigo et al. 1992). Organoleptic characteristics and preservation of the final product proved to be the same over at least 27 months when capers had been pretreated with 10, 15, or 20% NaCl at equilibrium (Alvarruiz et al. 1990). High salt concentrations inhibit both the growth of undesirable microorganisms and the activity of lactic acid bacteria. Lower NaCl brines (i.e., 5%) are more likely to permit growth of coliform bacteria, yeasts, and molds (Özcan and Akgül 1999a). Fermentation takes place at a higher rate when pickling small (≤ 8 mm) buds (Özcan and Akgül 1999a). Capers are also pickled in vinegar (at least 4% acidity as acetic acid) in a 1:1 (w/v) ratio (Reche Mármol 1967). Regular topping-up with vinegar ensures that all the capers remain covered. This pickling process lasts 30 days. Only 10% of vinegar is absorbed by the product, with the remainder being discarded at the end of the period. In Italy, growers arrange capers in cement tanks, PVC or wooden barrels, or open drums, between layers of solid salt (10–15% w/w). This promotes the extraction of water from the raw product by osmosis and generates a saturated brine. This treatment lasts 7–8 days. Then, the

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brine is removed and the capers are submitted to the same process once or twice more (Barbera 1991). 2. Industrial Treatment and Packaging. Following the completion of the curing period, the industrial processing is completed in three steps. First, capers are drained and rinsed with several changes of water to dislodge and remove all sediment. Second, damaged buds are disposed of, and then capers are carefully size-graded according to an international grading system (Table 4.4). Third, capers are prepared in a variety of ways and packed as a finished product. Pasteurization (80°C, 15 min) of the final product renders capers with good flavor and consumer acceptance and is recommended to prevent the development of microorganisms (Ranken 1988; Alvarruiz et al. 1990). Without pasteurization, 6–10% NaCl and 1% acidity as acetic acid (w/v) are required in the final product to avoid the risk of spoilage (Alvarruiz et al. 1990; Özcan and Akgül 1999b). In some cases, NaCl is avoided and covering capers with diluted acetic acid or distilled malt vinegar (4.3 to 5.9% acetic acid) serves as an alternative. In Italy, the final product is treated with dry salt. Such preparation decreases the cost of transportation and grants a more intensive flavor. In Spain, a similar treatment is carried out with capers of large diameter. Capers are drained and mixed with dry salt (20% maximum). The caper industry discontinued the use of olive oil in caper preparations due to its high cost. Other special preparations, including wine vinegar, with or without the addition of tarragon, Artemisia dracunculus L. (Vivancos Guerao 1948), are also expensive and exclusively utilized with capers of small diameter. Sweetening ingredients like sugar are added to those capers exported to Denmark or some northern European countries (González Soler 1973). Capers are generally packed in PVC or wooden barrels of 180–200 kg for the pickle industry but 40-kg barrels are used for packing “non pareil” and “surfine” capers, depending on the country importing them. For retail sale, capers are packed in various kinds of glass or plastic bottles containing 20 g to 5 kg, or translucent sachets of 0.1 to 1 kg. Fivekg flasks and sachets are usually sold to restaurants and coffee-shops. B. Caperberries Traditionally, caperberries are fermented by dipping them in water for 4–7 days. This immersion produces a strong fermentation accompanied by a color change (from green to yellowish) and loss of texture due to flesh breakdown and gas accumulation. This step affects the value of the

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product and has proven to be unnecessary (Sánchez et al. 1992). Lactic acid bacteria show faster growth rates at low NaCl concentrations (Sánchez et al. 1992) but, as for capers, undesirable microorganisms can grow in 5% NaCl brines (Özcan 1999a). In order to protect caperberries from spoilage during fermentation, 4–5% NaCl brines may be adequate (Sánchez et al. 1992), but fermentation must be continuously controlled (Özcan 1999a). Yields are increased by the use of 0.35% sodium acetate (Sánchez et al. 1992). Fermentation should last 20–25 days. Brines with 10% (Sánchez et al. 1992) to 15% (Özcan 1999a) NaCl at equilibrium create a favorable environment for pickled caperberry storage. Sorbic and benzoic acids, as well as their corresponding sodium and potassium salts, are used as preservatives during final packing. A method combining steam distillation (extraction) and HPLC determination has proven to be excellent for the analysis of both preservatives at low concentrations in caperberries (Montaño et al. 1995).

VI. COMPOSITION AND UTILIZATION A. Composition The chemical composition of caper plant, leaves, flower buds, fruits, and seeds is summarized in Table 4.5. As data were obtained using different genotypes, which were grown under various environmental conditions and analyzed using different experimental protocols, values can only be considered as approximations leading, at best, to the right orders of magnitude (Duke 1992). Important differences in lipid and mineral (P, Ca, Mg, Na, Fe, Zn) contents have been found in raw capers. In general, lower levels of water content, starch, and carotenoids and higher levels of ash, protein, and calcium were found in smaller capers (Rodrigo et al. 1992; Özcan and Akgül 1998). Significant differences in composition were also detected among genotypes and harvest dates (Rodrigo et al. 1992; Özcan and Akgül 1998). In caper plants, variations in organic acids (citric, tartaric, and oxalic acid), as well as in sugar components (glucose, glucuronic acid, arabinose, and xylose) and alkaloids have been considered to be evidence of taxonomic differences (Hammouda et al. 1975). High salt brine treatments greatly affect the composition of capers. Fiber and protein, as well as mineral (Mg, K, Mn) and vitamin (thiamine, riboflavine, ascorbic acid) contents drop during those preservation procedures, while ash increases due to the addition of NaCl (Nosti Vega and Castro Ramos 1987). A similar trend has been observed after

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Table 4.5. Phytochemical constituents and other variables in caper bush, capers, and caperberries. Data are on a fresh weight basis. Constituent

Organ

Content

Reference

Water

Leaf Flower bud

80.5% 69.6% 76.8–81.9%

Fruit Seed

88% 82.7% 6.48%

Katiyar et al. 1985 Duke 1992 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992; Özcan and Akgül 1998 Gorini 1981 Özcan 1999b Akgül and Özcan 1999

Ash

Leaf Flower bud

4.2% 13.2% 2.1% 1.33–1.84%

1.16–1.76%

Fiber

Protein

Fruit Seed

1.09% 1.73%

Leaf Flower bud

7.9% 2.04%

Fruit Seed

4.5–5.92% 3.13% 25.71%

Leaf Flower bud

3.85% 13.8% 3.2% 4.59–6.79%

4.81–7.27%

Amino Acids Alanine

Fruit Seed

3.34% 19–22%

Flower bud

3740 ppm

Katiyar et al. 1985 Duke 1992 Gorini 1981 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992 Özcan and Akgül 1998 Özcan 1999b Akgül and Özcan 1999 Duke 1992 Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan 1999b Akgül and Özcan 1999 Katiyar et al. 1985 Duke 1992 Gorini 1981 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992 Özcan and Akgül 1998 Özcan 1999b Duke 1992; Akgül and Özcan 1999 Nosti Vega and Castro Ramos 1987

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4. CAPER BUSH: BOTANY AND HORTICULTURE Table 4.5.

161

(continued )

Constituent

Organ

Content

Reference

Amino Acids (cont.) Aspartic Acid

Flower bud

6660 ppm

Glutamic Acid

Flower bud

7460 ppm

Glycine

Flower bud

1770 ppm

Isoleucine

Flower bud

3680 ppm

Leucine

Flower bud

4140 ppm

Lysine

Flower bud

4310 ppm

Methionine

Flower bud

910 ppm

Phenylalanine

Flower bud

3930 ppm

Proline

Flower bud

2110 ppm

Serine

Flower bud

1180 ppm

Threonine

Flower bud

2640 ppm

Valine

Flower bud

5410 ppm

Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987

Plant

0.22–0.5%z

Leaf

0.96% 0.71% 0.5% 0.47%

Lipids

Flower bud

1.51–1.77% 0.28–0.44% Fruit

Fatty Acids Palmitic Acidy

Seed

3.75% 0.84% 31.6–36%

Plant

0.076–0.084%z

Plant Flower bud

5.4–17.8% 23.9%

Mukhamedova et al. 1969 Katiyar et al. 1985 Rakhimova et al. 1978 Gorini 1981 Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan and Akgül 1998 Rakhimova et al. 1978 Özcan 1999b Pernet 1972; Duke 1992; Akgül and Özcan 1999 Mukhamedova et al. 1969 Katiyar et al. 1985 Nosti Vega and Castro Ramos 1987 (continues)

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162 Table 4.5.

G. O. SOZZI (continued )

Constituent

Organ

Content

Reference

Fruit Seed

31.9–32.4% 16.44% 13.2%

Flower bud

1.4%

Fruit Seed

1.4–8.9% 4.6%

Plant Flower bud

5.69–20.29% 7.4%

Fruit Seed

4.1–4.9% 2.7% 3.2%

Plant Flower bud

5.24–16.57% 5.9%

Fruit Seed

8.1–10.2% 29.7% 42–46%

Rodrigo et al. 1992 Özcan 1999b Akgül and Özcan 1999 Nosti Vega and Castro Ramos 1987 Özcan 1999b Akgül and Özcan 1999 Ahmed et al. 1972c Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan 1999b Akgül and Özcan 1999 Ahmed et al. 1972c Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan 1999b Hilditch and Williams 1964 Akgül and Özcan 1999 Ahmed et al. 1972c Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan 1999b Hilditch and Williams 1964 Akgül and Özcan 1999 Ahmed et al. 1972c Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992 Özcan 1999b Akgül and Özcan 1999

Fatty Acids (cont.)

Palmitoleic Acidy

Stearic Acidy

Oleic Acidy

49.9% Linoleic Acidy

Plant Flower bud

4.69–19.03% 14.9%

Fruit Seed

17.9–18.2% 29.9% 45–51% 25.2%

Linolenic Acidy

Carbohydrates Starch

Plant Flower bud

16.15–71.92% 35–37.5%

Fruit Seed

12.9% 1%

Leaf Flower bud

3.6% 0.83–1.24%

Katiyar et al. 1985 Özcan and Akgül 1998

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4. CAPER BUSH: BOTANY AND HORTICULTURE Table 4.5.

163

(continued )

Constituent

Organ

Content

Reference

Total Sugars Reducing Sugars

Fruit Flower bud Flower bud

0.61% 5.4% 2.62–4.69%

Pentosan

Fruit Flower

5.53% 40,000 ppm

Özcan 1999b Gorini 1981 Özcan and Akgül 1998 Özcan 1999b Duke 1992

Leaf Flower bud

290 ppm 1036 ppm

Carbohydrates (cont.)

Minerals P

166.5–264.5 ppm 591–806.4 ppm

S Ca

Fruit Flower bud

1167.9 ppm 180 ppm

Leaf Flower bud

1180 ppm 490.5–1344 ppm 1830 ppm 43.2–225.9 ppm

Mg

Flower bud

469–810.5 ppm

1118–1774 ppm K

Flower bud

4303–6135 ppm

Na

Fruit Flower bud

3269.3 ppm 59 ppm 190.5–285 ppm 24.3–36.7 ppm

Fe

Fruit Leaf Flower bud

121.4 ppm 150 ppm 13.7 ppm 9.25–21.1 ppm 1.59–4.68 ppm

Katiyar et al. 1985 Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan and Akgül 1998 Özcan 1999b Nosti Vega and Castro Ramos 1987 Katiyar et al. 1985 Rodrigo et al. 1992 Nosti Vega and Castro Ramos 1987 Özcan and Akgül 1998 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992 Özcan and Akgül 1998 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992; Özcan and Akgül 1998 Özcan 1999b Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan and Akgül 1998 Özcan 1999b Katiyar et al. 1985 Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan and Akgül 1998 (continues)

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164 Table 4.5.

G. O. SOZZI (continued )

Constituent

Organ

Content

Reference

Mn

Fruit Flower bud

5.45 ppm 2.9 ppm

Zn

Fruit Flower bud

7.18 ppm 7.6 ppm

Özcan 1999b Nosti Vega and Castro Ramos 1987 Özcan 1999b Nosti Vega and Castro Ramos 1987 Özcan and Akgül 1998 Nosti Vega and Castro Ramos 1987

Minerals (cont.)

1.17–2.59 ppm Cu Vitamins Thiamine (Vit B1)

Flower bud

3.4 ppm

Flower bud

0.72 ppm 0.698 ppm

Riboflavin (Vit B2)

Flower bud

0.89 ppm 2.16 ppm

Choline Ascorbic Acid (Vit C)

Leaf Flower bud

100 ppm 260 ppm 2300 ppm 156–324 ppm

Secondary Metabolites Total Alkaloids L-stachydrine Rutin

Pigments Carotenoids

Leaf Fruit Leaf Plant

200 ppm 740 ppm 100 ppm 0.02–0.026%z

Flower bud

0.28%

Flower bud

1.028 ppm 1.29–3.38 ppm

pH

z

Fruit

1.15 ppm

Flower bud

5.9–6.3 ppm

Fruit

4.32 ppm

Lemmi Cena and Rovesti 1979 Nosti Vega and Castro Ramos 1987 Lemmi Cena and Rovesti 1979 Nosti Vega and Castro Ramos 1987 Duke 1992 Lemmi Cena and Rovesti 1979 Nosti Vega and Castro Ramos 1987 Özcan and Akgül 1998 Ahmed et al. 1972c Ahmed et al. 1972c Duke 1992 Mukhamedova et al. 1969 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Özcan and Akgül 1998 Özcan 1999b Özcan and Akgül 1998 Özcan 1999b

Converted to a fresh weight basis considering 80% moisture. Each fatty acid is reported as the percentage of the total fatty acid content.

y

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caperberry preservation treatment (Özcan 1999b). This exchange of different components, i.e. ionic constituents, is not particular for capers and caperberries but a general trend when soaking fruits, probably enhanced by the presence of NaCl. Fatty acid analyses have revealed that capers are rich in linoleic and linolenic acids (Table 4.5). Moreover, seeds yield higher levels of oleic and linoleic acid and have lower contents of saturated acids than other Capparis species (Hegnauer 1961; Sen Gupta and Chakrabarty 1964). The lipid complex of the aerial part of caper bush has been extracted with chloroform-methanol and analyzed (Tolibaev and Glushenkova 1995). Many lipid products were obtained, including neutral lipids (mainly free fatty acids, triacylglycerols, and sterol and triterpenol esters), glycolipids (mainly digalactosyldiglycerides and sterol glycosides), and phospholipids (mainly phosphatidylglycerols, phosphatidylethanolamines, and phosphatidylcholines). In almost all classes of lipids, palmitic, oleic, linoleic, and linolenic acids were prevailing. Different flavonoids were identified in caper bush and capers: rutin (quercetin 3-rutinoside), quercetin 7-rutinoside, quercetin 3-glucoside7-rhamnoside, kaempferol-3-rutinoside, kaempferol-3-glucoside, and kaempferol-3-rhamnorutinoside (Rochleder and Hlasiwetz 1852; Zwenger and Dronke 1862; Ahmed et al. 1972a; Tomás and Ferreres 1976a,b; Ferreres and Tomás 1978; Artemeva et al. 1981; Rodrigo et al. 1992; Sharaf et al. 1997). Rutin and kaempferol-3-rutinoside are probably the most abundant flavonoids, followed by kaempferol-3-rhamnorutinoside in significantly lower concentrations (Rodrigo et al. 1992; Sharaf et al. 1997). Recently, Sharaf et al. (2000) identified a quercetin triglycoside (quercetin 3-O-[6’”-a-L-rhamnosyl-6”-b-D-glucosyl]-b-Dglucoside) in methanolic extract of the aerial part of caper bush. Guignard (1893b) first reported the presence of the enzyme myrosinase in C. spinosa. Brassicaceae are a major source of glucosinolates (Kjœr 1963; Kjœr and Thomsen 1963) whose hydrolysis to glucose, sulfuric acid, and isothiocyanates can be catalyzed by the enzyme myrosinase. The presence of glucosinolates is synapomorphic for members of this family and lends additional support to the new phylogenetic classification (Judd et al. 1999). In fact, the conclusion that Capparidaceae and Cruciferae should remain together, based on the presence of glucosinolates, was drawn almost 40 years ago (Hegnauer 1961; Kjœr 1963). Glucosinolates, their biochemistry, biological variations, and roles have been recently reviewed in detail (Rosa et al. 1997). Methyl glucosinolate (glucocapparin) is the most common glucosinolate occurring in the Capparis genus (Ahmed et al. 1972b) but others have also been detected in and isolated from caper plants. Those include 2-propenyl glucosinolate

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(sinigrin), 3-methylsulfinylpropyl glucosinolate (glucoiberin), indol-3ylmethyl glucosinolate (glucobrassicin), and 1-methoxyindol-3-ylmethyl glucosinolate (neoglucobrassicin) (Ahmed et al. 1972a). In leaves and stems, aliphatic glucosinolates seem to be prevailing (Kjœr and Thomsen 1963), but methanolic extracts from roots were found to contain 4methoxyindol-3-yl-methyl glucosinolate (4-methoxy-glucobrassicin) (Schraudolf 1989). Thus, there are qualitative and quantitative differences in glucosinolate composition in different caper tissues, as happens with most glucosinolate-containing species (Rosa et al. 1997). Methyl glucosinolate was reported to be present at levels in the range of 38–268 ppm in capers treated with dry salt, brine, or oil (Sannino et al. 1991). An interference in the determination of dithiocarbamate residues in capers has been reported and seems to be due to the presence of methyl glucosinolate (Sannino et al. 1991). However, thiocyanates and isothiocyanates (odoriferous breakdown products of glucosinolates), as well as other volatile compounds, do not interfere in those pesticide tests (Brevard et al. 1992). Brevard et al. (1992) identified 160 components of pickled caper flavor, including elemental sulfur (S8) and more than 40 sulfur-containing compounds, among them thiocyanates and isothiocyanates. These authors also detected “raspberry-like” constituents: a- and b-ionine, frambinone, frambinyl alcohol, and zingerone. Two different 1H-indole-3-acetonitrile glycosides, 1H-indole-3acetonitrile 4-O-b-glucopyranoside and 1H-indole-3-acetonitrile 4-Ob-(6’-O-b-glucopyranosyl)-glucopyranoside (capparilosides A and B, respectively), have been isolated in methanolic extracts of caperberries (Çalis ¸ et al. 1999). B. Utilization 1. Food Use. Capers are recognized as a safe product when used as a spice for natural seasoning (Simon et al. 1984). A site in the Internet (http://food.epicurious.com) offers more than 250 recipes that include capers (CondéNet 2000), most of them compiled from specialized journals (Gourmet, Bon Appetit). Capers have a sharp piquant flavor and are mainly used as a seasoning to add pungency to: (1) sauces (e.g., tartare, remoulade, ravigote, vinaigrette, sauce gribiche, tarragon sauce, and caper sauce, for serving with lamb or mutton); (2) salads (e.g., caponata, a cold eggplant salad with olives and capers) and dressings; (3) cold dishes (vithel tohnné), or sauces served with salmon, herring, whiting, or turbot; (4) pizzas and canapés; (5) cheeses (e.g., liptauer cheese); and (6) lamb, mutton, pork or chicken preparations (Hayes 1961; Knëz 1970;

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Machanik 1973; Nilson 1974; Baccaro 1978; Stobart 1980). An unusual and complex organoleptic profile is responsible for caper flavor (Brevard et al. 1992). Caperberries and tender young shoots of the caper bush are also pickled for use as condiments, as previously described. The unripe seeds or pickled buds of other species (Tropaeolum majus L., Caltha palustris L., Cytisus scoparius (L.) Link., Zygophyllum fabago L., Euphorbia lathyrus L.) are sometimes suggested as substitutes for capers (Redgrove 1933; Vivancos Guerao 1948; Seidemann 1970; Mitchell and Rook 1979; Stobart 1980; Bond 1990). 2. Ornamental Use. Caper foliage is attractive but the sweet-scented flowers, with delicate white petals and long-projecting staments, give the caper plant most of its ornamental value (Bailey 1927; Baccaro 1978; Foster and Louden 1980). Thus, caper plant is most commonly used in ornamental plantings, for terraces exposed to the sun, borders, rocky gardens, and walls (Coutanceau 1957). Caper bush may be used as part of a strategy for reducing potential or actual erosion hazard (Lozano Puche 1977) along highways or pronounced rocky slopes, locations for which control is often more difficult than on farmland because many species used for erosion control do not survive the stressful conditions of the C horizon or without irrigation. Caper plant has low flammability (Neyis¸çi 1987) and thus may play a vital role in preventing forest fires. 3. Medicinal and Cosmetic Value. Most of the organs of the caper plant have been extensively used as folk remedies—sometimes as part of polyherbal formulations—for various diseases (Pernet 1972; Kirtikar and Basu 1975; Boulos 1983; Duke 1983; Jain and Puri 1984; Abbas et al. 1992; Husain et al. 1992; Al-Said 1993; Ghazanfar and Al-Sabahi 1993; Ghazanfar 1994; Bhattacharjee 1998). Recent reports appear to confirm some claims of these traditional formulations. Liv.52, an Indian traditional medicine that contains different plant extracts, among them 24% of C. spinosa, is a “liver stimulant” with some protective action against hepatotoxic substances, radiation sickness, and dermatitis. This herbal formulation brings about an hepatoprotective action by inhibiting lipid peroxidation and improving antioxidant levels (Suja et al. 1997; Vijaya Padma et al. 1998; Sandhir and Gill 1999). Liv.52 has an hepatoprotective effect against ethanol in that it reduces the hepatic binding of both ethanol and acetaldehyde (Dhawan and Goel 1994) and accelerates acetaldehyde elimination (Dhawan and Goel 1994; Chauhan and Kulkarni 1991a,b). Studies with rats have shown that it also prevents the deleterious effects of maternal ethanol ingestion on the fetus

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during gestation (Gopumadhavan et al. 1993). In a chronic model using rats, Liv.52 normalized the blood ethanol and acetaldehyde levels in a dose-dependent manner (Chauhan et al. 1994). Liv.52 has a beneficial effect on the activity of superoxide dismutase and glutathione levels (Sandhir and Gill 1999). Nevertheless, Fleig et al. (1997) found that Liv.52 does not improve survival of patients with alcoholic cirrhosis. Different fractions of an ethanolic extract of the root bark of caper plant significantly reduce the hepatotoxic activity of carbon tetrachloride (CCl4) in rats (Shirwaikar et al. 1996). Furthermore, p-methoxy benzoic acid isolated from the aerial parts of caper bush was found to prevent the hepatotoxic effects of both CCl4 and paracetamol in vivo, as well as the hepatotoxic activity of thioacetamide and galactosamine in isolated rat hepatocytes (Gadgoli and Mishra 1995, 1999). Similar effects in rats were found using Liv.52. It impairs the CCl4-mediated reduction in aniline hydrochloride and p-aminopyrine N-demethylase activity (Thabrew et al. 1982), as well as cathepsin-B, acid phosphatase, glucose-6phosphatase, and ribonuclease activity (Kataria and Singh 1997). On the other hand, it prevents the CCl4-mediated increase in different serum and liver hepatotoxicity markers (alkaline phosphatase, alanine transaminase, and aspartate transaminase) (Dhawan and Goel 1994) as well as CCl4- and H2O2-induced lipid peroxidation (Pandey et al. 1994; Suja et al. 1997). Liv.52 was found to down-regulate the tumor necrosis factor in CCl4-treated rats (Roy et al. 1994). Liv.52-treated rats also showed less marked toxic effects when beryllium (Mathur 1994) and mercuric chloride (Rathore and Varghese 1994) were administered. The ingestion of Liv.52 reduced the number and mass of DMBA- and croton oil-induced skin papillomas in male Swiss albino mice (Prashar and Kumar 1994). Liv.52 also showed in mice some antiviral activity against the Semlike forest encephalitis virus and enhanced the protective activity of 6MFA, an interferon-inducing antiviral substance (Singh et al. 1983). Nevertheless, many of the effects of Liv.52 may be due to non-caper constituents and the active compounds and precise mechanisms of action are still not clear. Caper root bark and leaves may have some anticarcinogenic activity (Hartwell 1968; Khan et al. 1992). In fact, the hydrolysis products of indol-3-ylmethyl glucosinolates have anticarcinogenic effects (Rosa et al. 1997). Although the consumption of capers is low in comparison with the intake of other major dietary sources of glucosinolates (white cabbage, broccoli, and cauliflower; Dragsted 1999) it may contribute to the daily dose of natural anticarcinogens that reduces cancer risk. Glucosinolates are also known to possess goitrogenic (anti-thyroid) activity

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(Rizk 1986; Rosa et al. 1997). Some non-nutrient components of capers are antioxidant compounds, e.g., quercetin, rutin, and kaempferol (Miller 1996; Pietta et al. 1996). Rutin and quercetin may contribute to cancer prevention (Committee on Comparative Toxicity et al. 1996). Selenium, present in capers at high concentrations in comparison with other vegetable products (Herrero Latorre et al. 1987), has also been associated with the prevention of some forms of cancer (Committee on Comparative Toxicity et al. 1996 and papers cited therein). On the other hand, linoleic acid has been reported to enhance carcinogenesis; but at high levels such as those found in capers (>16%), a reduction in cell proliferation has been reported in the mammary gland (Committee on Comparative Toxicity et al. 1996). The decoction of C. spinosa has hypoglycemic properties and may be useful in antidiabetic therapy (Ageel et al. 1985; Yaniv et al. 1987). The oral administration of a caper root decoction or tincture to guinea pigs revealed strong desensitizing effects against various plant and animal allergens (Khakberdyev et al. 1968). Cappaprenol-12, -13 and -14 in ethanol extracts of caper leaves are antiinflammatory compounds (AlSaid et al. 1988; Jain et al. 1993). Methanolic extracts of C. spinosa showed some antimalarial activity when assayed in vitro against a multi-drug resistant strain of Plasmodium falciparum (K1) (Marshall et al. 1995). Extracts of the whole plant or its aerial part also exhibited variable degrees of antimicrobial activity against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris, Bacillus cereus, and Bacillus subutilis, as well as antifungal activity against Candida albicans, Candida pseudotropicalis, and Fusarium oxysporum (Nadir et al. 1986; Mahasneh et al. 1996). Nevertheless, Ali-Shtayeh et al. (1998) found that aerial plant extracts only have antimicrobial activity against S. aureus and Proteus vulgaris, but fail to display antimicrobial activity against C. albicans, E. coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Many spices and their derivatives have antifungal properties but Capparis flower bud extracts did not show inhibitory effects on Aspergillus parasiticus mycelial growth (Özcan 1998). On the other hand, the aqueous extracts of the aerial part of caper plant prevented the growth of Microsporum canis, Trichophyton mentagrophytes, and Trichophyton violaceum (Ali-Shtayeh and Abu Ghdeib 1998). Thus, antidermatophytic activity in caper extracts is comparable with that of griseofulvin preparations (often used as a standard in evaluating antibiotic potential), suggesting a possible use against dermatophytic infections in humans.

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The green parts of caper plant have been considered to be potentially irritating to the skin because of its glucosinolates (Mitchell 1974; Mitchell and Rook 1979; Cronin 1980; Foussereau et al. 1982). Caper leaf and fruit extracts, applied as wet compresses to inflamed skin, may produce acute contact dermatitis (Angelini et al. 1991). Nevertheless, Lemmi Cena and Rovesti (1979) pointed out that caper extracts may be used for treating enlarged capillaries and dry skin. Barbera (1991) suggested that they could be utilized for cosmetic preparations (creams, shampoo, lotions, and gels), due to the presence of some active principles: rutin and quercetin (flavonoids that produce effects similar to those of vitamin P), pectins (moisturizing and protecting effects), glucocapparin (rubefacient action), phytohormones, and vitamins.

VII. INTERNATIONAL TRADE A major obstacle to a satisfactory analysis of the economics of capers— as is the case with many other spices—is the lack of statistical information. Accurate production figures are lacking for most of the exporting countries. Moreover, export-import statistics do not include those capers that are marketed in other processed products, with loss of identity (G. Chironi, in Barbera 1991; Sozzi 2000). On the other hand, trade statistics are the only source of information on consumption in many regions of Europe, where capers are often produced for local or even household use. Caper commercial exchange involves more than 60 countries. Nowadays, the average annual production may be estimated to be around 10,000 t: 3,500–4,500 t are produced in Turkey, 3,000 t in Morocco, 500–1,000 t in Spain, and 1,000–2,000 t in other countries. The most complete time series reflecting the international trade is that of United States imports (Figs. 4.5 and 4.6). The United States is one of the most important consumer countries. Based on United States statistics, it may be concluded that: 1. Increasing amounts of capers are being consumed (Fig. 4.5), and this trend appears to be sustained for the next few years, the expanding ethnic populations and the interest in new tastes presumably accounting for most of the increase in caper consumption. 2. The Spanish and Italian production has increasingly been exposed to the international competitive influence of Turkey and Morocco and current prices have been on a downward trend. The decline in prices is more dramatic if inflation is taken into account, and the

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2400 2200

Turkey

2000

Spain

Tonnes

1800 1600

Morocco

1400

Others

1200 1000 800 600 400 200

99 19

97 19

95

93

19

91

19

89

19

19

87

85

19

83

19

81

19

19

79

77

19

75

19

73

19

19

19

71

0

Year Fig. 4.5. United States imports of capers from major producing countries (1971–1999). Prepared with data from the USDA, Foreign Agricultural Service (1973/2000).

8 1,000 Dollars per Tonne

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Spain Morocco Turkey Average

6 5 4 3 2 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 Year

Fig. 4.6. United States import prices for capers during the last decade. Prices are on a current basis. Prepared with data from the USDA, Foreign Agricultural Service (1991/2000).

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current prices are adjusted (i.e., using the consumer price index). On the other hand, caper quality and presentation are recognized by traders and higher prices are paid for the product made in Spain (Fig. 4.6) or Italy. In fact, the French and Greek products are generally even more expensive. Very small amounts are marketed and they are bought by traders concerned with securing a high-quality supply. 3. The caper trade is very dynamic, with imports and subsequent exports often combined. Turkey’s main markets for caper exports are Spain, the United States, France, Italy, Germany, The Netherlands, Brazil, United Kingdom, Belgium, Venezuela, Japan, Denmark, and Israel. Denmark does not produce capers but exports larger amounts to the United States than Italy. And the United States, whose production is negligible, exports some of its imports to other countries, e.g., Venezuela. Morocco also exports capers to Spain and Italy, traditional producers that devote most of their own production to exportation.

VIII. CONCLUDING REMARKS Caper bush has a unique and interesting biology but few scientific reports have been published to unravel its mechanisms of growth and survival in harsh and stressful environments. Apart from environmental conditions, success in caper bush cultivation depends mainly on five fundamental points: (1) biotypes of high quality and production; (2) adequate propagation; (3) good control of cultivation practices, particularly harvest; (4) adequate postharvest processing and storage; and (5) efficient marketing systems and strategies. In low-input systems with both low land and labor costs, the caper plant can provide the diversity required for sustainability. On the other hand, caper yields are much higher in irrigated plantings, with NPK fertilization, although much more research is required to determine the optimal cultivation conditions for this species. Diseases and pests do not seem to be a great problem in general but also need to be researched. Two major expenses are expected: implantation and harvesting. The latter may be the stumbling block in high-input systems, and the possibility of a semi-mechanical operation should be considered in order to remove this limiting factor. Moreover, further improvement in caper quality may be obtained by regulating harvesting dates. There is an assortment of opportunities for plant breeders to contribute to domestication of caper bush for agricultural purposes. Determination of the genetic bases for productivity, ease of propagation,

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absence of stipular spines, and flower bud quality and conservation are high-priority research needs. Finally, marketing research remains an area of high concern. Marketing of capers without a pre-arranged contract with processing or exporting companies could be very risky. Market promotion and the ability of handlers to provide a high-quality product at times that will yield a competitive price have become essential factors. Producers and handlers will be challenged to develop new and expanded markets for capers. Many ethnic foods have filtered into the diets of Americans and other developed countries. A global cuisine is coming, for which the world’s food diversity should be available to everybody. Caper plant, a simple drought-tolerant perennial bush that grows in semiarid areas, has a favorable influence on the environment, stabilizes eroding slopes, helps to prevent forest fires, benefits some rural economies, provides medicinal and cosmetic compounds, and brings a unique flavor to our meals, may play a significant role in the international spice trade in the future. LITERATURE CITED Abbas, J. A., and A. A. El-Oqlah. 1992. Distribution and communities of halophytic plants in Bahrain. J. Arid. Environ. 22:205–218. Abbas, J. A., A. A. El-Oqlah, and A. M. Mahasneh. 1992. Herbal plants in the traditional medicine of Bahrain. Econ. Bot. 46:158–163. Ageel, A. M., M. Tariq, J. S. Mossa, M. S. Al-Saeed, and M. A. Al-Yahya. 1985. Studies on antidiabetic activity of Capparis spinosa. Federation Proc. 44:1649 (7243). Ahmed, M. 1986. Vegetation of some foothills of Himalayan range in Pakistan. Pak. J. Bot. 18:261–269. Ahmed, M., and S. A. Qadir. 1976. Phytosociological studies along the way of Gilgit to Gopies, Yasin and Shunder. Pak. J. Forestry 26:93–104. Ahmed, Z. F., A. M. Rizk, F. M. Hammouda, and M. M. Seif El-Nasr. 1972a. Glucosinolates of Egyptian Capparis species. Phytochemistry 11:251–256. Ahmed, Z. F., A. M. Rizk, F. M. Hammouda, and M. M. Seif El-Nasr. 1972b. Naturally occurring glucosinolates with special reference to those of family Capparidaceae. Planta Med. 21:35–60. Ahmed, Z. F., A. M. Rizk, F. M. Hammouda, and M. M. Seif El-Nasr. 1972c. Phytochemical investigation of Egyptian Capparis species. Planta Med. 21:156–160. Akgül, A., and M. Özcan. 1999. Some compositional characteristics of caper (Capparis spp.) seed and oil. Grasas Aceites 50:49–52. Ali-Shtayeh, M. S., and S. I. Abu Ghdeib. 1998. Antifungal activity of plant extracts against dermatophytes. Mycoses 42:665–672. Ali-Shtayeh, M. S., R. M. R. Yaghmour, Y. R. Faidi, K. Salem, and M. A. Al-Nuri. 1998. Antimicrobial activity of 20 plants used in folkloric medicine in the Palestinian area. J. Ethnopharmacol. 60:265–271. Alkire, B. 1998. Capers. (http://www.hort.purdue.edu/newcrop/CropFactSheets/caper.html). Allen, B. (ed.). 1994. Food: an Oxford anthology. Oxford University Press, Oxford-New York. p. 43, 48.

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Schlein, Y., and R. L. Jacobson. 1994b. Mortality of Leishmania major in Phlebotomus papatasi caused by plant feeding of the sand flies. Am. J. Trop. Med. Hyg. 50:20–27. Schlein, Y., and R. L. Jacobson. 2000. Photosynthesis modulates the plant feeding of Phlebotomus papatasi (Diptera: Psychodidae). J. Med. Entomol. 37:319–324. Schlein, Y., and G. Muller. 1995. Assessment of plant tissue feeding by sand flies (Diptera: Psychodidae) and mosquitoes (Diptera: Culicidae). J. Med. Entomol. 32: 882–887. Schlein, Y., and A. Warburg. 1986. Phytophagy and the feeding cycle of Phlebotomus papatasi (Diptera: Psychodidae) under experimental conditions. J. Med. Entomol. 23:11–15. Schlein, Y., A. Warburg, and B. Yuval. 1986. On the system by which sandflies maintain a sterile gut. Insect Sci. Applic. 7:231–234. Schlein, Y., and B. Yuval. 1987. Leishmaniasis in the Jordan Valley. IV. Atraction of Phlebotomus papatasi (Diptera: Psychodidae) to plants in the field. J. Med. Entomol. 24:87–90. Schola Salernitana (School of Salerno). (9th?–13th? centuries). Regimen sanitatis: flos Medicinae Scholae Salerni. (in Italian and Latin). A. Sinno (transl.). U. Mursia, Milano, 1987. p. 160–161, 430–431. Schraudolf, H. 1989. Indole glucosinolates of Capparis spinosa. Phytochemistry 28:259–260. Scialabba, A., S. Fici, and M. Sortino. 1995. Capparis spinosa L. var. canescens Cosson in Sicily: seed ecomorphology and germination. Giornale Botanico Italiano 129(2):30. Seidemann, J. 1970. Kapern (Capparis spinosa L.). Quarterly J. Crude Drug Res. 10:1516–1523. Sen Gupta, A., and M. M. Chakrabarty. 1964. Composition of the seed fats of the Capparidaceae family. J. Sci. Food Agr. 15:69–73. Serres, O. (1539–1619). Le théâtre d’ agriculture et mesnage des champs. Société d’agriculture du département de la Seine, Paris, 1805. Sharaf, M., M. A. El-Ansari, and N. A. M. Saleh. 1997. Flavonoids of four Cleome and three Capparis species. Biochem. Syst. Ecol. 25:161–166. Sharaf, M., M. A. El-Ansari, and N. A. M. Saleh. 2000. Quercetin triglycoside from Capparis spinosa. Fitoterapia 71:46–49. Shirwaikar, A., K. K. Sreenivasan, B. R. Krishnanand, and A. Vasanth Kumar. 1996. Chemical investigation and antihepatotoxic activity of the root bark of Capparis spinosa. Fitoterapia 67:200–204. Simon, J. E., A. F. Chadwick, and L. E. Craker. 1984. Herbs: an indexed bibliography, 1971–1980: the scientific literature on selected herbs, and aromatic and medicinal plants of the temperate zone. Archon Books, Hamden, CT. p. 13. Singh, R. P., N. Bahar, and P. Chand. 1992. Autecology of Capparis spinosa Linn. in cold desert of Spiti Valley in Himachal Pradesh. Ann. Arid Zone 31:291–293. Singh, V. K., C. X. George, K. P. Gupta, and B. M. Gupta. 1983. Antiviral activity of plant extract Liv 52 in mice experimentally infected with Semliki forest encephalitis virus. Sci. Cult. (India) 49:354–356. Söyler, D., and N. Arslan. 1999. Effect of heat, light and dark treatments on seed germination of caper (Capparis spinosa L.) (in Turkish, summary in English). Anadolu 9:63–75. Sozzi, G. O. 2000. Alcaparras: un aderezo mundial. Énfasis Alimentación 6:104–110. Sozzi, G. O., and A. Chiesa. 1995. Improvement of caper (Capparis spinosa L.) seed germination by breaking seed coat-induced dormancy. Sci. Hort. 62:255–261.

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5 Water Relations and Irrigation Scheduling in Grapevine* M. H. Behboudian Institute of Natural Resources College of Sciences Massey University Palmerston North, New Zealand Zora Singh Department of Horticulture Muresk Institute of Agriculture Curtin University of Technology GPO Box U 1987 Perth, WA 6845, Australia

I. INTRODUCTION II. PHENOLOGY III. ASPECTS OF WATER RELATIONS A. The Soil-Plant-Atmosphere Continuum B. Plant Roots and Water Absorption C. Transpiration 1. Energy Supply and Leaf Interception 2. Vapor Pressure Deficit 3. Wind Speed 4. Resistance to Transpiration D. Development of Water Deficit: Measurement and Recovery E. Plant Responses to Water Deficit 1. Plant Water Status 2. Stomatal Conductance *We are grateful to Dr. Tessa Mills (HortResearch, New Zealand) for constructive discussion and for commenting on the manuscript. We thank Dr. Stephen Lawes (Massey University, New Zealand) for critical comments on the manuscript. Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 189

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3. Photosynthesis 4. Plant Growth F. Acclimation of Plants to Water Deficit 1. Osmotic Adjustment 2. Stomatal Behavior IV. IRRIGATION OF VINEYARDS A. Regulated Deficit Irrigation (RDI) 1. Timing of RDI Application 2. Early RDI: Budburst to Flowering 3. Mid-season RDI: Fruit Set to Veraison 4. Late-season RDI: Veraison to Harvest 5. Postharvest RDI 6. Degree of Deficit B. Partial Rootzone Drying (PRD) V. QUALITY ATTRIBUTES FOR WINE, DRIED, TABLE, AND JUICE GRAPES A. Wine Grapes B. Dried Grapes—Raisins C. Table Grapes D. Juice Grapes VI. FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION The grape, associated with humans since prehistory, spread throughout the world in antiquity both for fresh fruit and wine production (Smart and Coombe 1983). Grapevine, mainly Vitis vinifera but also V. labruska and other species, is now the most widely grown fruit plant in the northern and southern hemispheres (Grimes and Williams 1990). In 1999, grapes were grown on 7.5 million hectares worldwide, with production estimated at 58.7 million tonnes (Table 5.1). Generally Vitis is a temperate plant but is successfully grown in Mediterranean and subtropical climates and can be grown as an evergreen where the temperature is consistently in the range of 20°C to 30°C and where rainfall patterns have a high degree of reliability (Possingham 1994). There are approximately 35,000 ha of grapevines grown in the tropics and tropical viticulture is characterized by the production of two to three crops per vine each year (Araujo et al. 1999). At present, more than 80% of the world’s grapes are used for wine production (Mullins et al. 1992). The preferred wines are mainly cultivated in the areas with low annual precipitation. While the traditional areas of grape growing are non-irrigated, irrigation greatly increases yield. As an excess of moisture often decreases wine quality, many traditional wine growing areas in Europe have legally limited irrigation or even pro-

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Table 5.1. Area and production of grapes in different parts of the world in 1999. Source: FAO STAT 1999 (http://www.FAO.org/)

Continent Europe Asia South America North and Central America Africa Oceania Total (World)

Area (millions of ha)

Production (millions of tonnes)

4.5 1.7 0.5 0.4 0.3 0.1 7.5

29.7 13.2 5.2 6.5 3.0 1.1 58.7

hibited it both to insure wine quality and to reduce overproduction. At present, irrigation in many European areas may only be used in an emergency to save vines in times of drought. Nevertheless, the judicious use of irrigation in grape production is now an established practice in many non-European countries. Previous reviews related to water relations in grapevine and stress physiology include chapters in books on crop physiology or irrigation science. Plant and environmental factors affecting water relations in grapevine have been reviewed by Smart and Coombe (1983) and Williams and Matthews (1990), while stress physiology has been covered by Williams et al. (1994). The scope of this presentation is to review new information on basic and applied aspects of water relations in grapevine. It introduces some modern technologies used in studying water relations in general and specifically in grapevine, irrigation scheduling, and describes some of the known effects of deficit irrigation on fruit and wine quality. Except where specifically mentioned, all grapes refer to Vitis vinifera.

II. PHENOLOGY Phenology of the grapevine is similar to other deciduous fruit. In late summer or autumn the plant enters dormancy characterized by leaf senescence and abscission, and lack of visible bud growth, which allows the plants to survive cold winter temperatures. After exposure to sufficient chilling, growth is resumed the following spring (Lavee and May 1997). In the humid tropics grapevine behaves as an evergreen (Possingham 1994). In temperate and subtropical regions that have mild

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winters, budbreak is erratic and less uniform than in environments with lower winter temperature. Water uptake precedes rapid shoot growth and is followed by budbreak and by flower differentiation within the cluster primordia that continues up to anthesis. In California, anthesis occurs approximately eight weeks after budbreak (Williams et al. 1994). In contrast to many deciduous fruit trees, vegetative growth of grapevines precedes flowering and fruit growth. The degree of overlapping between vegetative growth and fruit growth varies among cultivars. Fig. 5.1 shows some growth parameters of ‘Thompson Seedless’ growing in the San Joaquin Valley of California as a function of growing degree days (GDD) (Williams and Matthews 1990; Williams 1987). Grape cultivars tend to develop at consistent rates relative to other cultivars regardless of seasonal conditions (Williams and Matthews 1990). Some irrigation scheduling is done according to the stages of fruit growth, a double sigmoid curve (Matthews et al. 1987a). Three growth stages are recognizable on the curve (Fig. 5.2). Stage I is the initial phase

Fig. 5.1. Changes in dry weight, leaf area, and soluble solids of ‘Thompson Seedless’ grapevines grown in the San Joaquin Valley of California as a function of growing degree days (GDD> 10°C). Average date of budbreak was March 9. Adapted from Williams and Matthews (1990) based on the data of Williams (1987).

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Fig. 5.2. Changes in fruit diameter for Vitis vinifera cv ‘Cardinal’ made on two berries (open and closed circles) for 70 days after anthesis. Stages I, II, and III are explained in the text. Adapted from Matthews et al. (1987a).

of rapid growth and stage II is the lag phase of slow or no growth. At the start of stage III, berries resume their final phase of growth and maturation. The transition from stage II to III (veraison) is characterized by many physiological changes, most of which could occur rapidly, i.e., within 24–48 hours.

III. ASPECTS OF WATER RELATIONS Grapevines are frequently grown in areas where water supply limits optimum growth and production. Thus, information on the physiology of water relations, the assessment of water status, and the basic plant responses to reduced water status is important to optimize water use. A. Soil-Plant-Atmosphere Continuum In the soil-plant-atmosphere continuum, water in liquid form moves through the soil to plant roots, is absorbed by the roots, and transported

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through the stem to the leaves, where it is lost to the atmosphere as vapor. Important processes in the pathway include: water absorption by plant roots, water movement within the plant, and loss of water vapor from the leaves to the atmosphere (transpiration). Along the pathway, water movement is driven by the gradients of water potential in that water moves from the phase of high to low potential and is retarded by a series of resistances present in the system (Cowan 1965). B. Plant Roots and Water Absorption Root factors that contribute to absorption efficiency are root density and distribution, maximum root depth, and rate of growth. The higher the root density, the larger the volume of soil water that can be utilized. The distribution of grapevine roots has been studied extensively for various grape-growing areas of the world (Richards 1983). The distribution depends both on species and on soil properties. For the majority of soils, most of the roots occur in the top 1000 mm, although individual roots might penetrate to depths of 6000 mm or more (Richards 1983). The fine lateral roots, which are thought to form the main absorptive area, occur in the top 100 to 600 mm of the soil. Barriers such as compacted layers, water tables, and saline and acid zones restrict root depth (Richards 1983). Mapfumo et al. (1994) found that in ‘Shiraz’ water flow into the main roots via the lateral roots is likely to be much smaller than that via the direct radial flow pathway through the main roots. Only about 1% of surface area of main roots is directly occupied by lateral roots, leaving the other 99% of main root surface area available for the direct radial flow pathway. Irrigation and mulching affect root distribution. In drip irrigated vines there is a confined soil wetted zone beneath the emitter that largely coincides with a confined and shallow root system, while furrow irrigated vines have a deeper and more widespread root system (Araujo et al. 1995). Black plastic mulch doubled the root:shoot ratio and berry weight of ‘Chenin Blanc’. The increased growth of mulched vines was attributed to improved weed control and to conservation of soil moisture, more uniform soil temperatures, and less soil compaction (Van der Westhuizen 1980). C. Transpiration Grape berries have stomata whose activity decreases with fruit age. On a surface area basis, leaf transpiration rate is 2.5–10 times higher than that of the grape berries (Blanke and Leyhe 1987). Various environ-

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mental and plant factors that control transpiration have resulted in different rates reported throughout the world. Over 100 days, between anthesis and harvest, the cumulative sap flow of 3-year-old ‘Chardonnay’ plants, which is presumed to be the same as the transpiration rate, was 461 ± 44 kg plant–1 or 124 ± 12 mm on a land area basis (Lascano et al. 1992). This was exclusive of soil evaporation, which was 77% of evapotranspiration (ET). Myburgh et al. (1996) found the same rate of transpiration for three-year-old ‘Pinot Noir’ growing in the coastal region of the Western Cape in South Africa. Schmid and Braun (1997) used the heat balance method for measuring the rate of transpiration of ‘Riesling’ on four different rootstocks and obtained higher values of 2 to 2.5 mm day–1, on a land area basis, in Germany, which is expected to have a lower evaporative demand than Texas and the Western Cape. Possibly the plants were older in Schmid and Brown’s experiment or daylengths were significantly longer. 1. Energy Supply and Leaf Interception. Solar radiation is the major source of the energy required to evaporate water from transpiring leaves and has a direct effect on stomatal aperture, photosynthesis, and leaf temperature, with the latter influencing vapor pressure deficit. In addition to direct solar radiation, other sources of energy include re-radiation from the soil and surrounding objects and sensible heat that flows between the leaf and the environment. In grapevines, seasonal changes in leaf area thus influence seasonal water loss through transpiration. Transpiration is low during the dormant period and becomes considerably higher during the active growth period, especially when leaves are fully developed and during fruit growth. Leaf area rapidly increases during the spring until reaching the maximum value, then becomes stable and later decreases (Fig. 5.1) as the leaves start to fall during autumn. The actual pattern of leaf area development in grape during the season, however, varies among cultivars and training systems used. 2. Vapor Pressure Deficit. Vapor pressure deficit (VPD), which is the driving force for water vapor to move from the leaf to the air, is the difference between the vapor pressure of the leaf and the vapor pressure of water in the bulk air (the humidity). Over the normal range of leaf water potentials, the vapor concentration of the air inside the leaf is nearly constant and very close to saturation (Wenkert 1983). A large change in cell water potential causes only a small change in its vapor pressure. For example, at 20°C, as the leaf water potential drops from 0 to –2.7 MPa, the vapor concentration drops from 100 to 98% of saturation (Wenkert

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1983). Thus VPD is largely determined by the water content of the outside air and the temperature difference between the leaf and the air. Temperature is important because a certain volume of the air can hold more water as its temperature increases. When other factors are equal, transpiration rate is proportional to the value of absolute humidity difference or vapor pressure difference between the leaf and the air (Kramer and Boyer 1995, page 210). In addition, VPD also has a direct effect on stomatal movement. Stomatal closure in response to the dryness of the air (high VPD) regardless of the leaf water potential has been observed in various species including V. vinifera. Williams et al. (1994) cited the literature covering the reactions of V. vinifera cultivars to VPD and state that these reactions are cultivar dependent and a high VPD seems to cause non-uniform stomatal closure in Vitis species. The effect of VPD on stomatal closure seems to be more severe if plants are undergoing water stress as shown in Fig. 5.3 (Williams et al. 1994). For ‘Thompson Seedless’ the higher stomatal conductance occurred when plants were watered at 100% of ET followed by those watered at 60% and 20% of ET (Fig. 5.3). For each watering regime stomatal conductance became

Fig. 5.3. The relationship between stomatal conductance of ‘Thompson Seedless’ grapevine and vapor pressure deficit for three irrigation treatments replacing 100% of ET (1.0), 60% of ET (0.6), and 20% of ET (0.2). Adapted from Williams et al. (1994).

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lower with increasing VPD. The lowering of stomatal conductance with increasing VPD might have implications for assimilation rates and the subsequent growth of the plants. However, there are not conclusive data in the literature to warrant such an assessment for grapevine. 3. Wind Speed. Wind seems to have opposite effects on transpiration. As wind speed increases, the thickness of the boundary layer surrounding the leaf or the canopy decreases, lowering the layer’s resistance and leading to a higher rate of transpiration. Wind may also increase transpiration rate by adding energy to evaporate water from the flow of sensible heat. However, wind also cools the leaves, resulting in a decrease in vapor pressure gradient from leaf to air. Studies carried out in the field and laboratory indicate that winds with a speed higher than 3 m s–1 will decrease stomatal conductance and transpiration (Williams et al. 1994). The effect on stomatal conductance could be both through increased production of abscisic acid (ABA) in the leaf and/or mechanical damage to the leaf. In addition to transpiration, wind influences assimilation, growth, and yield. The effect of a windbreak on ‘Chardonnay’ in California is shown in Fig. 5.4 (Williams et al. 1994). The vines in the control treatment, which were exposed to higher wind speeds, had lower stomatal conductance and lower assimilation rate than the vines protected by the windbreak. Dry and Botting (1993), in a study of the effect of windbreak on the performance of ‘Cabernet Franc’, found shoot length and pruning weight of sheltered vines averaged 59% more than exposed vines. The fruit yield increased by 15% in sheltered vines as a result of increased bunches per shoot and increased shoot and bunch number per row. 4. Resistance to Transpiration. The total resistance of a leaf to transpiration includes the resistance of a leaf’s air boundary layer and the leaf resistance, which is composed of stomatal and cuticular resistance. Air boundary layer resistance (ra) is the resistance to vapor diffusion across the laminar boundary layer. Water vapor from the leaf surface moves through the boundary layer mostly by diffusion and therefore it happens faster through a thinner layer (Jones et al. 1985). Boundary layer resistance of fruit orchards is usually lower than that of the field crops at a certain wind speed, due to the aerodynamically rough and nonhomogeneous surface of the fruit orchards (Jones et al. 1985). Stomatal resistance (rs), and its reciprocal stomatal conductance (gs), is regulated mainly by the size of stomatal aperture such that rs increases as the aperture decreases. Stomatal aperture is the major passage for

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Fig. 5.4. The effect of windbreak on the diurnal course of CO2 assimilation rate, stomatal conductance, and wind speed of ‘Chardonnay’ grapevine grown in the Salinas Valley of California. Adapted from Williams et al. (1994).

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water vapor movement out of the leaf and for CO2 movement from the air into the leaf. Because gs is affected by leaf water status, attempts have been made to correlate it with leaf water potential (ψ) for different species. For grapevine a linear relationship was observed between ψ and gs of ‘Thompson Seedless’ (Williams et al. 1994), although a clear relationship between ψ and gs could not be determined for ‘Colombard’ (Van Zyl 1987). For grapevine the relationship seems to depend on the cultivar, environmental conditions, and the rate at which water stress develops. This inconsistent relationship in grapevine might also be related to non-uniform stomatal closure over the leaf surface, i.e., stomatal patchiness, which is characteristic of grapevine in response to water stress (Downton et al. 1987). Düring and Loveys (1996) recommend that in measuring the gas exchange of grapevine leaf, areas that include both patches with open and closed stomata should be used to counteract the variability of gas exchange rates. The effect of chemical messages from roots on stomata during soil drying will also affect the relation between ψ and gs. The effectiveness of stomata in controlling canopy transpiration depends on the state of air boundary resistance. Stomatal movement affects transpiration only when the canopy is closely coupled to the atmosphere in terms of water vapor pressure (Hsiao 1990). A closely coupled canopy refers to the canopy in which the temperature and the water vapor in the boundary layer and the air stream outside the boundary layer are very similar, resulting in a very small gradient in temperature and water vapor pressure between the two locations. In contrast, a poorly coupled canopy has a relatively thick boundary layer; thus water vapor exchange between the canopy and the ambient air above the canopy does not occur rapidly and stomatal movements will not have an effective role in transpiration. Most fruit trees, including grape, are considered to have a closely coupled canopy. D. Development of Water Deficit: Measurement and Recovery The term water deficit implies that water status is less than the optimum value for plant growth and development. Plant water deficit occurs when water absorption lags behind transpiration. Thus excessive transpiration, slow absorption, or their combination can lead to plant water deficit. Plant water deficit is characterized by a decrease in plant water content, turgor, and total water potential resulting in wilting, partial or complete stomatal closure and a decrease in cell enlargement and plant growth (Hsiao 1973). Determination of plant water deficit can therefore

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be done through measurements of plant water content, plant water potential and its components, trunk diameter fluctuations, sap flow, and stomatal aperture. Generally, a plant routinely experiences water deficit both diurnally and seasonally. During the day, absorption often lags behind transpiration due to resistance to water flow in the system (Williams and Matthews 1990). The rate of replenishment at night is dependent upon water availability in the soil and the efficiency of water absorption and the water conducting system of the plant. In dry soils, the deficit persists and increases in magnitude with time as the plant is not recovered at night. Leaf water potential (ψ) and its components of turgor and osmotic potentials have been widely used as indicators of plant water status and as a measure of plant water deficit. A diurnal variation in ψ, which indicates the strong influence of evaporative demand on plant water status, has been observed in most plant species, including grapevine (Fig. 5.5). McCutchan and Shackel (1992) advocated the assessment of predawn ψ as the measure of water stress in plants due to the strong influence of environmental conditions on ψ during the day. However, the predawn ψ of the plant may not indicate the soil-water status over the entire root zone because it tends to be biased towards the water status of the wettest part (Jones 1990). In some cases leaf ψ has been found not to be sensitive enough as a measure of plant water status. Stem water potential (ψstem) has been suggested as an indicator of plant water status (Naor et al. 1995). Stem water potential can be measured by enclosing the leaf (inside the canopy) in a plastic bag and covering it with aluminum foil while it is attached to the plant. After the water status of the leaf and of the stem has reached an equilibrium, proposed to be 90 min, the leaf is removed and the water potential measured (Naor et al. 1995). Naor (1998) measured stomatal conductance, ψ, and ψstem on grapevine as well as apple and nectarine from early morning to midafternoon under several irrigation treatments and showed that ψstem was more sensitive to irrigation level than was ψ. Stem water potential correlated better with gs than did ψ. He proposed a model in which gs, ψ, ψroot, root signal intensity, and transpiration rate are linked in a feedback mechanism that leads to a higher correlation of gs with ψstem , and the correlation is higher than with ψ. Sipiora and Lissarrague (1999) reported that for ‘Tempranillo’ vines diurnal changes of gs and ψ were linearly related only when the midday data were analyzed separately from those obtained earlier and later in the day. They concluded that changes in ambient light and CO2 were involved in the observed stomatal responses. Due to the difficulty of relating ψ to metabolic processes, Sinclair and

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Fig. 5.5. Diurnal changes in water potential of leaves for fruiting (+F) and defruited (–F) ‘Riesling’ grapevine measured on two separate occasions: February 1, 1985 (a) and February 15, 1985 (b). Vertical bars represent average least significant differences between means at P = 0.05. Adapted from Downton et al. (1987).

Ludlow (1985) proposed that plant water status should be measured in terms of cell volume change, expressed as relative water content (RWC). The disadvantage of RWC is that it varies with plant species, age, and habitat; and it cannot be related to soil water status (Kramer 1988). Because measurements of both water potential and RWC are usually destructive and cannot be done continuously, other parameters that can

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be measured continuously and are non-destructive, such as stem diameter and sap flow, have been proposed as indicators of plant water status. Stem diameter, measured using a linear variable differential transducer (LVDT), changes rapidly in close correlation with plant water content (e.g., Simmonneau et al. 1993). Cohen et al. (1993) demonstrated that sap flow, measured by the heat-pulse method, is more sensitive to water deficit than is predawn ψ. Fluctuations in environmental conditions at the time of measurement may mask the effects of water stress on the above parameters. The measure of a plant’s discrimination against 13CO2 will be another tool to assess the extent of water stress. The discrimination is a measure of internal physiological and environmental parameters influencing the performance of the plant throughout the season and not subject to prevailing conditions at the time of sampling (Meinzer et al. 1991). This technique relies on the fact that due to discrimination against 13CO2 during photosynthesis, the ratio of 13C:12C in plants is lower than that found in the atmosphere. Organic matter is often depleted in 13C relative to the standard against which it is compared. Therefore values of δ13C (the notation used for expressing 13C discrimination) are negative because: δ13C = ((Rsample/Rstandard) – 1) × 1000,

[1]

where R is the ratio of 13C:12C. Rstandard is for Pee Dee belemnite (Farquhar et al. 1982). Assuming isotopic composition of atmospheric CO2 does not vary, a less negative figure represents less discrimination (higher 13C concentration in the tissue) against 13CO2 during photosynthesis. Water stress results in less discrimination against 13CO2, or less negative values of δ13C, as exemplified by apple leaves (Mills et al. 1998) and chickpea pods and seeds (Behboudian et al. 2000). For grapevine, values of δ13C have been reported only twice in the literature and those reported by Gaudillere et al. (1999) are related to water stress effects. They reported that for Angers and Bordeaux areas of France, the δ13C values in berry of non-stressed grapevines (cultivar not specified) were, respectively, –24.6 ± 0.8 and –25.0 ± 0.8. The corresponding values for the dry plots were –21.7 ± 0.6 and –23.3 ± 0.6. The authors found that different scion cultivars differed in δ13C values, as did different rootstocks. They concluded that 13C discrimination could be used for assessing water stress effects in grapevine, as has also been done for some other species. This technique could be of value when assessing long-term effects of different deficit irrigation regimes on grapevine. The effects of water deficit are enhanced with increased crop load as indicated for grapevine (Downton et al. 1987), ψ is lower in fruited

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grapevines compared with de-fruited vines, or in heavy crop load compared with light crop load (Fig. 5.5). The effects of crop level on tree water relations may be due to an increase in tree water use with the presence of fruit or with higher crop level. A concomitant increase in transpiration via increased gs also occurs when fruit is present. This fruit effect may also be due to decreased root growth in a heavily cropping vine. Roots will be receiving less assimilates because of competition by fruit leading to a reduction in plant water uptake capacity as suggested by Lenz (1986). If water deficit is not too long or too severe, plants generally are able to recover following full irrigation. The time and degree of recovery from water deficit upon re-watering varies among plant species and cultivars and depends on the degree and duration of deficit treatment. Plants with high hydraulic conductivity may be expected to recover faster than those with lower hydraulic conductivity (McAneney and Judd 1983). Gucci et al. (1996) found that recovery of gs, CO2 assimilation, and transpiration rate of a kiwifruit vine was complete within 36 h of re-watering after the first drought cycle, while the recovery was slower during the second drought cycle when the deficit was more severe. Net photosynthetic rate and ψ was found to recover only partially after re-watering of severely stressed olive trees (Angelopoulos et al. 1996). E. Plant Responses to Water Deficit Plant responses to water deficit are numerous and somewhat complex. A significant amount of research on such effects has been conducted over recent years, often in conjunction with the study of deficit irrigation (Behboudian and Mills 1997). Deficit irrigation may be defined as a system of managing soil water supply to impose specific periods of plant water deficit to elicit some desirable responses in plants. When reference to deficit irrigation is made throughout this section, it is assumed that plant water status was reduced during the deficit irrigation period. 1. Plant Water Status. As the rate of water loss often exceeds that of water uptake, especially during the middle of the day, the plant becomes increasingly exposed to water deficit leading to a decreased water status. Changes in evaporative demand that occur during the day may also cause short periods of water deficit. In cordon-trained ‘Roditis’ grape with north-south orientation of the rows, water potential was lower on the eastern leaves throughout the day, but the rate of photosynthesis and transpiration was higher than that of the western leaves (Patakas 1993).

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This was attributed to a maintenance of turgor potential by active accumulation of solutes in the eastern leaves through the process of osmoregulation. 2. Stomatal Conductance. It was initially thought that stomatal closure often observed with a decrease in ψ was caused by the effect of ψ on guard cell turgor. However, it now appears that the mechanism behind stomatal closure may be more complex. The involvement of hormones such as abscisic acid (ABA) (Tardieu and Davies 1993) and cytokinins (Golan et al. 1986) that are modified under conditions of water deficit may play a role. In ‘Concord’ grapevines (V. labrusca) leaf water potential in irrigated and non-irrigated plants was similar during the day (–1.0 to –1.6 MPa and –1.3 to –1.6 MPa, respectively) but assimilation rate and stomatal conductance of the non-irrigated vines were significantly lower than the irrigated controls (Naor and Wample 1994). Stomatal closure was attributed to root signals arising from the drier soil in the non-irrigated plants. 3. Photosynthesis. Some recent data indicate that a reduction in stomatal conductance does not always fully account for decreases in photosynthesis under water deficit (Flore and Lakso 1989). Additionally, the sensitivity of photosynthesis to reduced plant water status varies between and within species and appears dependent on the pre-treatment these plants have received. Quick et al. (1992) found that potted grapevines may show a non-stomatal response causing reduction in photosynthesis with the onset of water deficit. However, the detailed studies of Flexas et al. (1998) with field-grown ‘Tempranillo’ showed that stomatal conductance was the main reason for reduction in photosynthesis under water deficit. Based on their measurements of chlorophyll fluorescence and gas exchange rates, the authors concluded that photoinhibition and disruption of electron transport rate were not the main cause of reduction in photosynthesis of the vines undergoing mild water stress in the field. The discrepancy of results between these experiments might be due to the rate at which stress was developed in plants, which was slower in Flexas’s experiment than that of Quick. The cultivar difference could have also played a role, as exemplified by the study of Schultz (1996), who investigated the adaptive responses to water deficit of ‘Grenache’, of Mediterranean origin, and ‘Syrah’ of mesic origin. Stomatal conductance and photosynthesis were more sensitive to water stress in ‘Grenache’ than in ‘Syrah’. Chlorophyll fluorescence measurements showed higher sensitivity of the former cultivar to water stress than the latter.

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4. Plant Growth. Growth is the most sensitive plant parameter to water stress (Hsiao 1973). In grapevine the effects of water stress have been reviewed for vegetative and reproductive growth (Smart and Coombe 1983; Williams and Matthews 1990; Williams et al. 1994). The growth reactions to water stress may be modified by interaction with plant nutrient status. This was demonstrated by experiments with ‘Cabernet Sauvignon’ growing in pots under glasshouse conditions that received two irrigation schedules and two nutrient regimes (Ussahatanonta et al. 1996). The watering treatment involved adequate watering (WH) and intermittent water stress (WL) and nutrients were either adequate (NH) or insufficient (NL) involving one-fifth of the nutrients applied to the NH treatment. NL reduced major growth parameters such as shoot length, stem weight, and leaf weight more than did WL. Both WL and NL reduced node and leaf number, but only NL reduced internode length, leaf area, and weight. WL had a larger effect on growth reduction when combined with NH than when combined with NL. WL, especially when combined with NH, advanced maturity by 10 days. The authors concluded that advancement of maturity could be of potential value if intermittent water stress were combined with adequate nutrition in areas with a short season. Low nutrient soils might be desirable in areas where other factors are likely to induce strong growth. The effects of water stress may also be influenced by exogenously applied gibberellic acid (Williams et al. 1994). When GA3 was applied at berry set to ‘Thompson Seedless’, final yields were similar between the irrigated vines and the non-irrigated vines despite large differences in leaf area per vine at harvest. Water stressed vines that had reduced leaf area were able to mature a crop similar to that of irrigated vines, indicating that alterations in source/sink relationship may be able to overcome the detrimental effect of water stress. Berry growth rate of irrigated and non-irrigated vines was similar after Stage I despite differences in water status. The authors concluded that involvement of hormones, other than ABA, should be studied in plants under water stress situations. F. Acclimation of Plants to Water Deficit Because plants are often exposed to water deficits during growth and development, it is important that they be able to acclimate to the prevailing conditions so as to avoid permanent injury. Included in the mechanisms of adaptation to water deficit are drought escape, drought tolerance with low plant water potential, and drought tolerance with high plant water potential (Turner 1986). Drought escape requires that plants complete their life cycle before significant water deficit can

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develop. This is not applicable to deciduous trees. Drought tolerance on the other hand requires the plant to respond in some way as water deficit develops. Osmotic adjustment is an example of drought tolerance with low plant water potential. Drought tolerance with high plant water potential can be realized through the stomatal control of transpiration. A brief account of osmotic adjustment and stomatal control is given below. 1. Osmotic Adjustment. Osmotic adjustment is the lowering of osmotic potential by the net increase in intracellular solutes in response to a decreased plant water potential. Rodrigues et al. (1993) observed osmotic adjustment in leaves of grapevine and Matthews et al. (1987a) reported osmotic adjustment in grape berries. Schultz and Matthews (1993) showed that the occurrence of osmotic adjustment in leaves of ‘White Riesling’ is due to both accumulation of solutes and changes in cell wall elasticity. However, the results of Patakas and Noitsakis (1999) for ‘Victoria’ indicated that solute accumulation was the main mechanism of osmotic adjustment. The rate of stress development generally influences the ability of plants to demonstrate osmotic adjustment with the gradual imposition of water deficit favoring osmotic adjustment (Morgan 1984). There is also a difference in the ability of cultivars to undergo osmotic adjustment. Düring (1999) indicated that this difference did exist for wine grape cultivars and it was used as a basis for selection for drought tolerance in a breeding program in Germany, where irrigation of wine grapes is not generally permitted for quality wine production. 2. Stomatal Behavior. The maintenance of turgor potential via osmotic adjustment of the leaves may be expected to maintain stomatal opening despite a reduction in ψ (Turner and Jones 1980). However, stomatal conductance is not solely dependent upon leaf turgor potential. For example, Golan et al. (1986) demonstrated that stomatal response of sunflower and wheat was closely related to soil moisture due to the role of hormones produced in roots in dry soil. Additionally, Düring and Dry (1995) speculated that chemical signals such as ABA are synthesized in the roots and transported to the leaves, which act to induce stomatal closure. If roots osmotically adjust, this signal is suppressed and stomatal closure is reduced. Naor et al. (1995) showed a good correlation between stomatal conductance and ψstem and ψsoil but a poor correlation with ψleaf. A good correlation with ψsoil further indicates that root signals play an important role in the control of stomatal conductance. Such root:shoot communication highlights the complexity of stomatal response to water deficit. Tardieu and Davies (1993) emphasize that it is inadequate to dis-

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cuss the concept of stomatal control based only on chemical signals (via the roots) or solely on plant water relations. These influences are integrated within the plant under water deficit conditions. Irrespective of the mechanisms involved, the closure of stomata under water stress will result in maintenance of a high water potential providing adaptation to stress.

IV. IRRIGATION OF VINEYARDS Grapevines are often grown in dry areas where frequent irrigation may not be possible and water should be saved (Matthews et al. 1987b; Pire and Ojeda 1999). Grapes are drought tolerant (Van Zyl and Weber 1981) because of their low water consumption and an extensive root system that makes them able to withstand long periods without water (Saayman and Lambrechts 1995). Water use efficiency has been reported to be similar for well-watered and droughted vines, further indicating adaptation to dry conditions (Novello and de Palma 1997). Grapevine increases productivity under irrigation if water loss due to ET exceeds rainfall (Van Zyl and Weber 1981; McCarthy et al. 1983; McCarthy et al. 1997). Irrigation scheduling to avoid plant water deficit should therefore minimize periods of low water status in plants by replacing an adequate percentage of ET. Judicious irrigation therefore necessitates estimation or measurement of ET. Based on ET, the amount and timing of irrigation depend on both meteorological and crop factors (Williams and Matthews 1990). Meteorological factors influencing crop water use include radiation, temperature, VPD, and wind speed. Major crop factors influencing water use include stomatal response, leaf morphology, vine architecture, rootstock, crop load, and cultivar. The importance of meteorological factors affecting ET was first discussed by Penman (1948) and further developed by Monteith (1965), who included crop factors of importance in the estimation of ET. Several attempts to use the Penman-Monteith approach to calculate ET have been made in recent years for a number of crops (Green and McNaughton 1996; Mills et al. 1999), including the grapevine (Williams et al. 1992). The introduction of the FAO Penman-Monteith method for ET calculation is presented in Equation 2 adapted from Allen et al. (1998). This equation is an accurate and simple representation of the physical and physiological factors governing the ET process. It represents the reference evapotranspiration (ETo), and the crop coefficients (Kc) could be calculated by relating the measured crop evapotranspiration (ETc) with ETo because by definition Kc = ETc/ETo. Reference ET is

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defined as the transpiration from an extended surface of actively growing green grass cover of uniform height, completely shading the ground and not short of water. Crop coefficient is indicative of the effect of canopy size, canopy architecture and stomatal response on water use of a specific crop (Doorenbos and Pruitt 1975).

ETo =

900 u2 (es − ea ) T + 273 ∆ + γ (1 + 0.34u2 )

0.408∆(R n − G) + γ

[2]

where ETo Rn G T u2 es ea es-ea ∆ γ

reference evapotranspiration [mm day–1], net radiation at the crop surface [MJ m–2 day–1], soil heat flux density [MJ m–2 day–1], air temperature at 2 m height [°C], wind speed at 2 m height [m s–1], saturation vapor pressure [kPa], actual vapor pressure [kPa], saturation vapor pressure deficit [kPa], slope vapor pressure curve [kPa °C–1], psychrometric constant [kPa °C–1].

Values of Kc for grapevine given by Doorenbos and Pruitt (1975) range from 0.35 early in the season to 0.9 in mid season. Goodwin (1995) also showed crop coefficients are low early and late in the season but are maximum during the middle of the season. He indicated that for the hot Australian climate, Kc values were 0.1 at budburst, 0.25 at flowering, 0.5 at veraison to harvest, and 0.25 after harvest. Similar trends in changes of crop coefficient with time of season are reported in grapes by Grimes and Williams (1990). Evans et al. (1993) indicate that published crop coefficients may not be suitable when applied to local conditions. Ideally, crop coefficients should be calculated for each region. Oliver and Sene (1992) discuss the use of a constant Kc of 0.2 for a semi-arid region, well below the recommended values by Doorenbos and Pruitt (1975). Measurement of evaporation from an open water surface, such as a class A evaporation pan, has also been used as a guide for irrigation scheduling in many crops, including grape (Evans et al. 1993). Canopy models have been used for scheduling irrigation. Greenspan and Matthews (1996) used a micrometeorological approach employing on-site measurements and the use of a canopy energy budget model. Understanding the soil as the reservoir from which plants get their water is essential when developing a water management strategy

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(Wample 1997; Cass 1999). Soil moisture status may be used as an indicator of irrigation requirement. The conservation of water equation is a useful way to calculate expected water loss from the soil-plant system and thus to schedule irrigation (Mpelasoka et al. 1997). WU = I – ∆W – D

[3]

Where WU = crop water use, I = irrigation, ∆W = change in soil-water storage, and D = drainage volume. The use of the water balance equation for irrigation of grapes is illustrated by Caspari and Neal (1998). As soil water is depleted, plants become increasingly stressed and they are unable to extract all the water from the soil. The well-known terms “field capacity,” “stress point,” and “permanent wilting point” are used to describe critical points on the soil moisture release curve. Ideally, soil moisture would be kept within the range of field capacity and stress point. The amount of water present in the soil at these critical points varies depending on soil type. The size and nature of the soil particles dictate how tightly water is held by the soil. Therefore different soils have different amounts of water still held by the soil at the stress point. Saayman and Lambrechts (1995) illustrate the changes in available soil water with different soil types. Irrigation scheduling must take the storage capacity of a given soil into account (Wample 1996). Soil moisture measurement involves several techniques, including time domain reflectometry (TDR) (Green and Clothier 1995), Gypsum resistance blocks (Van Zyl and Weber 1981), neutron probes (Araujo et al. 1995), and tensiometers (Klein 1983; Saayman and Lambrechts 1995). Such sensors may be linked to an automatic irrigation system so that, once sensors measure a predetermined value of soil water, irrigation is supplied. Care must be taken when considering placement of such sensors and due consideration also given to variability of soil throughout the vineyard (Naor et al. 1993). Goodwin (1995) gives a practical guide to the use of soil moisture sensors. Periodic water deficit in plants may still occur even if soil is adequately moist. If evaporative demands from the atmosphere are higher than the rate of water uptake from the soil, plants will develop water deficit. Plant parameters may also be used as a basis for irrigation scheduling. Techniques such as measurement of plant water potential (Grimes and Williams 1990), diurnal changes in stem and/or fruit diameter using LVDT (Myburgh 1996), heat pulse method (Eastham and Gray 1998; Ginestar et al. 1998a), and heat balance method (Lascano et al. 1992) are commonly used by researchers to indicate irrigation requirements of vines. Such plant-based parameters are also useful as a cross check on

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the accuracy of models used to predict irrigation requirements. Lovisolo et al. (1999) used the Penman-Monteith model for predicting transpiration in ‘Nebbiolo’ grafted on ‘Kober 5BB’ and found that their prediction was accurate based on measurement of sap flow using the stem heat balance technique. Some studies recommend the use of plant-based indicators for irrigation scheduling because they have the potential to be more accurate (Ginestar et al. 1998b). However, some limitations to the use of these plant-based systems have been reported. For example, Braun and Schmidt (1999) reported that the heat balance method is not suitable for old vines with thick stems. Previous irrigation schedules may also influence the current one. For example, if irrigation water were always applied in small amounts frequently then roots are likely to be shallow. Such poor development of the root system means vines are unable to tap into water deeper in the profile and therefore require more frequent irrigation. Deep watering may encourage deep rooting but may also result in loss of irrigation water as it passes beyond the rootzone and is lost via leaching. A prolonged schedule of deficit irrigation will also limit total tree size and therefore water requirement. A. Regulated Deficit Irrigation Regulated deficit irrigation (RDI) is a technique of partial replacement of ET and therefore delivers less water than the actual plant requirement at selected times during the growing season, depending on the expected beneficial outcomes. It was initially developed to control vegetative growth of fruit trees in high-density plantings (Behboudian and Mills 1997). Control of vegetative growth is usually required in most deciduous fruit crops in order to maintain consistent production and to facilitate crop management. Excess vegetative growth of grapevines results in berry shading and reduced fruit quality (Dry and Loveys 1998). Additionally, disease problems, especially fungal disorders, are increased when the canopy is dense and air movement within the canopy is reduced. In 1997 McCarthy stated that RDI is becoming increasingly popular in Australian vineyards. The potential of RDI in reducing vegetative growth without detrimental effects on fruit growth and yield is based on at least two physiological principles. These are: differential sensitivities of tissues, organs, and processes to reduced plant water status, and phenological separation of shoot and fruit growth. Regulated deficit irrigation has been shown to effectively control vegetative vigor in grapevines (Caspari et al. 1997; Pire and Ojeda 1999).

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Reduced vegetative growth is often desirable to increase light and air movement through the canopy and to improve spray penetration. A decrease in vegetative growth allows greater exposure of fruit to solar radiation, which contributes to sugar accumulation and, for red varieties, color development (Calò et al. 1995). In addition to these indirect influences on fruit quality, deficit irrigation at specific phenological stages may also result in direct changes in fruit quality. However, the deficit level required to induce direct quality changes is generally quite severe. Goodwin (1995) states that the primary reason for the use of RDI in grapes is manipulation of fruit quality. Calò et al. (1995) also state that excessive watering can result in a dilution of fruit contents in grape and detrimentally alter quality, especially for wine grapes. With this in mind, Williams and Matthews (1990) state that grapevines in California are often over irrigated and therefore the potential to implement RDI and to improve irrigation practice is high. 1. Timing of RDI Application. As plants are only sensitive to water deficit during specific periods and because vegetative growth and fruit growth have somewhat separated periods of active growth (Fig. 5.1), there is the potential to manipulate targeted organs at specific times. Stevens et al. (1995) and Williams and Matthews (1990) report that vegetative growth is more sensitive to water deficit than fruit growth and similarly fruit composition is less sensitive than is overall fruit growth.. With moderate water deficit, some parameters may be affected but others remain essentially unchanged. Deficit irrigation may result in restricted root growth of fruit trees, as less root development was observed in dry soil (Behboudian and Mills 1997). However, Richards (1983) suggested that root growth is generally less sensitive to water deficit than vegetative growth. In contrast Dry and Loveys (1998) showed that deficit irrigation can result in a reduction in root growth of grapevines, which is coupled with reduced vegetative growth. The impact of any deficit irrigation on root growth must be considered when irrigation strategies are proposed. Fruit yield is often reduced under water stress either because fruit size is compromised or because flower development, fruit set, and berry number is reduced. A decrease in leaf growth allows greater exposure of fruit to the sun, contributing to sugar accumulation, color development (Calò et al. 1995), and a reduction in malate concentration (Stevens et al. 1995). These examples highlight the importance of the indirect consequences of reduced vine water status. In the following the impact of RDI is described for different stages of the growing season. Fig. 5.2 could be used as an approximate match for

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these stages. Although RDI at selected times may be beneficial, there are also phenological stages when it could be damaging. 2. Early RDI: Budding to Flowering. Although water requirements at this time are low, lack of water at this stage may lead to irregular bud burst, short shoots, and fewer flowers. Studies of deficit irrigation in grape indicate a reduction in vegetative growth if limited irrigation is applied between bud burst and flowering (Van Zyl 1984; Wample 1996). Bud break will be uneven during water deficit and flower development detrimentally affected. Water deficit during flowering and fruit set is generally associated with poor pollen and pistil viability and therefore poor fruit set. Goodwin (1995) reports yield losses of up to 50% if water deficit is induced at this time, mainly due to increased fruit abscission. Berry size is adversely affected if water deficit is induced during flowering (Van Zyl 1984). 3. Mid-season RDI: Fruit Set to Veraison. Following fruit set, it is ideal to avoid stress during cell division and initial cell enlargement of the fruit, as water stress during this time will reduce maximal berry size and thus yield (Smart and Coombe 1983; Wample 1996; McCarthy 1997). Creasy and Lombard (1993) showed that pre-veraison berries are very sensitive to water deficit and it appears as though the water requirements of the vine out-compete that of the fruit. This is supported by Goodwin (1995), who reports yield losses of up to 40% caused by water deficit at this time. Similarly, McCarthy (1997) found the largest reduction in yield if stress is imposed at this time, as compared to other fruit development stages. Trunk growth is reduced if water deficit is induced at mid-season (Myburgh 1996). In contrast, Caspari et al. (1997) reported that although water deficit was sufficient to reduce vegetative growth in ‘Sauvignon blanc’, fruit yield was not affected. Their study was carried out in a humid area in contrast to the above studies, which were done in dry regions. Fruit compositional changes due to water stress at this stage include increases in soluble solids (predominantly sugars) and titratable acidity. Fruit color may also be enhanced as anthocyanin development is encouraged with increased fruit sugar. 4. Late-season RDI: Veraison to Harvest. Grapevines are generally tolerant to reduced plant water status at this time (Van Zyl 1987). Deficit irrigation following veraison may cause senescence of lower leaves, leading to fruit exposure and sunburn resulting in reduced fruit quality (Wample 1996). Shoot growth is unlikely to be affected by water deficit following veraison, as minimal shoot growth occurs after this time,

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although there may be some abscission of shoot tips if the level of stress imposed is high (Wample 1996). Post-veraison water stress increases fruit pH (McCarthy et al. 1983; Ginestar et al. 1998b). Naor et al. (1993) indicate that a post-veraison water deficit reduced soluble solids in grape berries. Water deficit at this time appears to have a minimal impact on berry weight (McCarthy 1997). Some reports indicate that water deficit will accelerate fruit maturity, but reports are contradictory. Jackson and Lombard (1993) state excess water delays the onset of ripening, but water stress has not always been found to accelerate it (Matthews and Anderson 1988). Sipiora and Granda (1998) indicate a severe water stress will retard sugar accumulation and therefore maturity. Williams and Grimes (1987) also report that severe water deficit delays maturity. However, McCarthy et al. (1983) showed reduced irrigation did advance fruit maturity. Generally, drier soil during fruit ripening will improve fruit quality for wine grapes, but the level of stress imposed is important (Jackson and Lombard 1993). 5. Postharvest RDI. Water deficit following harvest and before vines become dormant for the winter may cause a reduction in root growth, which will further confound the effects of water stress in the future (Wample 1996). Increased low temperature hardiness has been reported in vines undergoing a post-harvest water deficit (Evans et al. 1993). This may provide an important management tool in areas where vines are exposed to low winter temperatures. Although grapevines do not transpire when dormant, roots still require damp soil. If drought is experienced late in the winter or in spring, bud burst may be detrimentally affected (Davidson 1998). Williams et al. (1991) report that a postharvest water deficit encourages earlier budbreak in the following season. The above information leads us to the conclusion that RDI could be applied for some benefits during the later stages of the growing season (veraison to harvest). These include minimal impact on berry weight (McCarthy 1997), acceleration of fruit maturity (McCarthy et al. 1983), and improvement of quality for wine grapes (Jackson and Lombard 1993). Care should be taken in the post-harvest application of RDI and for other parts of the growing season and only a mild measure of water deficit should be allowed if RDI were used. A promising approach to RDI whose timing spanned most of the growing season was taken by Hamman and Dami (2000) in Colorado. They irrigated ‘Cabernet Sauvignon’ at the rate of 192 (T1, control), 96 (T2), and 48 (T3) liters per vine per week from bud burst until veraison and then reduced all irrigation by 25% through to harvest. Soil water content was reduced in T2 and T3 compared with T1. The T2 treatment had the best canopy size, yield, and

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fruit and wine quality. Cold hardiness of buds and canes was unaffected by irrigation treatment. Canes matured earlier and were less susceptible to early fall frost in T2 and T3. The approach of Hamman and Dami (2000), which involved starting the RDI earlier in the season and then increasing the severity of deficit later in the season, seems feasible for repetition on other cultivars and localities combined with allowing various measures of water deficit to develop. Unfortunately the authors did not measure ψ for better comparison with other experiments. Some quantitative examples of deficit developed during RDI are given below. 6. Degree of Deficit. The benefits, or not, of manipulation of water at specific times for grapevines depends on the cultivar and the end use of the fruit. It also depends on the level of deficit that may be imposed. The level of deficit could be applied by replacing a certain percentage of ET or irrigating the vines after depletion of a measure of plant available water (PAW). Myburgh (1996) provides data on ‘Barlinka/Ramsey’ (scion/rootstock) that was irrigated at 10%, 40%, and 60% depletion of PAW in a field trial on sandy soil in South Africa. These treatments resulted in, respectively, midday ψ values of –0.93, –1.03, and –1.2 MPa. It was concluded that the most acceptable combination of growth, yield, berry size, and eating quality was obtained by irrigation at 40% PAW depletion. Van Zyl (1984) suggests that a rather severe deficit is required to bring about changes in fruit quality attributes in a direct way. Van Zyl and Van Huyssteen (1988) demonstrated how different types of irrigation influence the level of deficit achieved. Trickle irrigation may be very effective in some situations but it may cause severe water stress to develop when used on highly permeable soils. In these situations microjet, sprinkler, or furrow irrigation may be required. B. Partial Rootzone Drying Partial rootzone drying (PRD) is an irrigation protocol whereby at each turn of irrigation only a part of the root zone receives water and the other part would be allowed to dry. This has proven effective in inducing regulated deficit in several horticultural crops, including grapevine (Dry and Loveys 1998). This system works because roots are only active in moist soil (Elfving 1982; Behboudian and Mills 1997). Therefore, by inducing “dry spots” within the rootzone, the effective rooting volume is reduced. Additionally, chemical signaling from the dry roots to the vegetative portions of the vine brings about further physiological changes. Partial rootzone drying may be effective in limiting leaching

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and also reducing evaporation of irrigation water from the soil surface (Van Zyl and van Huyssteen 1988). Partial rootzone drying can be achieved via the use of trickle irrigation (Araujo et al. 1995) or by careful placement of other water emitters. Experiments by Van Zyl and Van Huyssteen (1988) indicate the use of trickle irrigation has the potential in improving fruit quality. A recent study by Dry and Loveys (1998) showed the effectiveness of PRD in the control of vegetative growth and the enhancement of berry quality. The theory behind PRD centers on the role that chemical signals, primarily ABA, originating in the roots, play in the control of shoot growth and transpiration. Stimulation of these signals through PRD can result in reduced vegetative growth and total vine water use, while maintaining crop yield and improving fruit quality indirectly (Loveys et al. 1997). Van Zyl (1987) provided interesting results from a trial in which only 50% of the rootzone was wet. The top 50% of the roots was irrigated rather than a split of the ground area occupied by the vine. Such a partial rootzone irrigation did not result in any significant changes in vine performance or vine water status. The lack of impact that shallow irrigation had was probably due to the exposure of a high proportion of absorptive roots to moist conditions that prevented the initiation of root signaling typical in PRD treatments. Poni et al. (1992) conducted split root experiments in pots using grapes and found that the above-ground portion of the plant equilibrates with the wet region of the root zone and very little change in vegetative growth, stomatal conductance, photosynthesis and overall water use was observed. However, the experiment of Poni et al. (1992) was short term and therefore there may have been insufficient time for differences to develop. Clearly PRD is a tool with great potential as a management strategy to save water, decrease leaching, reduce soil evaporation, decrease vegetative growth, and possibly improve berry quality.

V. QUALITY ATTRIBUTES FOR WINE, DRIED, TABLE, AND JUICE GRAPES A. Wine Grapes As a general rule, wine grapes require less water than table grapes because of quality concerns. Wine grape irrigation often comprises an RDI regime to control vegetative growth and to improve wine quality (Sipiora and Granda 1998). In wine grapes, one desirable attribute is a

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specific level of soluble solids (SS). If SS is too low, the wine will lack sugar and if it is too high the grapes are termed overripe. Both titratable acidity and pH of the wine are critical quality parameters. As berry SS increases, pH also increases, which may result in a wine that has bland flavor and is, therefore, less desirable (Mullins et al. 1992, page 138). Freeman (1983) reports an increase in titratable acidity with irrigation and a reduction in anthocyanin development. Although deficit irrigation is reported to increase grape SS levels, the results are somewhat contradictory (Jackson and Lombard 1993; Naor et al. 1993). Titratable acidity may decline with reduced irrigation (Van Zyl and Van Huyssteen 1988; Pire and Ojeda 1999). Grapes grown for wine have distinct quality requirements that differ with cultivar. The changes in fruit quality effected by modified plant water status can give wine some fundamental characteristics and distinct flavors that are associated with region and cultivar, as reviewed by Jackson and Lombard (1993), who emphasized that these characteristics give each region and wine its uniqueness. When reference is made to a vintage year for wine, it is a description of a season that has encouraged the optimization of fruit quality attributes for a specific wine. It is suggested that irrigation management is a major contributor to wine quality. Jackson and Lombard (1993) reported that ‘Müller-Thurgau’ grown in pots and deficitirrigated from veraison to harvest produced wine that was rated as “fruity, fragrant, and elegant.” Plants fully irrigated in this period produced wine that was “full-bodied and less elegant.” Preferred wines were from vines that were irrigated until veraison and then deficit-irrigated, and least preferred were from the vines deficit-irrigated until veraison and then fully irrigated. B. Dried Grapes—Raisins ‘Thompson Seedless’ is the most widely planted grape variety for raisin production in California (Williams and Matthews 1990). Seedlessness, large size, and high sugar content are important quality attributes for raisin production. Both size and sugar content are altered with irrigation management (Jackson and Lombard 1993). There are similarities between the irrigation requirements of grapes grown for raisin production and for wine grapes. During the grape-ripening period it has been recommended that the soil moisture supply be gradually lessened and the irrigation cut-off should be early enough to slow vine growth and to provide for adequate soil surface drying in areas where terraces are made under the vines for fruit drying (Williams and Matthews 1990).

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C. Table Grapes Quality attributes of table grapes differ from those required of wine grapes. Visual factors such as berry size, color, texture, and the condition of the waxy bloom on the fruit surface are all important. Table grapes are harvested at a lower SS level than those used for wine or raisin production (Williams and Matthews 1990). Texture is a particularly important attribute of table grapes, and a firm and crisp texture is essential (Jackson and Lombard 1993). Generally, the level of irrigation required for table grapes is higher than that required for raisin or wine production. Therefore, a higher percentage of ET should be replaced in deficit irrigation of table grapes as exemplified by the experiment of Srinivas et al. (1999), who replaced 75% of the ET for ‘Anabe-e-Shahi’ and reported increases in bunch weight and berry size. Irrigation of vines following harvest of table grapes is also important. Generally, fruit is harvested well before leaf fall. The vines, although not carrying a current crop, are at this stage laying the foundations for the next season. Adequate irrigation at this time is critical to ensure good flowering and fruit set in the coming season (Williams and Matthews 1990). These authors also indicate that many vineyards producing table grapes use grass between rows to control dust and to influence the light exposure of bunches. Pieri et al. (1999) reported that nitrogen was strongly taken up by the grass early in the season in two vineyards of ‘Cabernet franc’ in France, but vines did not suffer from such competition for water. The root system adjusted and also changed significantly according to soil characteristics. Grass therefore might not have a major influence on the irrigation requirement of vineyards.

D. Juice Grapes The grape juice industry, especially in the United States, is mainly based on the Concord grapevine (Vitis labruska) (Lakso and Dunst 1999). Information on the effects of irrigation on juice quality is scant for V. labruska, whereas more information, although not conclusive, is available for V. vinifera. For the latter, Hamman and Dami (2000) showed that total soluble solids (TSS) decreased with a late-season reduction of irrigation, while the results of Wample (1997) showed an increase. Balo et al. (1999) reported that irrigated and non-irrigated vines had the same aroma volatiles in the must for ‘Chardonnay’ and a sensory panel did not find any significant qualitative differences for the wine from the two treatments. For Concord vines, Poni et al. (1994) found that at pre-dawn

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leaf water potentials of lower than –1.1 MPa, juice Brix was reduced in berries and this was aggravated with heavier crop loads. Lakso and Dunst (1999) showed that irrigation increased berry juice Brix in both minimally pruned and balance-pruned Concord vines. It is therefore doubtful that deficit irrigation would be of any advantage to V. labruska. Wample (1997) states that Concord vine may require irrigation in many areas where it is growing and it requires a uniform level of soil moisture throughout the growing season. He further indicates problems that V. labruska faces if water-stressed at different times during the growing season in Washington State. One problem is the development of “black leaf” for mid-season stress, which could result in defoliation of vines. He concludes that maintaining a higher and more uniform soil moisture for American grapevine varieties may be more critical than for European varieties.

VI. FUTURE PROSPECTS Recent advances in vine management indicate that high-quality wines may be produced under irrigation. However, in many arid areas the increased use of irrigation brought about by the demands of rising populations has greatly increased the cost of water. Consequently, there has been increasing interest in scheduling irrigation to reduce usage, the rationale of regulated deficit irrigation. Although this technique can enhance fruit quality in grape, insufficient irrigation water can cause problems equally as damaging as an excess. Thus, the threshold whereby the detrimental effects of reduced water outweighs the advantages should be determined precisely for all cultivars. Given the different fruit quality requirement of wine, juice, table, and dried grapes, future research should concentrate on formulating irrigation schedules specifically targeted to each end product based on the cost of water. More research is needed on the physiological and product quality impacts of PRD, which seems to be a promising irrigation strategy based on the available information. Gaps remain in various aspects of grapevine water relations. Despite the fact that grapevine is extensively grown in dry areas, the plant reaction to VPD has not been researched to a satisfactory conclusion. The physiology of recovery from water stress such as occurs after re-watering of deficit-irrigated vines, has not been investigated as extensively as for other crops. Although phytohormones, such as ABA, have been studied as they relate to the water relations of grapevines, others, such as gibberellins, have been under-researched.

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Finally, there are other approaches to water management that need to be explored. One is the genetic solution, i.e., breeding rootstocks with improved drought tolerance. The discovery that water channel proteins (aquaporins) facilitate water transport across membranes, i.e., control water flux through tissues (Kjellbom et al. 1999), suggests that molecular approaches need to be explored in rootstock improvement. Breeding scion cultivars for increased drought tolerance is another solution. Düring (1999) has reviewed the important criteria for selecting drought tolerance, which includes higher water use efficiency based on higher rates of photosynthesis to transpiration, and increased ability to undergo osmotic adjustment. However, industry conservatism in changing wine grape cultivars is a limiting factor arrayed against this approach.

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Sipiora, M. J., and J. R. Lissarrague. 1999. Diurnal changes in vine water status and gas exchange parameters of Vitis vinifera L. cv. ‘Tempranillo’ grapevines as influenced by irrigation and pruning level. Acta Hort. 493:109–116. Smart, R. E., and B. G. Coombe. 1983. Water relations of grapevines. p. 137–196. In: T. T. Kozlowski (ed.), Water deficits and plant growth. Volume VII. Academic Press, New York. Srinivas, K., S. D. Shikhamany, and N. N. Reddy. 1999. Yield and water-use of ‘Anabee.Shahi’ grape (Vitis vinifera L.) vines under drip and basin irrigation. Indian J. Agric. Sci. 69:21–23. Stevens, R. M., G. Harvey, and D. Aspinall. 1995. Grapevine growth of shoots and fruit linearly correlate with water stress indices based on root-weighted soil matric potential. Aust. J. Grape and Wine Res. 1:58–66. Tardieu, F., and W. J. Davies. 1993. Interpretation of hydraulic and chemical signalling in the control of stomatal conductance and water status of droughted plants. Plant Cell Environ. 16:341–349. Turner, N. C. 1986. Adaptation to water deficits: a changing perspective. Aust. J. Plant Physiol. 13:175–190. Turner, N. C., and M. M. Jones. 1980. Turgor maintenance by osmotic adjustment. In: N. C. Turner and P. J. Kramer (eds.), Adaptation of plants to water and high temperature stress. John Wiley, New York. Ussahatanonta, S., D. I. Jackson, and R. N. Rowe. 1996. Effects of nutrient and water stress on vegetative and reproductive growth in Vitis vinifera L. Aust. J. Grape and Wine Res. 2:64–69. Van der Westhuizen, R. H. 1980. The effect of black plastic mulch on growth, production, and root development of Chenin Blanc vines under dryland conditions. S. Afric. J. Enol. Vitic. 1:1–6. Van Zyl, J. L. 1984. Response of Colombar grapevine to irrigation regards quality aspects and growth. S. Afric. J. Enol. Vitic. 5:19–28. Van Zyl, J. L. 1987. Diurnal variation in grapevine water stress as a function of changing soil water status and meteorological conditions. S. Afric. J. Enol. Vitic. 8:45–52. Van Zyl, J. L., and L. Van Huyssteen. 1988. Irrigation systems—their role in water requirements and the performance of grapevines. S. Afric. J. Enol. Vitic. 9:3–8. Van Zyl, J. L., and H. W. Weber. 1981. The effect of various supplementary irrigation treatments on plant and soil moisture relationships in a vineyard (Vitis vinifera var. Chenin Blanc). S. Afric. J. Enol. Vitic. 2:83–99. Wample, R. L. 1996. Issues in vineyard irrigation. Wine East (July–Aug): 8–21. Wample, R. L. 1997. When understanding irrigation—many things are to be considered. Vineyard & Winery Mgmt (Nov–Dec) 72:1–7. Wenkert, W. 1983. Water transport and balance within the plant: an overview. p. 137-172. In: H. M. Taylor, W. R. Jordan, and T. R. Sinclair (eds.), Limitations to efficient water use in crop production. American Society of Agronomy, Madison, WI. Williams, L. E. 1987. Growth of ‘Thompson Seedless’ grapevines: I. Leaf area development and dry weight distribution. J. Am. Soc. Hort. Sci. 112:325–330. Williams, L. E., N. K. Dokoozlian, and R. Wample. 1994. Grape. p. 85–133. In: B. Schaffer and P. C. Andersen (eds.), Handbook of environmental physiology of fruit crops. Volume I, Temperate crops. CRC Press, Boca Raton, FL. Williams L. E., and D. W. Grimes. 1987. Modelling vine growth-development of a data set for a water balance subroutine. p. 169–174. In: T. Lee (ed.), Proc. 6th Aust. Wine Ind. Tech. Conf. Adelaide, Aust. 14–17 July 1986. Australian Industrial Publ., Adelaide.

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Williams, L. E., and M. A. Matthews. 1990. Grapevine. p. 1019–1055. In: B. A. Stewart and D. R. Nielsen (eds.), Irrigation of agricultural crops. American Society of Agronomy, Madison, WI. Williams, L. E., R. A. Neja, J. L. Meyer, L. A. Yates, and E. L. Walker. 1991. Postharvest irrigation influences budbreak of ‘Perlette’ grapevines. HortScience 26:1081. Williams, L. E., D. W. Williams, and C. J. Pheves. 1992. Modelling grapevine water use. Proc. 8th Aust. Wine Indust. Tech. Conf. (8):29–33.

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6 Physiology and Biochemistry of Superficial Scald of Apples and Pears Morris Ingle Division of Plant and Soil Sciences West Virginia University Morgantown, West Virginia 26506

I. INTRODUCTION II . SCALD SYMPTOMS AND CELL CHANGES III. BIOCHEMISTRY OF SCALD A. Volatiles 1. α-Farnesene 2. Conjugated Trienes B. Antioxidants 1. Lipid Soluble 2. Water Soluble C. Anthocyanins IV. PHYSIOLOGY OF SCALD A. Cultivar, Maturity, and Year B. Temperature C. Storage Atmosphere D. Ethylene E. Scald-inhibiting Materials V. A MODEL OF SCALD DEVELOPMENT A. Step 1 B. Step 2 C. Step 3 D. Step 4 VI. PROSPECTS A. Predicting Scald 1. Maturity 2. Chlorophyll Fluorescence 3. Preharvest Temperatures B. Future Research LITERATURE CITED

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I. INTRODUCTION Superficial scald, a serious postharvest storage disorder of apple and pear, has received a marked increase in research activity in the last decade. The accepted dogma regarding the physiology and control of scald was summarized by Ingle and D’Souza (1989) as follows: (1) scald results from damage to hypodermal cells by the accumulation of αfarnesene oxidation products; (2) scald incidence and severity are influenced (or regulated) by harvest dates, maturity, preharvest air temperatures, preharvest treatments, geographical location, cultivar, rootstock, ethylene in storage, storage periods, and storage conditions; (3) diphenylamine (DPA) and 6 ethoxy-1,2-dehydro-2,2,4-trimethyl quinoline (ethoxyquin) are commercially effective scald inhibitors. Since that was written, restrictions have been placed on the use of those compounds and alternates have been sought. In this review, evidence that has accumulated in the last 10 years affecting those statements will be examined and a model will be offered that integrates what is now known. Although the model will be incomplete, it will emphasize what needs to be learned. Emonger et al. (1994) have reviewed preharvest conditions that affect scald development.

II. SCALD SYMPTOMS AND CELL CHANGES Scald is recognized as discoloration of the fruit surface ranging from light tan to dark brown (Ingle and D’Souza 1989). Sometimes the discolored lesions or patches appear wrinkled and sunken and may be rough to the touch, which is the result of the collapse of the hypodermal cells (Bain and Mercer 1956, 1963). Epidermal cells in affected areas appear normal, while the hypodermal cells beneath the epidermis are usually collapsed and filled with dark materials that are usually said to be polyphenols (Bain and Mercer 1956, 1963). The lesions usually are not visible until fruit have been in refrigerated storage (RS) for over 100 days, depending on cultivar and harvest date (D’Souza 1991; Mir et al. 1998; Meir and Bramlage 1988). Lesions will appear after less RS if fruit is held at 20°C. During that holding, lesions develop linearly (Ju et al. 1996; Mir et al. 1998). The scald lesions on ‘Law Rome’, ‘Delicious’, ‘Gala’, and ‘Cortland’ increased in area and became darker during holding at 22°C. The chromaticity values L* and hue were not influenced (Mir et al. 1998b). Minimal and maximal chlorophyll fluorescence (Fo and Fm) were reduced as lesions developed; however, photochemical efficiency did not change. The reduction in Fo and Fm suggests a loss in hypodermal

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chloroplast structure and function. ‘Cortland’ apples that had been treated with DPA did not develop scald and there was no change in chlorophyll fluorescence during holding at 22°C. The epidermis of apple fruits is covered by a cuticle that is covered by a layer of wax that is mainly composed of C25-C30 alkanes and alcohols but also some ketones, aldehydes, and free fatty acids. Cutin forms the thickest layer of the cuticle and is made up of fatty acid chains linked by ester bonds to hydroxyl and epoxide groups. These fatty acid polymers are usually embedded in wax. In the lower or inner layer of the cuticle, cutin and waxes blend with the polysaccharide of the outer epidermal cell walls. This part of the cuticle is less hydrophobic (lipophilic) than the outer parts.

III. BIOCHEMISTRY OF SCALD A. Volatiles 1. α-Farnesene. Brooks et al. (1923) proposed that scald was initiated or regulated by volatile substances produced by the fruit. Scald was reduced by wrapping apples and pears in paper impregnated with mineral oil, which was believed to absorb volatiles. Scald was also reduced by increasing the rate of ventilation, which could remove or dilute volatiles. α-Farnesene (C15H24, M=204[3E,6E]-3,7,11-trimethyl-1,3,6,10dodecatetraene) was the first volatile identified as a constituent of the waxy coating or cuticle apple (Murray et al. 1964). Huelin and Murray (1966) then reported the alpha isomer that was present in several apple and pear cultivars and it was subsequently shown that α-farnesene was evaporated or volatilized from apples and accumulated in oil wraps during storage (Huelin and Coggiola 1968, 1970a). There seemed to be more α-farnesene in scald-susceptible cultivars and less mature fruit and no other volatiles were known to be produced by the peel of apples or pears. Though there were no statistical analyses, it seemed that there was a relation between α-farnesene concentrations and scald incidence, although there were many exceptions (Huelin and Coggiola 1968; Meigh and Filmer 1969); however, α-farnesene as the cause of scald became uncertain when nonsignificant correlations were found between concentration at removal from RS and scald after holding an additional 7 days at 20°C (Huelin and Coggiola 1970b). Until 1995, the concentrations of α-farnesene and conjugated trienes (CTs, oxidation products of α-farnesene) were determined by absorbance at selected wavelengths of hexane or pentane extracts made by immersing

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intact fruits. The concentration of α-farnesene was estimated from absorbance at 232 nm while the concentration of CTs were estimated from absorbance at 269 nm or 282 nm (Huelin and Coggiola 1969; Anet 1972). Since it is now known that several other compounds absorb at these wavelengths (Rowan et al. 1995; Rupasinghe et al. 1998; Whitaker et al. 1997), spectrophotometric measurements need to be treated with some reserve, but not disregarded. High performance liquid chromatography (HLPC) and gas chromatography-mass spectrometry (GC-MS) are undoubtedly more accurate and precise than spectrophotometry, but the few studies using the former methods conform with the earlier reports (Meigh and Filmer 1969; Rowan et al. 1995; Whitaker et al. 1997). Production of α-farnesene may be monitored by measuring the headspace concentration above fruits or tissue slices that have been enclosed in static or flow through chambers or vials by solid phase microextraction (Mir et al. 1999; Ju and Curry 2000). α-Farnesene and other volatiles are absorbed on fibers coated with polydimethylsiloxane, which are then inserted into the injection port of a gas chromatograph for separation and measurement. Since there is variability in thickness and composition of epicuticular waxes (Belding et al. 1998), volatile release into the gas phase may be incomplete and uncertain, and thus confirmatory extraction may be necessary (Ju and Curry 2000). It is the α-farnesene and CTs in the wax covering and cuticle and perhaps the epidermal and outer hypodermal cells that are extracted when whole fruit are immersed in hexane or pentane. Some researchers have used “peel” (or “skin,” depending on authors) that has been removed from fruit. Peel is taken to be cuticle, epidermis, and hypodermis. Most workers seem to have used a vegetable or fruit peeler or razor blade that removed the outer 1–3 mm of the fruit and included some cortical tissue attached to the peel (Barden and Bramlage 1994a; Whitaker et al. 1997). In some studies, researchers have tried to scrape off the cortical cells attached to the peel. The definition and method for removing peel is far from standardized. In some recent studies the enzymatically isolated cuticle has been assayed for several constituents (Ju and Bramlage 1999a,b). Concentrations are given in a number of dimensions, e.g., nmole cm–1, µgm cm–1, or some unit unique measurement system (Ju and Curry 2000). Most of the α-farnesene has been found in the fruit coating or cuticle. After extraction of whole ‘Granny Smith’ fruit with hexane, fruit were peeled and peel pieces extracted with hexane. At harvest, both extracts contained 0.2 µg. cm–2 (Huelin and Coggiola 1968). The maximum concentrations were reached after 12 weeks RS, 32 µg cm–2 in the coating

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and 7 µg cm–2 in the cortex. After the maximum, concentrations α-farnesene decreased at about the same rate in both extracts such that the ratio of content in the coating versus that in the cells ratio stayed about the same. There are differences in α-farnesene concentrations in fruit from different parts of trees. When whole fruit extracts of ‘Cortland’ apples were measured after 1 month RS at 0°C, fruit from the exterior of trees harvested on Sept. 18 and 30 contained 20% more α-farnesene than fruit from the interior of the same trees. The internal ethylene concentration increased between harvests but more in the exterior fruit so that they finally contained 4-fold more than the internal fruit (Meir and Bramlage 1988). The nonblushed side of ‘Rome’ contained 1.66-fold as much αfarnesene as the blushed side (D’Souza 1991). There is now evidence that α-farnesene is synthesized by the isoprenoid pathway, starting with acetate. Trace amounts of [2-14C]-acetic acid and [5-3H]-mevalonate are incorporated into α-farnesene by ‘Red Delicious’ apple peel pieces (Rupasinghe et al. 1998), but there is no record of incorporation by a cell-free system. The committed, regulated step is the conversion of hydroxymethylglutaryl coenzyme A (HMG) to isoprenoid pyrophosphates by HMG reductase (HMGR). These isoprenoids are synthesized to farnesyl pyrophosphate. HMGR can be inhibited in both animals and plants by a number of compounds, including Lovastatin (Chappell 1995). As nontreated preclimacteric ‘Golden Supreme’ apple accumulated ethylene and α-farnesene during storage at 20°C, prestorage treatment with 1.25 and 2.50 nmole L–1 Lovastatin nearly eliminated α-farnesene without affecting ethylene (Ju and Curry 2000). The addition of 0.25 nmole L–1 delayed α-farnesene accumulation for 12 days, again without affecting ethylene. The effects of Lovastatin was the same on fruit that had been stored at 0° before treatment and transferred to 20°C. While the application of ethephon to preclimacteric fruit stimulated α-farnesene and ethylene production, addition of Lovastatin suppressed α-farnesene production. The incorporation of labeled trans, trans-farnesyl pyrophosphate into α-farnesene by ‘Delicious’ peel pieces has also been reported (Rupasinghe et al. 1998). There was no conversion by cortex tissue. Low activity was observed at harvest, even though there was no α-farnesene present, and increased rapidly to a peak at 20 weeks of RS, sometime after the concentration of α-farnesene had begun to decline (Rupasinghe et al. 2000) A cell-free extract of ‘Delicious’ peel has been prepared that transfers 14C label from farnesyl pyrophosphate to α-farnesene (Rupasinghe et al. 2000). This activity is ascribed to α-farnesene synthase.

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Total enzyme activity in the cytosol fraction was 16-fold greater than in the microsome fraction. The enzyme was purified 70-fold and many of its characteristics described. The molecular weight is approximately 100,000 Kd and the enzyme is composed of 2 or 3 subunits. Maximum activity was at 10°–20°C, with half-maximal activity at 0°. There is an absolute requirement for a metal ion, Mg+2 or Mn+2, which is common to other sesquiterpene cyclases (Vogeli et al. 1990). The enzyme from apple shows allosteric kinetics. The incorporation of radioactivity into α-farnesene from acetate, mevalonate, and farnesyl pyrophosphate, the α-farnesene synthesis-suppressing effect of the HMGR inhibitor Lovastatin, and the presence of an α-farnesene synthase in apple peel combine to strongly support that α-farnesene is indeed synthesized via the isoprenoid pathway in apple peel. During the commercial harvest period in Massachusetts, ‘Cortland’ peel α-farnesene concentrations ranged from 5–37 nmole cm–2 in 1991 (Du and Bramlage 1993; Barden and Bramlage 1994c). Measurements made from the same orchard were not started until after 1 month RS (Meir and Bramlage 1988). ‘Delicious’ contained 4.0 nmole cm–2 (Barden and Bramlage 1994c). In experiments conducted at West Virginia, ‘Delicious’ contained an average of 83.9 nmole cm–2, with small variations around the fruit. Fruit peel of ‘Golden Delicious’ from the same orchard contained 51.4, ‘York Imperial’ 56.2, and ‘Rome’ 62.0 nmole cm–2 (D’Souza 1991). The blushed side in red cultivars contained more αfarnesene than the nonblush side. ‘Granny Smith’ contained 13.8 nmole cm–2 which appears to be much higher than concentrations found in New Zealand fruit of that cultivar (Watkins et al. 1995). Between 1988 and 1990, the concentration of α-farnesene ranged from 17.75 to 80.85 nmole cm–2 in the peel of ‘Rome’ apples harvested from the same block 167 days after full bloom (DAFB) (D’Souza, 1991). There was little difference in α-farnesene concentrations of ‘Cortland’ apples between 1989 and 1990 (Watkins et al. 1993), but two studies with ‘Cortland’ and ‘Delicious’ from the same University of Massachusetts orchard showed a 3-fold concentration range for both cultivars (Barden and Bramlage 1994c; Du and Bramlage 1993). As the effects of maturity and environment are reviewed, it will be realized that these comparisons of cultivars must be viewed with caution. A number of studies have established a pattern of α-farnesene concentration increase with time on the tree (maturity), which continues during storage at any temperature, with peaks being reached after 50–60 days RS, followed by a decline (Huelin and Murray 1966; Meigh and Filmer 1969; Huelin and Coggiola 1968a,b; Du and Bramlage 1993, 1994;

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Barden and Bramlage 1994a; Watkins et al. 1993; Whitaker et al. 1997), but exceptions can be found (Barden and Bramlage 1994b). In ‘Rome’ apples the peel α-farnesene concentration increased between 83 and 97 DAFB and then declined to 1.5 µg cm–2 at 139 DAFB, after which there was an increase to a maximum of 77 µg cm–2 on the blush side at 169 DAFB, which is about the commercial harvest time in West Virginia. No correlation or regression coefficients between fruit age and α-farnasene concentration have been found in the literature; however, a significant relation between hours of exposure to hours

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