Fruit Development and Seed Dispersal

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ANNUAL PLANT REVIEWS VOLUME 38 Fruit Development and Seed Dispersal

Edited by

Lars Østergaard John Innes Centre, Norwich Research Park, Norwich, UK

A John Wiley & Sons, Ltd., Publication

Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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This edition first published 2010 C 2010 Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing programme has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom Editorial office 9600 Garsington Road, Oxford, OX4 2DQ, United Kingdom 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell. The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988. 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 or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on 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 should be sought. Library of Congress Cataloging-in-Publication Data Fruit development and seed dispersal / edited by Lars Østergaard. p. cm. – (Annual plant reviews ; v. 38) Includes bibliographical references and index. ISBN 978-1-4051-8946-0 (hardback : alk. paper) 1. Fruit–Development. 2. Seeds–Dispersal. I. Østergaard, Lars. II. Series: Annual plant reviews; v. 38. SB357.283.F78 2010 631.5–dc22 2009020417 A catalogue record for this book is available from the British Library. Annual plant reviews (Print) ISSN 1460-1494 Annual plant reviews (Online) ISSN 1756-9710 R Set in 10/12 pt Palatino by Aptara Inc., New Delhi, India Printed in Singapore

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2010

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Annual Plant Reviews A series for researchers and postgraduates in the plant sciences. Each volume in this series focuses on a theme of topical importance and emphasis is placed on rapid publication. Editorial Board: Prof. Jeremy A. Roberts (Editor-in-Chief), Plant Science Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK; Dr David Evans, School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, OX3 0BP; Prof. Hidemasa Imaseki, Obata-Minami 2419, Moriyama-ku, Nagoya 463, Japan; Dr Michael T. McManus, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand; Dr Jocelyn K.C. Rose, Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA. Titles in the series: 1. Arabidopsis Edited by M. Anderson and J.A. Roberts 2. Biochemistry of Plant Secondary Metabolism Edited by M. Wink 3. Functions of Plant Secondary Metabolites and their Exploitation in Biotechnology Edited by M. Wink 4. Molecular Plant Pathology Edited by M. Dickinson and J. Beynon 5. Vacuolar Compartments Edited by D.G. Robinson and J.C. Rogers 6. Plant Reproduction Edited by S.D. O’Neill and J.A. Roberts 7. Protein–Protein Interactions in Plant Biology Edited by M.T. McManus, W.A. Laing, and A.C. Allan 8. The Plant Cell Wall Edited by J.K.C. Rose 9. The Golgi Apparatus and the Plant Secretory Pathway Edited by D.G. Robinson 10. The Plant Cytoskeleton in Cell Differentiation and Development Edited by P.J. Hussey 11. Plant–Pathogen Interactions Edited by N.J. Talbot 12. Polarity in Plants Edited by K. Lindsey 13. Plastids Edited by S.G. Moller 14. Plant Pigments and their Manipulation Edited by K.M. Davies 15. Membrane Transport in Plants Edited by M.R. Blatt

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16. Intercellular Communication in Plants Edited by A.J. Fleming 17. Plant Architecture and Its Manipulation Edited by CGN Turnbull 18. Plasmodeomata Edited by K.J. Oparka 19. Plant Epigenetics Edited by P. Meyer 20. Flowering and Its Manipulation Edited by C. Ainsworth 21. Endogenous Plant Rhythms Edited by A. Hall and H. McWatters 22. Control of Primary Metabolism in Plants Edited by W.C. Plaxton and M.T. McManus 23. Biology of the Plant Cuticle Edited by M. Riederer 24. Plant Hormone Signaling Edited by P. Hadden and S.G. Thomas 25. Plant Cell Separation and Adhesion Edited by J.R. Roberts and Z. Gonzalez-Carranza 26. Senescence Processes in Plants Edited by S. Gan 27. Seed Development, Dormancy and Germination Edited by K.J. Bradford and H. Nonogaki 28. Plant Proteomics Edited by C. Finnie 29. Regulation of Transcription in Plants Edited by K. Grasser 30. Light and Plant Development Edited by G. Whitelam 31. Plant Mitochondria Edited by David C. Logan 32. Cell Cycle Control and Plant Development Edited by D. Inzé 33. Intracellular Signaling in Plants Edited by Z Yang 34. Molecular Aspects of Plant Disease Resistance Edited by Jane Parker 35. Plant Systems Biology Edited by Gloria M. Coruzzi and Rodrigo A. Gutiérrez 36. The Moss Physcomitrella Patens Edited by Celia Knight 37. Root Development Edited by Tom Beeckman 38. Fruit Development and Seed Dispersal Edited by Lars Østergaard

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CONTENTS

Contributors Preface 1 Carpel Evolution Aurélie C.M. Vialette-Guiraud and Charlie P. Scutt 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11

The importance of having carpels Hypotheses of carpel origin A phylogenetic framework for studies of carpel evolution A morphological portrait of the ancestral carpel The genetic control of carpel development in the first flowering plants A major role for the E-function in the origin of the carpel? Carpel specification in monocots Gene duplication and carpel evolution in the core eudicots The A-function finds a role in fruit development The multiple origins and mechanisms of syncarpy in the angiosperms A fruit by any other name: evolutionary convergence between angiosperms and gymnosperms References

2 Gynoecium Patterning in Arabidopsis: A Basic Plan Behind a Complex Structure Eva Sundberg and Cristina Ferrándiz 2.1 Introduction 2.2 The basic plan in lateral organs 2.3 The Arabidopsis gynoecium 2.4 Genetic and hormonal factors controlling gynoecium development 2.5 Conclusion Acknowledgements References 3 The Ins and Outs of Ovule Development Raffaella Battaglia, Monica Colombo and Martin M. Kater 3.1 Introduction 3.2 Origin of the ovule

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vi Contents 3.3 3.4 3.5 3.6 3.7 4

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Ovule development in Arabidopsis Sporophytic tissues Gametophytic tissue Interaction between the female gametophyte and the maternal sporophyte Ovule identity determination References

Fertilisation and Fruit Initiation Sara Fuentes and Adam Vivian-Smith 4.1 Introduction 4.2 Pollination 4.3 Female receptivity and the cessation of gynoecial growth 4.4 Additional restraints on flower development and fruit initiation 4.5 Fertilisation 4.6 Hormonal cues during fruit initiation 4.7 RNA silencing during fruit initiation 4.8 Signal transduction from ovule to carpel and vascular canalisation 4.9 Current models of fruit initiation 4.10 Concluding remarks Acknowledgements References Arabidopsis Fruit Development Antonio Mart´ınez-Laborda and Antonio Vera 5.1 Introduction 5.2 Morphology of the Arabidopsis silique 5.3 Determining the boundary between valve and replum: valve margin genes 5.4 The making of valves and replum requires repression of valve margin genes 5.5 Suppressors of the rpl phenotype: setting up territories 5.6 A model for patterning the mediolateral axis of the Arabidopsis silique 5.7 Auxin: a signaling molecule for the mediolateral axis? 5.8 A biotechnological view Acknowledgements References Long-Distance Seed Dispersal Frank M. Schurr, Orr Spiegel, Ofer Steinitz, Ana Trakhtenbrot, Asaf Tsoar and Ran Nathan 6.1 6.2

Introduction Six generalizations on LDD mechanisms

72 73 81 91 93 97 107 107 110 113 115 117 123 141 145 147 150 151 151 172 172 174 177 179 182 185 192 195 196 196 204

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Contents vii

6.3 6.4

A vector-based perspective on the evolution and predictability of long-distance seed dispersal Future directions Acknowledgements References

7 Seed Dispersal and Crop Domestication: Shattering, Germination and Seasonality in Evolution Under Cultivation Dorian Q. Fuller and Robin Allaby 7.1 Introduction 7.2 Loss of natural seed dispersal in wheat and barley: archaeobotanical evidence 7.3 Non-shattering in other cereals: rice, pearl millet and maize 7.4 The genetics of non-shattering cereals 7.5 Reduction in seed dispersal aids 7.6 Non-cereal alternative: appendage hypermorphy in fibre crops 7.7 Loss of natural seed dispersal in pulses and other crops 7.8 Germination traits in domestication: the importance of loss of dormancy 7.9 The genetic basis for dormancy and germination 7.10 Germination and seedling competition: changes in seed size 7.11 The genetics of seed size 7.12 Seasonality controls: photoperiodicity and vernalization 7.13 Discussion: evolution and development of domesticated seed traits References 8 Factors Influencing the Ripening and Quality of Fleshy Fruits Cornelius S. Barry 8.1 Introduction 8.2 Control of fruit ripening 8.3 Transcription factors serve as master regulators of fruit ripening 8.4 Hormonal control of fruit ripening 8.5 The influence of light on fruit quality 8.6 The discovery of aroma and flavour genes in fruit 8.7 Cell wall changes influence fruit quality 8.8 The cuticle influences fruit quality and postharvest longevity

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238 239 240 246 249 252 254 255 257 260 261 271 273 278 280 296 296 297 298 302 306 307 309 310

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viii Contents 8.9 8.10

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Genomics Resources Conclusions and future perspectives Acknowledgements References

Parthenocarpy in Crop Plants Tiziana Pandolfini, Barbara Molesini and Angelo Spena 9.1 Introduction 9.2 Parthenocarpy 9.3 Auxin-synthesis parthenocarpy 9.4 Parthenocarpy via auxin signal transduction 9.5 Parthenocarpy via gibberellin signal transduction 9.6 Aucsia-silencing parthenocarpy 9.7 Auxin sensitivity and parthenocarpy 9.8 Apetalous parthenocarpy and the role of other floral organs 9.9 Stenospermocarpy 9.10 Parthenocarpy in perennial crop plants 9.11 Parthenocarpy and fruit crop breeding 9.12 From green plants to fruit crop plants References

Index Color plate section appears at the start of the book, before Chapter One

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CONTRIBUTORS

Robin Allaby Warwick HRI, University of Warwick, Wellesbourne, Warwick, United Kingdom Cornelius S. Barry A32 Plant and Soil Sciences, Michigan State University, East Lansing, MI, USA Raffaella Battaglia Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, Milano, Italy Monica Colombo Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, Milano, Italy Cristina Ferrándiz Instituto de Biolog´ıa Molecular y Celular de Plantas (UPV-CSIC), Campus de la Universidad Politécnica de Valencia, Avda de los Naranjos s/n, Valencia, Spain Sara Fuentes Department of Crop Genetics, John Innes Centre, Norwich, United Kingdom Dorian Q. Fuller Institute of Archaeology, University College London, London, United Kingdom Martin M. Kater Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, via Celoria 26, Milano, Italy Antonio Mart´ınez-Laborda ´ de Genética, Universidad Miguel Hernández, Campus de San Division Juan, Ctra. de Valencia s/n, 03550-San Juan de Alicante, Spain

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x Contributors Barbara Molesini Dipartimento Scientifico e Tecnologico, Università di Verona, Strada Le Grazie 15, Verona, Italy Ran Nathan Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel Tiziana Pandolfini Dipartimento Scientifico e Tecnologico, Università di Verona, Strada Le Grazie 15, Verona, Italy Frank M. Schurr Plant Ecology and Conservation Biology, University of PotsdamMaulbeerallee 3, Potsdam, Germany Charlie P. Scutt Reproduction et Developpement des Plantes, ENS de Lyon, 46 allee d’Italie, Lyon Cedex, France Angelo Spena Dipartimento Scientifico e Tecnologico, Università di Verona, Strada Le Grazie 15, Verona, Italy Orr Spiegel Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel Ofer Steinitz Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel Eva Sundberg Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden Ana Trakhtenbrot Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel Asaf Tsoar Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel

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Contributors xi

Antonio Vera ´ de Genética, Universidad Miguel Hernández, Campus de San Division Juan, Ctra. de Valencia s/n, 03550-San Juan de Alicante, Spain Aurélie C. M. Vialette-Guiraud Reproduction et Developpement des Plantes, ENS de Lyon, 46 allee d’Italie, Lyon Cedex, France Adam Vivian-Smith Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64, AL Leiden, The Netherlands

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PREFACE

The reproduction system of flowering plants (angiosperms) provides an extraordinary advantage, which has led to the massive dominance of this division over any other plant division on the planet. At the centre of angiosperm reproduction is the carpel that protects and nurtures the seeds developing inside until they are ready for dispersal. Different species have developed different strategies for efficient seed dispersal involving both short-range and long-range aims allowing widespread colonization. While angiosperms represent an evolutionary success story by themselves, fruit and the seed that they produce have been crucial for human civilization and particularly for crop domestication during the Neolithic revolution. Plant research over the last couple of decades has beautifully demonstrated how fruit provide excellent model systems to move crop improvement even further to increase the yield. Such advances are required now more than ever in order to maintain a sustainable production while meeting the increasing demands from an expanding population. Fruit are, however, not only interesting from an agronomic point of view. Both flowers and fruit are also ideal systems to study cell differentiation processes and tissue-specific development that can answer general questions regarding the development of multicellular organisms. In this book, the aim is to cover the recent impressive advances of research into fruit development and seed dispersal. Moreover, the objective is also to review the history of crop domestication as well as the most novel discoveries in optimizing the development of fruit of significant agricultural importance. The opening chapter describes the evolution of carpels and fruit among the flowering plants and how this has led to a tremendous biodiversity of forms in different angiosperm lineages. The following chapters describe in detail the genetic, hormonal and molecular knowledge on the different stages of development from the unfertilized flower till the mature fruit including gynoecium development, ovule development, fruit initiation and the postfertilization fruit development. Chapter 6 reports on exciting new insight to long distance seed dispersal, while Chapter 7 emphasizes the importance of controlling seed dispersal for the establishment of agriculture and human civilization. The eighth chapter provides a compilation of the most recent discoveries in development and ripening of the agricultural important fleshy fruit and finally, Chapter 9 shows how precision technology can be employed to create seedless varieties in certain crop plants.

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Preface xiii

The topics that are covered in this volume are intended to provide in-depth knowledge of a wide scope of aspects relating to fruit biology. As such, this book contains information that should be of interest to scientists ranging from experimental biologists to agronomists pursuing technology to exploit the knowledge for crop improvement purposes. Lars Østergaard

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Plate 1 The Arabidopsis gynoecium. The different axes of polarity and morphological terms are indicated (a) scanning electron micrograph of the mature gynoecium at anthesis. (b) Chloral hydrate cleared anthesis gynoecium to reveal vascular patterns. Primary and secondary bifurcations of the medial veins are indicated with arrows. (c) Cross section of the ovary at anthesis.


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Plate 2 Confocal laser scanning microscopy (CSLM) images of unfertilized and fertilized ovules of Arabidopsis expressing the synthetic auxin-responsive reporter gene DR5rev::eGFP. (a) Unfertilized anthesis ovule with minimal GFP expression. (b) Post-fertilized ovule at 5 h. The first nuclear endosperm division has occurred and GFP expression is observed in the endothelium, the chalazal domain and adjacent to the funiculus vascular strand. (c) Ovule after the third endosperm division (9 h post-fertilization) with eight endosperm nuclei and an elongated zygote. Strong GFP expression occurs in the endothelium, the chalaza and funiculus. Weaker expression is observed in the outer integument. (d) Treatment of detached pistils with NAA (50 µM) for 1 h, with subsequent washing for 7 h, induces strong GFP activation in the funiculus and chalaza, and moderate activation in the inner integument and weaker expression in the outer integument. a, antipodal cells; cc, central cell; cr, chalazal region; e, egg cell; en, endosperm; f, funiculus; fv, funiculus vascular tissue; g, generative cell; ii, inner integument; m, micropyle; p, pollen tube; pn, polar nucleus; oi, outer integument; rv, replum vascular tissues; s, synergid cell; sp, sperm cells; t, endothelium; z, zygote.

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Spatial scale, x (m) Plate 3 (a) The total dispersal kernel (TDK, thick black line) for a hypothetical plant population dispersed by four vectors (different orange tints). The inset shows the unequal distribution of seed loads (Q) on a log scale. The lines at the bottom indicate the range of distances of all seeds dispersed by each vector. (Figure modified from Nathan et al., 1071.) (b) The relative contribution of each vector to the TDK as a function of spatial scale x, showing that the vectors dominating long-distance dispersal of seed can be very different from those dominating seed dispersal at the typical scales of empirical studies.

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Plate 4 In vitro development of parthenocarpic DefH9-iaaM tomato fruit. (a) (Left panel) Flower buds from auxin-synthesis parthenocarpic plants collected at pre-anthesis and cultivated in medium not supplemented with auxin. (Middle panel) Ovaries growth after 10 days of in vitro cultivation. (Right panel) Mature fruits after approximately 30 days of in vitro cultivation. (b) (Left panel) Ovaries present in pre-anthesis (stage a) wild-type (wt) flower buds as compared with the ovaries present in DefH9-iaaM flower buds. (Right panel) DefH9-iaaM pre-anthesis flower bud showing enlarged ovary.

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Annual Plant Reviews (2009) 38, 1–34 doi: 10.1002/9781444314557.ch1

www.interscience.wiley.com

Chapter 1

CARPEL EVOLUTION Aurélie C.M. Vialette-Guiraud and Charlie P. Scutt Laboratoire de Reproduction et Développement des Plantes (CNRS UMR 5667-INRA-ENSL-UCBL), Université de Lyon, Lyon Cedex, France

Abstract: The carpel is the progenitor organ to the fruit and a defining feature of the flowering plants, or angiosperms. This organ has evolved in the angiosperms to generate a wide diversity of forms, often related to breeding strategies and seed distribution mechanisms. In this chapter, we focus on a number of key stages in the evolution of the carpel and fruit, about which something can be said of the molecular mechanisms underlying evolutionary change. In particular, we describe hypotheses for the evolutionary origin of the carpel in the first flowering plants and attempt to reconstruct the history of its structural diversification in various major angiosperm groups. In doing so, we concentrate on the genes and mechanisms whose presence can be deduced at key evolutionary stages in the angiosperms, and on molecular-evolutionary processes such as neo- and sub-functionalization, which have moulded these genes and the developmental processes they regulate. We also review the literature on the evolution of syncarpy – a phenomenon of enormous adaptive significance in the angiosperms. Lastly, we describe some examples of convergent evolution that have led to the development of fruit-like structures both within and outside the flowering plants. Keywords: carpel; fruit; evolution; development; angiosperm; syncarpy

1.1 The importance of having carpels The carpel is the female reproductive organ that encloses the ovules in the flowering plants, or angiosperms. By contrast, the ovules of the remainder of the seed plants, the gymnosperms, are most frequently naked structures borne in the axils of leaf-like organs. The carpel is thought to confer several major selective advantages on the flowering plants. Firstly, carpels protect, both physically and biochemically, the ovules within them: many classes of carpel-specific genes encode proteins associated with defence against insects or micro-organisms (Scutt et al., 2003). Secondly, highly efficient systems have evolved to facilitate pollen capture and pollen tube guidance in angiosperm carpel tissues, which probably represent considerable improvements over Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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2 Fruit Development and Seed Dispersal the mechanisms which bring about fertilization in gymnosperms. Thirdly, during the phase of pollen germination and growth, the carpel provides a site for the operation of self- and inter-specific incompatibility mechanisms which may confer important evolutionary advantages. Accordingly, self-incompatibility prevents close inbreeding, while inter-specific incompatibility prevents too wide hybridizations that may lead to infertile offspring. Fourthly, after fertilization, carpel tissues undergo further developmental changes to form fruits, which protect the developing seeds within them and, at maturity, contribute to the dissemination of these by a wide variety of mechanisms. Carpels and fruits have evolved to generate a tremendous biodiversity of forms in different angiosperm lineages. The novel carpel and fruit structures thus generated are often linked to diversification in factors including pollinators, breeding systems, seed structure and seed dispersal mechanisms. In numerous angiosperm groups, carpels have fused together to form a syncarpic pistil. A syncarpic arrangement provides a single stigmatic surface, giving a common point of access to all the ovules in the flower. Syncarpy may also allow for heavier pollination vectors and larger fruits with more elaborate seed dispersal mechanisms. For all of the above reasons, carpels and fruits were almost certainly of key importance in the evolutionary success of the angiosperms, which arose from an unknown common ancestor living some 160 MYA (million years ago) (Davies et al., 2004) to generate over 300 000 species alive today. The immense biodiversity of carpel and fruit development in the extant angiosperms means that no thorough or comprehensive treatment of this subject can realistically be undertaken. In this chapter, we will therefore concentrate on a few key stages in the evolution of carpels and fruits, about which something can be said of the molecular mechanisms underlying evolutionary change. Accordingly, we first describe a number of hypotheses for the evolution of the first carpels and fruits in the flowering plant lineage and review the literature on the likely state of the female reproductive structures in the last common ancestor of the extant angiosperms. We then describe the molecular and morphological differences between carpel development which have arisen following speciation events at two key stages in angiosperm evolution: the last common ancestors of the euangiosperms (including monocots and eudicots) and of the core eudicots (including rosids, asterids and Caryophyllales). We particularly concentrate on such molecular-evolutionary processes as sub- and neo-functionalization, as these apply to the genes of carpel and fruit development. As syncarpy represents an evolutionary change within the angiosperms that was clearly of enormous adaptive significance, we also review the literature on this subject, again concentrating on examples on which something can be said of the molecular mechanisms involved. Lastly, we look briefly at fruit-like structures that, to a botanist, are not fruits. Examples of convergent evolution that has generated structures resembling fruits can be found both within and outside the flowering plants.

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Carpel Evolution 3

1.2 Hypotheses of carpel origin The origin of the carpel is intimately linked to that of the flower – a structure for which several technical definitions have been proposed. For example, Arber and Parkin (1907) suggest ‘an ampisporangiate strobilius of determinate growth and with an involucrum of modified bracts’. According to this definition, the flower equates to a single, compacted developmental axis, or strobilus, rather than a multiply branched system in which each floral organ would correspond to a reduced strobilus. The male and female reproductive organs of the flower are separated into two zones: a central gynoecium, containing the carpels, is typically surrounded by an androecium, containing the stamens. A number of hypotheses have been formulated to account for the unique features of the flower (Fig. 1.1). As our interest here centres on the carpel, we will concentrate on what these hypotheses have to say about that organ type. It should first be mentioned that a hypothesis proposed by Goethe, the German philosopher, poet and dramatist (von Goethe, 1790), which is now well supported by molecular genetic evidence (Honma and Goto, 2001), regards all plant lateral organs, including carpels, as mutually homologous. Such lateral organs, which form on the flanks of the stem apical meristem or floral meristems, can accordingly be regarded as variants of a basic leaf-type developmental ground plan. Though carpels may be homologous to leaves, these reproductive organs are almost certainly more directly related to leaflike organs in the reproductive structures of gymnosperms. On this subject, the existing hypotheses for flower origin divide conceptually into two types, depending on whether they regard the carpel as derived by the modification of male or female structures in the presumed gymnosperm-like ancestor of the flowering plants. The Mostly Male Theory (MMT) (Frohlich and Parker, 2000; Frohlich, 2003) postulates the flower to be mainly derived from the male strobili, or male cone-like structures, of a gymnosperm-like ancestor. According to this hypothesis (Fig. 1.1a), the ancestor of the flowering plants would first have generated ectopic ovules on (male) microsporophylls, which would thereby have become bisexual. The MMT postulates that ectopic ovules were concentrated on sporophylls near the apex of the strobilus, and that the sporophylls bearing these ovules subsequently lost their ability to produce microsporangia, thus becoming female. These newly female sporophylls would then have closed around the ovules to form, in effect, the first carpels. In subsequent evolutionary steps, the residual, entirely female strobili of these proto-flowering plants would have been lost, leaving only bisexual reproductive axes containing apical carpels and basal microsporophylls which would later become stamens. The MMT is based on evidence from a number of sources, including molecular evidence linked to the LEAFY (LFY) gene, which acts upstream of genes that specify the identities of floral organs in typical angiosperm flowers. In at

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4 Fruit Development and Seed Dispersal

Figure 1.1 Hypotheses for the origin of the flower and its carpel. (a) According to the Mostly Male Theory (Frohlich, 2003), ectopic ovules developed on previously male sporophylls. In a second step, these sporophylls lost their (male) microsporangia and closed around the ovule to form the carpel. The outer integument of the angiosperm ovule (thick line) was formed from a pre-existing female cupule structure. (b) According to the Out-of-Male hypothesis (Theissen et al., 2002), the basipetal movement of male-determining, MADS-box B-function gene expression (shaded area) in a male strobilus left female structures at the apex, which later became carpels. (c) According to the Out-of-Female hypothesis (Theissen et al., 2002), the acropetal movement of MADS-box B-sister gene expression (shaded area) in a female strobilus left male structures at the base, which later became stamens. Female structures at the apex became carpels. (d) According to the Baum and Hileman hypothesis (Baum and Hileman, 2006), a temporal switch in the transcriptional regulation of B- and C-function MADS-box genes by LEAFY (LFY) occurred in an ancestor of the flowering plants. This change generated high concentrations of C-function-rich MADS-box protein complexes at late developmental stages, causing the patterning of the strobilus into apical female and basal male reproductive structures, which later became carpels and stamens, respectively.

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least certain gymnosperm taxa, a paralogue of LFY, termed NEEDLY (NLY), appears to be expressed principally in female cones (Mouradov et al., 1998). The orthologue of NLY seems to have been lost from the angiosperm lineage, after its separation from that of the living gymnosperms. The MMT postulates that the loss of NLY was accompanied by a more extensive loss of female-specific developmental programmes during the evolution of the flower. Hence, the MMT regards the carpel and the rest of the flower, with the exception of the ovule, as historically male. It should be noted that several studies have brought into question the proposed sex-specific expression of LFY and NLY in gymnosperms (Carlsbecker et al., 2004; Dornelas and Rodriguez, 2005). Vazquez-Lobo et al. (2007) have recently postulated a rather different partitioning of functions between these genes into early and late roles in reproductive development. However, it should also be noted that the hypothesized sex-specific expression of LFY and NLY in the (unknown) ancestor of the flowering plants is not an absolute requirement for the MMT. Rather, the loss of NLY is correlative evidence for the MMT, of which the key postulate is that of ectopic ovule development, which leads to the evolution of carpels from previously male sporophylls. An attractive feature of the MMT is that it provides an explanation for several unique aspects of reproductive development in angiosperms. For example, this hypothesis is formulated with fossil gymnosperms of the extinct order Corystospermales in mind as potential ancestors of the flowering plants. Corystospermales produced ovules enclosed within cupules, which were borne on unisexual female axes. According to the MMT, such female cupules would have become the outer integument of the ovule – a developmental feature which, like the carpel, is specific to the angiosperms. Several further hypotheses of flower origin have been proposed, which differ from the MMT in that they postulate the bisexuality of the flower to have arisen by a spatial or temporal change in factors governing the sex of reproductive organs. Hence, these hypotheses do not, in contrast to the MMT, postulate the extensive loss of female developmental programmes during angiosperm evolution. It follows that these various hypotheses would regard the carpel as homologous to the female reproductive structures in gymnosperms. The Out-of-Male (OOM) hypothesis (Theissen et al., 2002) proposes the bisexual flower to have evolved by the basipetal movement of male-promoting, B-class MADS-box gene expression in a previously male strobilus, leaving female structures at the apex (Fig. 1.1b). A sister hypothesis to the OOM hypothesis, the Out-of-Female (OOF) hypothesis (Theissen et al., 2002), postulates a sex-determining role for B-sister MADS-box genes, whose expression is proposed to have moved acropetally in a female strobilus to leave male structures in basal positions (Fig. 1.1c). The identification of the function of a B-sister gene in Arabidopsis thaliana, which proves to determine coloration in the outer integument (Nesi et al., 2002), has hardly provided support for the OOF variant of the above two hypotheses. However, it is certainly possible that B-sister genes played a much more central role in

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6 Fruit Development and Seed Dispersal female organ identity in early stages in the evolution of the flowering plants, and may continue to do so in basal flowering plant lineages. In general, therefore, the OOM and OOF hypotheses look below the level of LFY and NLY in the hierarchical control of gene expression, and postulate a spatial change in MADS-box gene expression, forming a boundary of B- or B-sister class gene expression in a previously unisexual strobilus, which would thereby have become bisexual. Baum and Hileman (2006) have formulated a further, nameless, hypothesis (we will call it the B&H hypothesis), to account for the evolution of the first flowers (Fig. 1.1d). Like the MMT, the B&H hypothesis proposes a central role for LFY in the origin of the flower, but postulates that the origin of floral bisexuality was caused, not by the loss of female-specific developmental programmes, but by a temporally generated switch in the response to LFY. According to this hypothesis, LFY protein builds up over time in the meristems of developing reproductive axes and, at a certain threshold of LFY concentration, these meristems switch from the production of (male) microsporoplylls to (female) megasporophylls. This hypothesized switch may involve the action of LFY cofactors, such as the ancestors of the Arabidopsis F-box protein UNUSUAL FLORAL ORGANS (UFO), and transcription factor WUSCHEL (WUS). Whatever the precise mechanism, the B&H hypothesis proposes that a difference occurred in the relative response to LFY of Band C-class MADS genes during early flower evolution. Accordingly, C-class proteins are proposed to have predominated at high LFY concentrations, encountered at the apex of the strobilus at late developmental stages, resulting in MADS-box complexes that were rich in C-class proteins. These proteins would have formed C-rich complexes which would then have specified the development of megasporophylls at the apex of the strobilus. The above hypotheses are, to some extent, testable. Baum and Hileman (2006), for example, propose a list of predictions that could be tested in basal angiosperm and gymnosperm lineages to support or refute their hypothesis. The MMT stands out from the other hypotheses in proposing the extensive loss of female developmental programmes during early flower evolution. This prediction might provide a means to eliminate either the MMT, or the other contending hypotheses, from consideration. Essentially, if the MMT were correct, we might expect to find numerous classes of genes with femalespecific expression patterns in gymnosperms, whose orthologues have apparently been lost from the angiosperm lineage. Gymnosperm genes with male-specific expression patterns should not be affected in this way. The fullscale testing of the MMT by this method has yet to be performed. However, one question mark concerning such a test relates to the degree to which male and female developmental programmes in gymnosperms might be based on different sets of genes, rather than on subtle differences in the expression patterns of a common set of genes. If the latter is predominately the case, this relatively simple method of hypothesis testing may be unavailable. All of the hypotheses discussed above for the evolution of the flower concentrate to a large extent on the origin of bisexuality, with only the MMT

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explicitly accounting for the origin of the carpel. Additionally, it must be admitted that even the MMT does not go into detail on the molecular mechanism of carpel closure. Thus, even if substantial evidence were to favour one of the above hypotheses, elevating it to the status of a theory, many of the mechanistic gaps would still have to be filled in concerning carpel evolution.

1.3 A phylogenetic framework for studies of carpel evolution Evolutionary studies of the carpel and other unique features of the flowering plants are possible in part due to the contribution of molecular data to phylogenetic analyses of the flowering plants and their relatives. Studies in the 1990s (Goremykin et al., 1996; Winter et al., 1999) indicated the extant seed plants to divide into the two monophyletic groups of the extant angiosperms and gymnosperms, respectively (Fig. 1.2). These findings replaced an earlier hypothesis, based on morphological data, that the small gymnosperm order Gnetales might form a sister group to the flowering plants, as discussed by Donoghue and Doyle (2000). Rather, it would seem that the living gymnosperms, including Gnetales, are monophyletic and share a more distant common ancestor with the flowering plant lineage, which would have lived some 300 MYA (Savard et al., 1994; Goremykin et al., 1997). According to the current consensus view of seed plant phylogeny, the living gymnosperms can be divided into five groups, with Cycadales in the most basal position, followed by the monotypic Ginkgoales, represented only by Ginkgo bioloba. In the crown group of living gymnosperms, most molecular phylogenetic studies split the conifers into two, placing Pinaceae in a sister position to Gnetales, as discussed by Kuzoff and Gasser (2000). This split leaves a clade of remaining conifers (Fig. 1.2) that, following the classification of Page (1990), would be composed of Araucariaceae, Cephalotaxaceae, Cupressaceae, Phyllocladaceae, Podocarpaceae, Sciadopityaceae, Taxaceae and Taxodiaceae. Molecular phylogenetic analyses have also clearly identified the firstdiverging lineages within the angiosperm clade, as reviewed by Kuzoff and Gasser (2000). According to these studies, three extant lineages, Amborellales, Nymphaeales and Austrobaileyales, collectively known as the ANA grade, would have diverged from a remaining common lineage at an early stage in the evolution of the flowering plants (Fig. 1.2). Amborellales contains the single species Amborella trichopoda, a small tree which is endemic to the tropical island of New Caledonia in the Southern Pacific. Nymphaeales contains three families of herbaceous aquatic plants: Nymphaeaceae, Cabombaceae, and the recently added Hydatellaceae (Saarela et al., 2007). Austrobaileyales contains the four families of Austrobaileyaceae, Illiciaceae, Schissandraceae and Trimeniaceae, which are mostly shrubs, climbers or small trees. There is good evidence, both from phylogenetic analyses and from INDEL (insertion/deletion) mutations (Aoki et al., 2004; Stellari et al., 2004), that Amborellales

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Figure 1.2 The phylogeny of the seed plants, based on a consensus of molecular phylogenetic studies. The numbers of species in major clades are given in parentheses, while approximate dates of divergence are taken from Davies et al. (2004), based on a calibration of the molecular clock using fossil data. The positions of some of the taxa referred to in the text are indicated as follows: Am, Amborella trichopoda; An, Antirrhinum majus; Ar, Arabidopsis thaliana; Ca, Cabomba; Cr, Catharanthus roseus; Il, Illicium; Ze, Zea mays (maize); Pe, Petunia hybrida; Or, Oryza sativa (rice).

and Nymphaeales diverged from the remaining angiosperm lineage before Austrobaileyales. However, the relative order of divergence of Amborellales and Nymphaeales remains unclear. Most molecular phylogenies have placed Amborellales alone in the most basal position (e.g. Zanis et al., 2002), while others have grouped it together with Nymphaeales in a first-diverging clade (Qiu et al., 2001). The remaining angiosperm lineage, after the divergence of the ANA grade, diversified to give five groups with living representatives: Cerratophyllum, Chloranthanceae, eumagnoliids, monocots and eudicots (Fig. 1.2). Of these,

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Carpel Evolution 9

the eudicots and monocots together account for over 95% of the estimated 300 000 extant angiosperm species, while some 6500 species of eumagnolids are known (Davies et al., 2004). The short internal branches connecting the five groups of euangiosperms, compared to their long terminal branches, mean that even very large DNA data sets have failed to convincingly resolve their relative points of divergence within the flowering plants (Moore et al., 2007). Within the eudicots, molecular phylogenetic analyses have clearly identified a number of early-diverging lineages, of which Ranunculales occupies the most basal position. The divergence of these basal lineages leaves a crown group eudicots that includes the major clades of the rosids, asterids and Caryophyllales (Fig. 1.2). The last common ancestor of these ‘core eudicots’ seems to have been a critical stage in angiosperm evolution, perhaps of equal evolutionary significance as the origin of the flowering plants itself. Indeed, many of the genes controlling flower development in model eudicots seem to be derived from a large-scale or whole-genome duplication that occurred in an ancestor of the core eudicots (Litt and Irish, 2003; Vandenbussche et al., 2003; Kramer and Hall, 2005). Phylogenetic studies do not of themselves explain the origin of the flower and its carpel. However, these studies provide an essential framework in which we may attempt to answer these questions, which Charles Darwin famously described as an ‘Abominable Mystery’. Part of the mystery surrounding the origin of the flower is the evolutionary distance of the flowering plants from their nearest living relatives, the extant gymnosperms, with no continuum of intermediate forms known from the fossil record. Molecular clock estimates, calibrated from well-documented fossil divergences within the angiosperms, suggest the last common ancestor of this group to have lived some 160 MYA (Davies et al., 2004). This date is in reasonable agreement with the first appearance of angiosperm fossils, corresponding to the Lower Cretaceous period, some 130 MYA (Friis et al., 2005). We must therefore conclude that the origin of the carpel, among other unique angiosperm features, occurred some 140 MY after the last common ancestor of the seed plants. It seems likely that the flower resulted from a combination of molecular and ecological or environmental factors. On the molecular level, a large-scale gene or whole-genome duplication seems to have preceded the radiation of the angiosperms (De Bodt et al., 2005), from which many pairs of paralogous genes that function in reproductive development have been retained. This hypothesized duplication may therefore have provided the raw material for neo-functionalization events on a large scale, which may have been necessary to generate such a novel structure as the flower. However, the generation of polyploids, corresponding to whole-genome duplications, is relatively common in plants (much more so than in animals). It therefore seems probable that the appearance of flowering plants in the Late Jurassic or Early Cretaceous, rather than at any earlier time since the last common ancestor of the seed plants, occurred not only because of a whole-genome duplication, but also in response to specific ecological or environmental conditions. Among such

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10 Fruit Development and Seed Dispersal ecological factors, co-evolution with novel groups of insects that acquired roles as pollinators seems likely to be of significance (Barrett and Willis, 2001). Certain novel features that contributed to the success of the early flowering plants may have been external to the flower itself. It has been proposed, for example, that the first flowering plants were shrubs or small trees, with a short lifespan compared to their gymnosperm-like ancestors (Feild et al., 2003, 2004). These somewhat ephemeral species are proposed to have found a successful ecological niche in disturbed soils along shaded riverbanks. Other suggestions for ecological factors that may have helped select for the flowering plants include a change in the feeding behaviour of herbivorous dinosaurs, related to a change in their jaw anatomy, and a change in atmospheric carbon dioxide concentration, as discussed by Barrett and Willis (2001). These conjectures serve to illustrate that the fullest possible explanation for the origin of the angiosperms will be achieved only by combining molecular data with the results of morphological and ecological studies and, if possible, with key evidence from fossil groups. In the absence of a living, close relative to the flowering plants, at least some novel fossil evidence would seem to be essential if we are to fill in the exasperating gap of 140 MY of evolution which preceded the origin of the flower.

1.4

A morphological portrait of the ancestral carpel

Morphological comparisons of ANA grade angiosperms (Fig. 1.3a–f) have enabled a number of conclusions to be made on the likely state of the flower, and of the carpel, in the last common ancestor of the living flowering plants (Endress and Igersheim, 2000; Endress, 2001). Accordingly, the flowers of this ancestral species were probably small, bisexual and protogynous. Its carpels were likely to have been simple (apocarpic), rather than fused together into a syncarpic pistil. The stigmatic tissues that permitted the capture of pollen grains in the angiosperms’ ancestor were likely to have been covered in multicellular protrusions and would probably have secreted a sticky liquid to hold and supply water to pollen grains during germination. Pollen tubes would then have grown towards the ovary through a canal or aperture in the carpel, which would have contained substances secreted from the carpel margins. Self-incompatibility (SI) systems operating between female tissues and pollen grains are present in some ANA angiosperms, including Austrobaileya scandens (Prakash and Alexander, 1984) and Trimenia moorei (Bernhardt et al., 2003). However, it is not yet clear whether these SI systems in distantly related Austrobaileyales are homologous, and still less certain that such a system would be ancestral in the entire angiosperm clade. Interestingly, Amborella, the only representative of the likely most basally diverging angiosperm lineage, Amborellales, avoids inbreeding by dieocy, rather than through an SI mechanism. However, female Amborella flowers contain a nonfunctional stamen, or staminode, which would seem to indicate Amborella to be descended from a bisexual ancestor.

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Figure 1.3 A comparison of carpel structures in ANA grade and core eudicot taxa. (a) Longitudinal section of an Amborella trichopoda (ANA grade, Amborellales, Amborellaceae) carpel (cr) showing its single anatropous ovule (o) and stigmatic crest (sc) of ridged tissue, which harbours an aperture through which pollen tubes may grow. (b) Transverse section of an A. trichopoda female flower bud showing the five separate carpels (cr) of the apocarpic gynoecium. (c) Longitudinal section of a Cabomba aquatica (ANA grade, Nymphaeales, Cabombaceae) flower bud showing two anatropous ovules (o) attached to the placenta (p) of one of the three carpels (cr) present. (d) Longitudinal (slightly oblique) section of a C. aquatica flower bud showing the secretion-filled canal (ca) in the style, through which pollen tubes may grow. (e) Transverse section of a C. aquatica flower bud showing the ovary tissues of the three separate carpels (cr). One or both ovules (o) are visible in each carpel. (f) Transverse section of a C. aquatica flower bud showing the secretion-filled canal (ca) in the style of each carpel. (g) Longitudinal section of the syncarpic gynoecium (gy) of Petunia hybrida (core eudicots, asterids, Solanaceae) showing many ovules (o) attached to an axile placenta (p). (h) Transverse section of the P. hybrida gynoecium showing its two fused carpels, axile placentation (p) and many ovules (o). (i) Transverse section of the syncarpic gynoecium (gy) of Arabidopsis thaliana (core eudicots, rosids, Brassicaceae), which is divided into two locules by the post-genital development of a vertical septum (vs). Placentation (p) is parietal. All scale bar represent 250 µm.

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12 Fruit Development and Seed Dispersal The carpel of the ancestral angiosperm probably contained a single ovule of anatropous placentation. This ovule would probably have been covered by two integuments that enclosed an embryo sac and a large nucellus (for more on ovule development, see Chapter 3 of this issue). Double fertilization, leading to the production of an embryo and a bi-parental endosperm, also appears to be a pleisiomorphic feature of the angiosperms. However, the likely cellular arrangement of the embryo sac in the last common ancestor of the angiosperms, and the ploidy of its endosperm tissue, remain open to question. In the majority of flowering plants, the embryo sac arrangement is of the Polygonum type, which contains seven cells, one of which, the central cell, is binucleate (Fahn, 1982). The two nuclei of the central cell combine with one sperm nucleus on fertilization of the Polygonum-type embryo sac to generate a triploid endosperm. In Nuphar (Williams and Friedman, 2002) and Hydatella (Friedman, 2008) of Nymphaeales and in Illicium (Williams and Friedman, 2004) of Austrobaileyales, the embryo sac contains only four cells, including a uninucleate central cell. Double fertilization in these ANA grade species generates a diploid embryo and a diploid, rather than a triploid, endosperm. However, studies of Amborella, which represents the likely most basally diverging ANA grade lineage, indicate a different embryo sac arrangement. The Amborella embryo sac contains eight cells, including a binucleate central cell that produces a triploid endosperm after fertilization (Friedman, 2006). The extra cell in the Amborella embryo sac, by comparison to the Polygonum type, is in the egg apparatus, which thus contains four cells in Amborella. Interestingly, Hydatella produces a perisperm, or embryo-nourishing tissue derived from maternal cells, which develops significantly prior to fertilization (Friedman, 2008). This feature resembles the arrangement in gymnosperms, in which entirely maternal tissues fulfil the role of nourishing the embryo, and in which substantial reserves are laid down before fertilization is effected. In the absence of a clear conclusion on the embryo sac arrangement in the last common ancestor of the extant angiosperms, the multiplicity of arrangements in ANA grade angiosperms has been interpreted as a sign of early diversification, prior to the selection of features which became standard in the majority of flowering plant groups (Friedman, 2006).

1.5

The genetic control of carpel development in the first flowering plants

Using molecular techniques to compare ANA grade angiosperms with model plants, we can now begin to describe the mechanisms likely to have controlled carpel development in the ancestor of the living flowering plants. Carpel development in the model taxa Arabidopsis and Antirrhinum is specified, according to the ABC model of flower development, through the expression of Cfunction MADS-box transcription factors, in the absence of B-function factors (Coen and Meyerowitz, 1991). The molecular genetic ABC model has more

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recently been extended to an ABCE model, in which E-function MADS-box factors form various higher order complexes with those of the A-, B- and C-functions (Pelaz et al., 2000; Honma and Goto, 2001; Theissen and Saedler, 2001). According to this model, carpel development is specified by tetramers containing two molecules of E-function, and two molecules of C-function, MADS-box transcription factors (Fig. 1.4a). The unisexual reproductive structures of gymnosperms appear to be specified by the expression of orthologues of B- and C-clade MADS-box genes (Tandre et al., 1995; Becker et al., 2000; Jager et al., 2003; Zhang et al., 2004), certain of which show similar activities to their Arabidopsis orthologues in transgenic Arabidopsis plants (Tandre et al., 1998; Winter et al., 2002; Zhang et al., 2004) (Fig. 1.4b). These data strongly suggest that some basic elements of the regulation of flower development have been conserved since the last common ancestor of the living seed plants, well before the origin of the flower. Phylogenetic analyses of the MADS-box family in ANA grade angiosperms and in gymnosperms clearly indicate that a duplication event took place in the C-function MADS-box lineage prior to the last common ancestor of the living flowering plants (Kim et al., 2005). As a result of this duplication, the ancestors of the clade-defining genes AGAMOUS (AG) from Arabidopsis thaliana, and FLORAL BINDING PROTEIN7 (FBP7) from Petunia hybrida (reviewed by Kramer et al., 2004) were generated. The AG clade contains angiosperm C-function genes, whereas the FBP7 clade contains genes involved in ovule development in diverse taxa, including Petunia, Arabidopsis and rice. The role of FBP7-like genes in ovule development has been defined as the D-function (Angenent et al., 1995; Colombo et al., 1995). This function is postulated to be necessary for ovule development, and its inactivation leads to supernumerary carpels that develop ectopically in the place of ovules. Interestingly, the FBP7 (D-function) clade appears to have been lost from Ranunculales (Kramer et al., 2004) and, as will be discussed later, has evolved to share its ovule development function with genes of the AG clade in some eudicots, including Arabidopsis. Both of these observations suggest a degree of functional fluidity between MADS-box genes of the related C- and D-clades. In addition to the C- and D-functions, two clades of SEPALLATA (SEP) genes, which encode E-function MADS-box proteins, have been found in basal angiosperms. The genes SEP1, SEP2 and SEP4 from Arabidopsis appear to be orthologous to one of these ANA grade SEP-clades, while SEP3 appears to be orthologous to the other (Zahn et al., 2005). The expression of C-function genes in ANA grade angiosperms is mostly limited to the third and fourth floral whorls, while the E-function genes of these species are expressed in all floral organs (Kim et al., 2005) (Fig. 1.4c). These expression patterns closely resemble those of the corresponding genes in Arabidopsis, suggesting important elements of the control of carpel identity to have been conserved in distinct lineages throughout angiosperm evolution. Kim et al. (2005) did however note some expression of C-function genes in the perianth organs of the ANA grade angiosperms Amborella (Amborellales)

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(d) Rice

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(a) Arabidopsis thaliana

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OsMADS1 (E- function)

Figure 1.4 The ABCE model of flower development in Arabidopsis, and its derivatives in other taxa. (a) In Arabidopsis, A-, B-, C- and E-function floral homeotic genes, expressed in overlapping domains (horizontal bars) of the floral meristem, control the identities of floral organs in a combinatorial manner: A + E specifies sepal development in the first whorl, A + B + E specifies petal development in the second whorl, B + C + E specifies stamen development in the third whorl, and C + E specifies carpel development in the fourth whorl. In addition, the C-function causes an arrest of organ proliferation (the ‘STOP’ function) in the fourth whorl. AG, AGAMOUS; AP1, APETALLA1; AP2, APETALA2; AP3, APETALA3; PI, PISTILLATA; SEP1–4, SEPALLATA1–4. (b) In gymnosperms, B- and C-clade MADS-box genes are expressed in a combinatorial manner in male (B + C) and female (C alone) reproductive structures, resembling the expression of their Arabidopsus orthologues in male and female floral organs. (c) In ANA grade angiosperms, B- and C-clade MADS-box gene expression resembles that of the respective Arabidopsis orthologues, though with less well-defined boundaries. Strong B-clade gene expression is generally detected in the outer floral whorl of ANA grade angiosperms, possibly reflecting an absence of developmental differentiation between whorls 1 and 2. A-clade MADS-box gene expression differs radically between ANA grade angiosperms and Arabidopsis, extending throughout the flower and into leaves. (d) In rice flowers, A-, B- and E-function genes are expressed in similar patterns to those of their Arabidopsis orthologues to specify specialized perianth organs (paleas, lemmas and lodicules) and stamens. Two paralogous C-clade MADS-box genes show a partial sub-functionalization between the third and fourth whorls, with one paralogue playing a major role in stamen development in the third whorl, while the other plays a major role in the ‘stop’ function in the fourth whorl (thick arrows, major roles; thin arrows, minor roles). The YABBY gene DROOPING LEAF (DL) plays a major role in carpel development that is independent of C-clade MADS-box gene expression. DL may act directly on carpel development (solid arrow), or indirectly by limiting the inner boundary of B-function gene expression (dashed arrow), or both of these.

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and Illicium (Austrobaileyales), in contrast to the more strictly delimited expression patterns of C-function genes in model eudicots. As pointed out by these authors, this observation may reflect the rather gradual transition of floral organ types that is apparent in ANA grade angiosperms, rather than a more fundamental difference in the regulation of carpel development between early and later diverging plant lineages. In addition to MADS-box genes, the expression patterns of several YABBY transcription factors have been analyzed in ANA grade angiosperms. In Arabidopsis, these factors participate in the specification of abaxial cellular identity in lateral organs by defining the side of these organs that faces away from the developmental axis (Bowman, 2000). CRABS CLAW (CRC) is a YABBY gene that is expressed only in the abaxial tissues of the gynoecium and in the nectaries of Arabidopsis flowers (Bowman and Smyth, 1999). AmbCRC, a putative CRC orthologue from Amborella, shows a similar pattern of expression in carpels to that of CRC in Arabidopsis (Fourquin et al., 2005), suggesting these genes to have conserved a common developmental role since the speciation event that separated their lineages at the base of the flowering plants. CRC is a direct target of AG in Arabidopsis (Gomez-Mena et al., 2005), though it is not yet known whether such a direct control relationship exists between the Amborella orthologues of these two genes. INNER NO OUTER (INO) represents a further YABBY gene with a very specific role in female reproductive development in Arabidopsis. INO is expressed in the outer ovule integument, and its inactivation causes the loss of this angiosperm-specific tissue (Villanneva et al., 1999). A putative INO orthologue from the ANA grade angiosperm Nymphaea alba is expressed in both ovule integuments and in the suspensor (Yamada et al., 2003). The broadly similar expression patterns of INO orthologues between the ANA grade angiosperm Nymphaea and the eudicot Arabidopsis suggest the conservation of a role in integument development since the last common ancestor of the flowering plants. CRC and INO are unusual in the YABBY family in showing very specific expression profiles: the other members of this family in Arabidopsis are more generally expressed in the abaxial zone of both vegetative and reproductive plant lateral organs (Bowman, 2000). The carpel and outer integument, in which CRC and INO are respectively expressed, represent pleisiomorphic features of the angiosperms. The relationships of these structures to reproductive organs in gymnosperms could therefore be highly informative of the mechanism by which the flower evolved. Hence, it would be extremely interesting to know whether CRC and INO orthologues exist in gymnosperms, and if so, to determine their exact expression patterns. In general, the search for carpel development genes in ANA grade angiosperms has highlighted several instances of the broad conservation of gene functions since the common ancestor of the last flowering plants. In particular, mechanisms involving C- and E-function genes, that specify carpel development at a high level in gene hierarchies, seem to be conserved. However,

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16 Fruit Development and Seed Dispersal much work remains to be done in this field. Many families of transcription factors of known importance to carpel development in Arabidopsis, including the auxin response factor (ARF), basic Helix–Loop–Helix (bHLH), MYB and NAC families, have yet to be examined in any detail in ANA grade angiosperms. As major elements of the ABCE model appear to be conserved between angiosperms and gymnosperms, it is at a lower level in regulatory hierarchies, perhaps involving the families listed above, that we might discover the molecular changes that were responsible for the evolution of the carpel in early flowering plants. EST resources are proving very useful for the analysis of flower development orthologues in numerous seed plant groups (Brenner et al., 2003; Albert et al., 2005; Brenner et al., 2005; Pavy et al., 2005, 2007). However, in any given investigation, the use of such database resources must frequently be complemented by onerous approaches such as cDNA library screening and reverse-transcriptase PCR. With the advent of novel, high-throughput sequencing technologies, the sequencing of the Amborella genome from the ANA grade, which has a c-value of 0.89 pg (Leitch and Hanson, 2002), has recently been proposed (Soltis et al., 2008) (The c-value indicates the mass of DNA in one haploid chromosome complement and is thus a convenient measure of genome size.) The sequencing of the Amborella genome and other genomes from the ANA grade would certainly provide an invaluable resource for studies of the origin of the flowering plants. The complete sequences of one or more gymnosperm genomes would also be extremely useful in this regard, though this seems still some way off due to the large genome sizes of this group. Gymnosperm genomes range in size from the relatively modest (for a gymnosperm) c-value of 2.25 pg for Gnetum ula (Ohri and Khoshoo, 1986) to the very large value of 36.00 pg for Pinus ayacahuite (Grotkopp et al., 2004). In particular, the cycads, which are of special interest for their basal position in the gymnosperms, all have large genomes. For example, Cycas revoluta has a c-value of 12.75 pg (Ohri and Khoshoo, 1986), some 80-fold larger than that of Arabidipsis thaliana (Bennett et al., 2003).

1.6

A major role for the E-function in the origin of the carpel?

E-function MADS-box genes play fundamentally important roles in flower development in Arabidopsis. The transcription factors encoded by these genes are hypothesized to act together with combinations of A-, B- and C-function proteins in quaternary complexes that specify the type of floral organ that develops in each whorl of the flower (Pelaz et al., 2000; Honma and Goto, 2001; Theissen and Saedler, 2001). As discussed above, two E-function clades seem to have been present in the last common ancestor of the extant flowering plants (Zahn et al., 2005), perhaps not very long after the origin of the flower. Interestingly however, no SEP-like genes have been found in gymnosperms

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(Becker and Theissen, 2003). It might therefore seem reasonable to postulate that the origin of the E-function, leading to the generation of quaternary MADS-box complexes, was of fundamental importance for the origin of the flower. Theissen and Melzer (2007) have discussed the possibility that, before the flower, dimers of C-function genes may have specified the development of female reproductive organs, and that the evolution of quaternary MADSbox protein complexes, incorporating both C- and E-function proteins, may thus have represented a key step in the evolution of the carpel. More precisely, the evolution of such quaternary complexes is hypothesized to have caused transcription factor binding to two distinct MADS-box binding motifs, which are termed CArG boxes, in the cis-acting control regions of their downstream target genes. According to this hypothesis, the newly evolved binding behaviour of quaternary MADS-box complexes, also involving Aand B-function proteins in different combinations, would have generated the necessary multiplicity of interactions to specify at least three organ types in early flowers, carpels, stamens and tepals, with the possible later division of tepals into distinct whorls of petals and sepals. Quaternary complexes might also have led to a positive cooperativity of binding to multiple sites in target gene promoters, which might in turn have generated a steeper gradient of transcriptional response between MADS-box genes and their direct targets. Such a steep gradient of response could have generated sharper transitions between the reproductive organs and produced the concomitant compaction of the axis that is apparent in the angiosperm flower. In discussing the evolutionary role of the E-function, it should be noted, however, that the origin of this function is, like that of the angiosperms, rather mysterious. The four SEP genes of Arabidopsis form an E-function clade that is closely related to the Arabidopsis gene AGAMOUS-LIKE6 (AGL6). Orthologues of AGL6, rather than those of the combined AGL6 + SEP-clade, appear to be present in gymnosperms (Carlsbecker et al., 2004). If this phylogenetic interpretation is correct, the SEP-clade would seem to have been lost from the extant gymnosperm lineage, rather than generated by a gene duplication event in the angiosperm lineage. In this case, the origin of the angiosperm SEP-clade could not have correlated with that of the flower. Even if the above phylogenetic interpretation is incorrect, and SEP genes did originate specifically in the angiosperm lineage, the presence of two distinct SEP-lineages in ANA grade angiosperms suggests the SEP-clade to have existed for some time prior to the origin of the flower. If indeed the SEP-clade considerably predates the flower, the formation of quaternary MADS-box complexes involving SEP proteins may also be far more ancient than the flower itself, and hence perhaps not a key factor in flower origin. Furthermore, the formation of quaternary MADS-box complexes may not involve SEP proteins in all plant groups. For example, AGL6 proteins, as close relatives of SEP proteins, might participate in quaternary MADS-box complex formation in gymnosperms. Careful attention should now be paid to the formation of complexes of MADS-box proteins in both angiosperms and gymnosperms

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18 Fruit Development and Seed Dispersal to attempt to address these questions. At present, it is not clear whether the origin of quaternary MADS-box protein complexes correlated with the origin of the flower.

1.7

Carpel specification in monocots

The monocots form a large, monophyletic group of angiosperms whose lineage diverged after the divergence of the ANA grade lineages, probably around 145 MYA (Davies et al., 2004). The functional comparison of carpel development genes in monocot and eudicot models provides evidence of differences in the molecular mechanisms specifying carpel development in these two groups. Genes controlling floral organ identity have been analyzed at a functional level in rice and maize of the Poaceae or grass family. Phylogenetic analyses suggest at least one major gene duplication event to have occurred in the MADS-box C-clade prior to the separation of the rice and maize lineages, with an additional duplication in one of the two sub-clades generated in that duplication, specifically in the maize lineage. Accordingly, the rice C-clade gene OsMADS58 appears orthologous to the maize gene ZAG1, while OsMADS3 from rice appears orthologous to both ZMM2 and ZMM23 from maize (Yamaguchi et al., 2006). The phenotypes associated with mutations in C-clade genes have been investigated in both rice and maize, though more thoroughly in the former of these species. The inactivation of OsMADS58 in rice leads to defects in carpel development, though it does not eliminate carpels (Yamaguchi et al., 2006) (Fig. 1.4d). In addition to abnormal carpels, osmads58 mutants show reduced floral determinacy, indicating a major contribution of this gene to the so-called ‘stop’ function, which arrests the proliferation of organs in the fourth whorl. Whereas OSMADS58 appear to act mainly in the fourth floral whorl, the inactivation of its paralogue OsMADS3 has little or no effect on either carpel development or floral determinacy. Instead, stamen development is eliminated in osmads3 mutants (Kang et al., 1998; Yamaguchi et al., 2006). Rice plants in which both OsMADS3 and OsMADS58 have been inactivated produce aberrant carpels, similar to those of the osmads58 mutant, suggesting OsMADS3 to make no unique contribution to carpel development (Yamaguchi et al., 2006). In maize, zag1 mutants show a defect in floral determinacy, indicating functional conservation of ZAG1 with its rice orthologue OsMADS58. It seems, therefore, that C-clade genes in the grass family have undergone significant sub-functionalization, following a monocot-specific gene duplication. The multiple roles of the single Arabidopsis gene AG in carpel development, stamen development and floral determinacy are thus shared in a whorl-specific manner between two and three C-clade genes in rice and maize, respectively. Additionally, in these monocot species, carpel development can occur independently of C-clade MADS-box genes, suggesting that some other factor may be involved in the specification of carpel development. The functions of a paralogous pair of D-clade genes, OsMADS13 and OsMADS21, have been investigated in rice (Dreni et al., 2007). Of these,

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Carpel Evolution 19

OsMADS13 shows ovule-specific expression and appears to play a classical D-function role in ovule development: inactivation of OsMADS13 results in the ectopic conversion of ovules into internal carpelloid organs. Interestingly, OsMADS21 appears to make no significant contribution to the D-function, and is expressed more widely in female reproductive tissues. It is thus tempting to speculate that OsMADS21 might show genetic redundancy in the control of carpel development with rice C-clade MADS-box genes. Double and triple knockout mutants between osmads21, osmads58 and osmadsS3 would be required to address this question. Though the inactivation of C-clade genes does not lead to the loss of carpels in rice flowers, carpels are entirely replaced by ectopic stamens in mutants in which the YABBY gene DROOPING LEAF (DL) has been inactivated (Yamaguchi et al., 2004) (Fig. 1.4d). dl mutants also show a developmental defect in leaves, which consequently lack a mid-rib. DL is the likely rice paralogue of CRC, though it clearly shows a considerable functional difference from CRC in Arabidopsis. DL expression is maintained in the carpels of rice plants in which both OsMADS3 and OsMAD58 have been inactivated (Yamaguchi et al., 2006), demonstrating its action to be independent of these C-function genes. This may represent a difference from the situation in Arabidopsis, in which CRC is a direct target of AG (Gomez-Mena et al., 2005). It is not yet clear whether carpel development depends on DL expression per se, or whether DL is mainly responsible for preventing B-function gene expression in the fourth floral whorl. Experiments that combine B-clade, C-clade and dl mutations in rice might help to evaluate the role of DL in floral patterning, and/or in carpel specification. The conservation of expression patterns of CRC orthologues between Arabidopsis and ANA grade angiosperms (Fourquin et al., 2005), as discussed above, suggests the distinct roles of DL in carpel identity and leaf development (Yamaguchi et al., 2004) to have arisen specifically in the monocot lineage. Experiments in which Arabidopsis crc-1 mutants were rescued by transformation with CRC orthologues from various species (Fourquin et al., 2007), however, showed the DL coding sequence to be capable of restoring near wild-type carpel development when expressed from the Arabidopsis CRC promoter. These experiments indicate the CRC and DL coding sequences to show similar activities in carpel development, suggesting that upstream factors may be largely responsible for the novel function shown by DL in carpel specification, or in the definition of the inner limit of the third whorl, in rice. It is not yet clear whether changes to the DL coding sequence, in addition to changes to its regulatory region, may have been necessary for the evolution of the leaf development function of DL in rice. Transformation of dl mutants with constructions containing eudicot CRC coding sequences may help to answer this question. SEP genes, which are necessary for carpel development in eudicots, are also known from monocots. OsMADS1 from rice corresponds to the LEAFY HULL STERILE1 locus, and groups within the same clade as SEP1, SEP2 and SEP4 from Arabidopsis (Zahn et al., 2005). Outer whorl floral organs in

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20 Fruit Development and Seed Dispersal osmads1 loss-of-function mutants take on a leaf-like appearance, whereas the inner whorl floral organs of these mutants are partially converted to paleas and lemmas, which are normally found only in the outer two whorls of rice flowers (Agrawal et al., 2005). These results suggest OsMADS1 to be a principal component of the E-function in rice (Fig. 1.4d), while the functions of the four remaining rice SEP genes, OsMADS5, OsMADS7, OsMADS8 and RMADS217 (Zahn et al., 2005), remain to be determined. In general, carpel and ovule development in the highly derived Poaceae of the monocots seem to depend on the orthologues of regulatory genes that are known to play key roles in these processes in Arabidopsis and other eudicots. However, duplications have taken place in several MADS-box gene lineages in Poaceae, including the C-, D- and E-function lineages, in some cases leading to sub-functionalization events between paralogous genes. The precise limits of this sub-functionalization have not yet been defined, which might explain the currently hidden component of the specification of carpel identity in monocots.

1.8

Gene duplication and carpel evolution in the core eudicots

The core eudicots form a monophyletic group that contains several very successful molecular genetic model species, including Arabidopsis thaliana, Antirrhinum majus and Petunia hybrida. This group is estimated to descend from a last common ancestor that lived around 110 MYA (Davies et al., 2004). Analysis of the Arabidopsis genome sequence has provided evidence of a largescale duplication event that probably occurred not long before the divergence of the main core eudicot lineages, the rosids, asterids and Caryophyllales (De Bodt et al., 2005). As discussed above, several classes of MADS-box genes in core eudicots contain pairs of paralogues that are orthologous to single genes in basal eudicots, such as Ranunculales, and which may therefore have been generated in the hypothesized core eudicot genome duplication event. The retention of many of these pairs of paralogues over long periods of evolutionary time would appear to be a clear indication that sub- and neofunctionalization processes have occurred, rendering both copies of each pair essential or advantageous to survival. In the core eudicots, two C-clades are present in place of an ancestral paleoAG clade in basally diverging eudicot lineages. In Arabidopsis, the euAG clade contains the AG gene itself, while the PLENA (PLE) clade (Fig. 1.5), contains a pair of paralogous genes termed SHATTERPROOF1 and SHATTERPROOF2 (SHP1/2), which resulted from a more recent duplication in the Arabidopsis lineage. In Antirrhinum majus, the probable orthologue of AG is termed FARINELLI (FAR), while that of SHP1/2 is the clade-defining gene PLE. Interestingly, the non-orthologous genes AG and PLE are responsible

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Carpel Evolution 21

(a) Arabidopsis thaliana AG sub-clade (AG)

PLE sub-clade (SHP1 and SHP2)

Stamen Carpal identity identity

(b) Antirrhinum majus AG sub-clade (FAR)

Fruit dehiscence zone development

PLE sub-clade (PLE)

Carpel identity Stamen Pollen identity fertility STOP function

STOP function

Ovule identity Funiculus and seed abscission layer development

FBP7 sub-clade (STK)

(c) Petunia hybrida AG sub-clade (PMADS3)

PLE sub-clade (FBP6)

FBP7 sub-clade (not characterized)

(d) Phylogeny of C-clade MADS-box genes

STOP

Stamen function identity Carpel identity

FBP-clade (D)

AG-clade (C) PLE-clade (C) Ovule identity

Pre-angiosperm duplication FBP7 sub-clade (FBP7 and FBP11)

Core-eudicot duplication

Figure 1.5 Fluidity in the functionalization of C- and D-function MADS-box genes in the core eudicots. (a–c) Venn diagrams representing the functions of genes from the MADS-box clades AG (C-function), PLE (C-function) and FBP7 (D-function) in three species of core eudicots. Overlapping regions represent functional redundancy between genes in wild-type genetic backgrounds. AG, AGAMOUS; FAR, FARINELLI; FBP, FLORAL BINDING PROTEIN; PLE, PLENA; SHP, SHATTERPROOF. (d) The sequence of duplications that generated of the eudicot AG, PLE and FBP7 MADS-box gene clades.

for specifying the C-function in Arabidopsis and Antirrhinum, respectively (Davies et al., 1999; Kramer et al., 2004) (Fig. 1.5). FAR, by contrast, is redundantly involved in stamen development and is required for pollen fertility in Antirrhinum. In an example of neo-functionalization, the paralogues SHP1 and SHP2 play a novel role in Arabidopsis fruit development (Liljegren et al., 2000). In Petunia hybrida, which, as a member of the asterids, is more closely related to Antirrhinum than to Arabidopsis (Fig. 1.2), a further case of sub-functionalization is apparent. The Petunia AG orthologue, PMADS3, is principally responsible for stamen development (Kapoor et al., 2002), though it also plays a redundant role with the PLE orthologue FLORAL BINDING PROTEIN6 (FBP6) in carpel development and floral determinacy (Kramer et al., 2004).

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22 Fruit Development and Seed Dispersal Though sub-functionalization between the paralogous AG and PLE clades in Arabidopsis has left AG playing the major C-function role, elegant experiments involving multiple mutants show that the SHP genes have retained a capacity for C-function activity, perhaps reflecting the C-function role of the common ancestor, prior to the radiation of the core eudicots, which these genes share with AG. Ectopic carpelloid organs develop in the first floral whorl of Arabidopsis ag mutants, conditionally on the inactivation of APETALLA2 (AP2), a gene which contributes to the A-function (Bowman et al., 1991). This effect is thought to occur because AP2 is responsible for downregulating C-clade MADS-box genes in the outer floral whorls of wild-type plants. In the case of ag/ap2 double mutants, the C-function activity responsible for specifying ectopic carpel development in the first whorl is provided by SHP1 and SHP2, as evidenced by the fact that first whorl organs of quadruple ap2/ag/shp1/shp2 mutants are devoid of all carpelloid features (Pinyopich et al., 2003). These data indicate a subtle effect of functional overlap between paralogous gene clades, which does not equate to simple genetic redundancy: the SHP genes adopt novel C-function-like roles in ap2/ag mutants which they do not play in wild-type plants. The fluidity of functions among duplicated genes in the core eudicots is further illustrated by an exchange of function between C- and D-clade MADSbox genes. Two paralogous D-function genes in Petunia, FBP7 and FBP11, are redundantly essential for ovule development (Angenent et al., 1995). The probable Arabidopsis orthologue of these genes, SEEDSTICK (STK), is also involved in ovule development, though STK shares this role redundantly with the C-clade genes SHP1 and SHP2 (Fig. 1.5). Accordingly, the Arabidopsis stk/shp1/shp2 triple mutant (Pinyopich et al., 2003), like the Petunia fpb7/fpb11 double mutant (Angenent et al., 1995), produces supernumerary carpels in the place of ovules within the gynoecium. In addition to its redundant role in ovule specification, STK plays non-redundant roles in the development of the funiculus and in seed abscission in Arabidopsis (Pinyopich et al., 2003). The combined C+D-clade in the eudicots, whose members were derived from duplication events that occurred both before and after the radiation of the angiosperms, therefore represents a complex situation in which diverse evolutionary processes have taken place. These processes include subfunctionalization between paralogous genes, exchanges of function between paralogous genes, exchanges of function between non-paralogous genes, and, finally, neo-functionalization to generate novel fruit shattering mechanisms (Fig. 1.5).

1.9

The A-function finds a role in fruit development

A further likely consequence of the hypothesized genome duplication at the base of the core eudicots is the generation of a second sub-clade of MADS-box genes within the A-clade (Litt and Irish, 2003). The A-function MADS-box gene APETALLA1 (AP1) plays roles in floral meristem patterning and in the

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Carpel Evolution 23

specification of perianth (petal and sepal) organ identity in Arabidopsis. This latter role corresponds to the A-function, as defined by the ABC and ABCE models. However, gene duplications in the core eudicots have provided further A-clade sequences, one of which appears to have been recruited to carpel and fruit development somewhere along the lineage leading to Arabidopsis. Accordingly, the Arabidopsis A-clade MADS-box gene FRUITFULL (FUL) is involved in the patterning of the gynoecium and fruit wall (Gu et al., 1998). FUL is known to act in a network involving a large number of genes (Roeder et al., 2003; Liljegren et al., 2004), including the MADS-box genes SHP1 and SHP2 (Ferrandiz et al., 2000) that also function redundantly with STK in ovule development, as described above. Gene duplication in the A-function clade of MADS-box genes has thus resulted in novel fruit shattering mechanisms in the Brassicaceae by the process of neo-functionalization. An interesting feature of gene-duplication in the A-clade is the evolution of a distinct C-terminal protein motif in AP1 genes, apparently produced by a frame-shift mutation that occurred towards the 3 -extremity of the coding sequence in an ancestor of the core eudicots (Litt and Irish, 2003). This frameshift created a farnesylsation site in the encoded protein that is known to be post-translationally modified in vivo in Arabidopsis and which is required for wild-type AP1 protein activity (Yalovsky et al., 2000). Other frame-shift mutations in duplicated genes are present in the B- and C-function MADS-box clades of the eudicots (Vandenbussche et al., 2003). However, the conserved motifs generated in these cases are distinct from that of the AP1 lineage and do not contain farnesylation sites. The novel C-terminal motifs present in certain lineages within the eudicot A-, B- and C-clades of MADS-box genes have been conserved over a long period, clearly indicating their functional significance. However, it is not yet known whether the functions of these novel motifs are connected with biochemical processes in common, such as the higher order assembly or sequestration of MADS-box transcription factor complexes (Vandenbussche et al., 2003).

1.10

The multiple origins and mechanisms of syncarpy in the angiosperms

Most angiosperm flowers possess more than one carpel. As discussed above, the carpels of species from the early-diverging ANA grade lineages are typically separate structures that occur in a spiral arrangement at the centre of the flower (Fig. 1.3a–f). Such an arrangement, with separate carpels, is termed apocarpic and, from its presence in early-diverging lineages, appears to represent the pleisiomorphic condition of the angiosperms. However, more than 80% of extant angiosperm species are syncarpic: their carpels are fused into a single female structure in the centre of the flower (Endress, 1982) (Fig. 1.3g–h). Various morphological sequences have been described which lead to carpel fusion in syncarpic species (Verbeke, 1992). These developmental processes

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24 Fruit Development and Seed Dispersal can be conceptually divided into two types, based on the timing of the fusion event involved. Accordingly, in cases in which carpels are fused from the earliest emergence of carpel primordia, the organ fusion is termed ‘congenital’, whereas in cases in which carpels are observed to fuse together during development, the fusion is termed ‘post-genital’. Congenital carpel fusion is the most common type, whereas post-genital carpel fusion is known from only a handful of angiosperm families (Lolle and Pruitt, 1999). Arabidopsis, which typifies gynoecium development in the Brassicaceae, is a good example of congenital carpel fusion. At developmental stage 6 of Arabidopsis flowers (Smyth et al., 1990), a central dome emerges from the flower meristem which will become the syncarpic gynoecium. By developmental stage 7, this dome begins to invaginate by a reduction in growth rate at its centre to generate a slot, which forms in line with the pair of lateral stamen primordia of the Arabidopsis flower bud. The tissues on either side of this slot, perpendicular to the lateral stamen primordia, then grow out to meet in the middle, eventually forming a vertical septum that divides the ovary of the Arabidopsis gynoecium into two locules. The gynoecium wall undergoes considerable differentiation at the extremities of the vertical septum to define the dehiscence zones that will permit pod shattering in the mature fruit. Interestingly, though Arabidopsis is a clear example of a species showing congenital syncarpy, it is not absolutely clear how many carpels are fused together in the Arabidopsis gynoecium. Most recent authors (Okada et al., 1989) have interpreted this structure as containing two carpels, corresponding to the two locules of the (secondarily) divided ovary. This view is to some extent supported by molecular genetic studies, which have succeeded in isolating a number of mutants in which carpel fusion is affected, resulting in the division of the Arabidopsis gynoecium into two separate carpelloid organs. However, the ovary wall contains four vascular traces in Arabidopsis, two of which occur at either extremity of the vertical septum, and two in positions corresponding to the valves of the ovary. Hence, it is entirely possible that there are four carpels in the wild-type Arabidopsis gynoecium (Lawrence, 1951). According to this view, the valve carpels would have become sterile, whereas the carpels at either extremity of the vertical septum, corresponding to the positions of the placentae, would have remained fertile. Postgenital syncarpy has been best characterized in Catharanthus roseus (The Madagascar Periwinkle) of the Apocynaceae. In this species, two separate carpel primordia are initiated and grow until their inner surfaces come into contact (Walker, 1978; Siegel and Verbeke, 1989; Verbeke, 1992). The already differentiated epidermal cells of these surfaces then begin to interlock and redifferentiate into parenchyma. This redifferentiation is dependent on diffusible, water-soluble substances produced by the carpels themselves and takes a total of about 9 h (Siegel and Verbeke, 1989). Even before epidermal cell redifferentiation has terminated, plasmodesmata can be observed to form between the surface layers of the fusing carpels (Vanderschoot et al., 1995). Walker (1978) has shown that the pollen tubes are then able to cross between

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Carpel Evolution 25

the carpels, thus fulfilling one of the principal theoretical reasons for the evolution of syncarpy, which are explained in greater detail below. The major evolutionary advantage of syncarpy is probably to allow a regular repartition of pollen tubes within the gynoecium by the formation of a ‘compitum’, which consists of tissues that permit pollen tube transfer between carpels (Endress, 1982). Accordingly, a visit to a syncarpic by a single pollinator might result in full seed set. Another potentially important advantage of syncarpy results from the enhanced competition between pollen tubes that this phenomenon produces, which may act as a filter of fitness by selecting for vigorous male parents. Syncarpy also allows the production of larger fruits, with potentially more complex and efficient seed dispersal mechanisms (Walker, 1978; Endress, 1982; Armbruster et al., 2002). A further advantage of syncarpy may stem from its potentially lesser requirement for cell wall synthesis, compared to an apocarpic gynoecium of similar total size. Though most representatives of basally diverging angiosperm lineages are morphologically apocarpous, Endress (1982) has noted several unusual constructions that confer a degree of functional syncarpy. In Nymphaea (Nymphaeaceae), for example, large quantities of mucilage are secreted from the stigmatic tissues of the separate carpels. Pollen tubes are able to grow through the resulting mucilage layer, which is sufficiently extensive as to form a bridge between the carpels. In Tambourissa of the eumagnolid family Monimiaceae (Laurales), a ‘hyperstigma’ is generated by the entire female flower, which forms a cup that fills with mulicage into which pollen grains may fall and germinate (Endress, 1982). The separate carpels of Tambourissa develop in this floral cup, and are thus potentially accessible to any pollen grain present. Functional syncarpy in the ANA grade genus Illicium (Austrobaileyales) is achieved by the growth of pollen tubes in a groove around a central axis which connects the separate carpels (Williams et al., 1993). By mapping different arrangements of morphological and functional syncarpy onto a phylogeny of the angiosperms, Armbruster et al. (2002) estimated that there have been at least 17 independent transitions from apocarpy to syncarpy during angiosperm evolution. Only two instances, by contrast, of a likely change from syncarpy to morphological apocarpy were noted. As syncarpy seems to have arisen several times independently in the angiosperms, it is possible that distinct molecular mechanisms have been recruited to bring about carpel fusion in distantly related syncarpic groups. In Arabidopsis, numerous mutations, including aintegumenta, crabs claw, ettin, leunig, spatula and tousled, generate various degrees of carpel separation, either singly or in double mutant combinations (Sessions and Zambryski, 1995; Roe et al., 1997; Alvarez and Smyth, 1999; Liu et al., 2000). It is not, however, clear whether these genes played any role in the evolution of syncarpy in the Arabidopsis lineage. Rather, the inactivation of such key regulators of carpel development might disrupt the delicate and labile process of carpel fusion, which perhaps evolved through other, unrelated molecular changes. One genetic system that may potentially have been responsible for the generation of

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26 Fruit Development and Seed Dispersal congenital syncarpy, however, does seem worthy of particular mention. The genes CUP-SHAPED COTYLEDON1 and 2 (CUC1/2) (Aida et al., 1997) are members of the NAC transcription factor family which promote, in a partially redundant manner, organ separation in a number of positions in the plant, including the cotyledons, floral meristems and leaf margins. Both CUC1 and CUC2 are, together with five other NAC genes, negatively regulated by the MIR164 family of microRNAs, which contains three genes in Arabidopsis. Interestingly, both the triple mutant mir164abc (Sieber et al., 2007), and plants transformed with a miR164-resistant allele of CUC2 (Nikovics et al., 2006), show complete carpel separation, thus implicating the negative regulation of CUC2 by miR164 in the process of carpel fusion in Arabidopsis. The CUC and MIR164 system may represent a conserved developmental module that has been recruited many times independently over the course of angiosperm evolution to modify certain highly variable traits in the angiosperms, such as leaf dissection (Nikovics et al., 2006). It is therefore possible that this developmental module has also been independently recruited to generate syncarpy in distinct angiosperm lineages. The transformation of diverse syncarpic species with miR164-resistant alleles of their own native CUC genes could help to shed some light on this question. A clue to the potential molecular mechanism of post-genital carpel fusion in C. roseus and other taxa comes from the Arabidopsis mutant fiddlehead (fdh). In fdh mutants, all above-ground organ types, including leaves, stems, sepals, petals and stamens, tend to fuse together on contact (Lolle et al., 1992). In addition, wild-type Arabidopsis pollen can readily germinate and emit pollen tubes into leaf and other non-carpel tissues of fdh mutants (Lolle and Cheung, 1993). FDH encodes an enzyme necessary for the generation of the waxy cuticle (Pruitt et al., 2000). Inactivation of FDH makes the cuticle much more permeable to small molecules, and this effect also seems to be conserved in Antirrhinum majus (Efremova et al., 2004). Interestingly, the transfer of small water-soluble molecules is known to be involved in post-genital carpel fusion in C. roseus (Siegel and Verbeke, 1989). It thus seems plausible that carpel fusion in C. roseus and other taxa showing post-genital fusion might depend on the down-regulation, specifically in the contacting surface of developing carpels, of a gene such as FDH that is necessary for cuticle formation.

1.11

A fruit by any other name: evolutionary convergence between angiosperms and gymnosperms

The fruit is a pleisiomorphic character of the angiosperms, and to use this term to describe non-angiosperm seed bearing structures might be regarded as botanical heresy! However, it is interesting to note that the reproductive structures of several groups of living gymnosperms have evolved to superficially resemble angiosperm fruits. For example, the aril of the yew, Taxus baccata (Taxaceae), is red and fleshy at maturity, and contributes to

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seed dissemination by forming a food source for birds. Arils in Taxaceae and Taxodiaceae develop from ovuliferous scales that grow around the seed after pollination. These structures are thus quite distinct from angiosperm fruits, which are formed by the modification of pre-existing carpel tissues. Similarly, in Gnetum of the gymnosperm order Gnetales, a fruit-like structure is generated from two extra tissue layers that form around the unitegmetic ovule. In the case of Gnetales, the production of fruit-like structures represents just one of a long list of evolutionary convergences with angiosperms (Donoghue and Doyle, 2000). For example, a form of double fertilization is present in this group, though this leads to the production of a second, inviable embryo, rather than endosperm tissue (Friedman, 1998). Other evolutionary convergences of Gnetales with angiosperms include an apical meristem that is divided into a tunica and corpus, vessel elements in xylem tissues, a lack of archegonia, net-veined leaves (in Gnetum), morphologically bisexual male strobili (in Gnetum and Welwitschia), and even insect pollination (in some Gnetum spp.) (Kato et al., 1995). Structures playing the role of fruits, but which are not derived from carpels, have also evolved in some angiosperm groups. Examples of such false fruits include the ‘pome’, which develops from the receptacle in Rosaceae including Malus and Pyrus, and the false berry of Vaccinium spp.

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34 Fruit Development and Seed Dispersal Yalovsky, S., Rodriguez-Concepcion, M., Bracha, K., Toledo-Ortiz, G. and Gruissem, W. (2000) Prenylation of the floral transcription factor APETALA1 modulates its function. Plant Cell 12, 1257–1266. Yamada, T., Ito, M. and Kato, M. (2003) Expression pattern of INNER NOOUTER homologue in Nymphaea (water lily family, Nymphaeaceae). Development Genes and Evolution 213, 510–513. Yamaguchi, T., Lee, D.Y., Miyao, A., Hirochika, H., An, G.H. and Hirano, H.Y. (2006) Functional diversification of the two C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa. Plant Cell 18, 15–28. Yamaguchi, T., Nagasawa, N., Kawasaki, S., Matsuoka, M., Nagato, Y. and Hirano, H.Y. (2004) The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa. Plant Cell 16, 500–509. Zahn, L.M., Kong, H., Leebens-Mack, J.H., Kim, S., Soltis, P.S., Landherr, L.L., Soltis, D.E., de Pamphilis, C.W. and Ma, H. (2005) The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout Angiosperm history. Genetics 169, 2209–2223. Zanis, M.J., Soltis, D.E., Soltis, P.S., Mathews, S. and Donoghue, M.J. (2002) The root of the angiosperms revisited. Proceedings of the National Academy of Sciences of the United States of America 99, 6848–6853. Zhang, P.Y., Tan, H.T.W., Pwee, K.H. and Kumar, P.P. (2004) Conservation of class C function of floral organ development during 300 million years of evolution from gymnosperms to angiosperms. Plant Journal 37, 566–577.

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Chapter 2

GYNOECIUM PATTERNING IN ARABIDOPSIS: A BASIC PLAN BEHIND A COMPLEX STRUCTURE Eva Sundberg1 and Cristina Ferrándiz2 1

Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden 2 Instituto de Biolog´ıa Molecular y Celular de Plantas (UPV-CSIC), Campus de la Universidad Politécnica de Valencia, Avda de los Naranjos s/n, Valencia, Spain

Abstract: The Arabidopsis gynoecium consists of two congenitally fused carpels that, at maturity, form a bilocular chamber protecting the ovules and placentae produced by the meristematic regions of the carpel margins. This meristematic region also gives rise to a style capped with stigmatic papillae at the apical end of the developing gynoecia and a transmitting tract that connects the stigma to the ovule-bearing chambers. Most data point towards a common evolutionary origin of leaves and carpels and suggest that leaves can be transformed to carpels by expressing only a few carpel identity genes. In this review, we have therefore approached the carpels from the leaf-like organ hidden within by stressing the parallels between leaf and carpel development. Many of the genes with a role in leaf development were first identified by the effect their mutations cause in carpel development, suggesting that the regulatory networks may be more robust in leaves than in the more complex and evolutionary younger carpels. Similar genetic networks ensure the maintenance of adaxial–abaxial, proximal–distal and medial–lateral dichotomies in leaves and carpels. Data have emerged showing that crosstalk and redundancies are characteristics of these pathways, as well as a general interplay of hormonal balances, with auxin as a major morphogen. Keywords: gynoecium; fruit; patterning; hormones; auxin; development

2.1 Introduction In 1735, the Swedish botanist Carolus Linneaus published the first version of Systema Naturae where he presented a sexual system for classifying Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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36 Fruit Development and Seed Dispersal flowering plant species according to the number of male and female organs, a system both easily accessible and useful, although not completely correct in evolutionary terms. Linnaeus became fascinated by the complexity, beauty and the romantic aura of the plant reproductive system and in an essay, shockingly to some of his contemporaries, he portrayed the more or less enclosed space formed by the petals as a bridal chamber and the gynoecium, the female reproductive structure and most complex organ of the flower, as a bride. This extraordinary bride consists of one or more ovule-bearing organs, so-called carpels, whose structures have contributed to their evolutionary success and have been used to define the group of flowering plants, which are termed angiosperms in Greek, meaning ‘seed enclosed in a vessel’ (angion, vessel and sperma, seed). The ingenious bridal organ protects the ovules, assists in the selection of male gametophytes and aids pollen tube growth for successful pollination. After fertilization of the ovules, the gynoecium develops into a fruit that initially protects and subsequently disperses the seeds. All present data are consistent with Goethe’s early hypothesis that floral organs originate from ancestral vegetative leaf-like organs (Goethe, 1790). For example, ectopic expression of a set of floral organ identity genes is sufficient to convert leaves to floral organ-like structures (Pelaz et al., 2001; Castillejo et al., 2005), and, accordingly, loss of function of SEPALLATA 1 (SEP1), 2, 3 and 4 results in the transformation of all floral organs into vegetative leaves (Honma and Goto, 2001; Ditta et al., 2004). The hypothesis that carpels have evolved from leaves, probably spor-producing sporophylls, is therefore widely accepted. However, it is still debated which ancestral type of sporophyll was recruited and how it evolved into a closed gynoecium (reviewed in Scutt et al., 2006 and Chapter 1 in this book). Molecular and genetic research in the model plant Arabidopsis thaliana has provided us with basal knowledge of the frameworks controlling gynoecium development. Reviews picturing what is known about the regulation of carpel and fruit development in Arabidopsis have recently been published (Dinneny and Yanofsky, 2005; Roeder and Yanofsky, 2005; Balanzá et al., 2006). Here, we aim to complement those reviews and to summarize the recent progress in the field. As in Balanzá et al. (2006), we have attempted to approach the carpels from the leaf-like organ hidden within by stressing the parallels between leaf and carpel development. We have also highlighted the role of hormones in gynoecium morphogenesis now that the view on hormone action in relation to development has rapidly become more comprehensive.

2.2

The basic plan in lateral organs

Most data point towards a common evolutionary origin of leaves and floral organs. It is likely that the pathways regulating the basic organ plan inherited from the leaf-like ancestral organs are active still during floral organ

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Gynoecium Patterning in Arabidopsis 37

development, although perhaps in a slightly modified manner downstream of the floral identity genes. In strong support of this hypothesis, the majority of floral organ patterning mutants are also defective in the development of the basic plan of leaves. So, how is patterning of lateral organs, formed at the flanks of the shoot apical meristem (SAM), regulated? Huge progress has recently been made in discerning genetic networks promoting leaf initiation and patterning. Several in-depth reviews have recently been published in this field of plant development (Tsukaya, 2003, 2006; Byrne, 2005, 2006; Fleming, 2005; Piazza et al., 2005; Aida and Tasaka, 2006; Kepinski, 2006; Shani et al., 2006; Barkoulas et al., 2007) and the aim in this section is only to give a brief overview of how the basic plan of lateral organ develops in Arabidopsis, which will be used in later sections as a framework to present the gynoecium. 2.2.1 Lateral organ initiation The first questions we would like to address is: How are lateral organs initiated? And what changes are required at the initiation zone to allow for organ primordia to be formed? Lateral organ primordia are established in the peripheral zone (PZ) of the SAM, a process preceded by the local downregulation of SAM promoting factors (Fig. 2.1a). Maintenance of the undifferentiated state of the meristematic cells in the SAM requires the activity of transcription factor class I KNOTTED1-like homeobox (KNOX) family members (Hake et al., 2004) partially acting by modulating hormonal balances (Jasinski et al., 2005; Yanai et al., 2005). High cytokinin levels induce CyclinD-mediated cell division and low gibberellin represses cell expansion, two characteristics important for undifferentiated cells (Riou-Khamlichi et al., 1999; Dewitte et al., 2007). KNOX proteins directly regulate the level of these hormones by binding the promoter and repressing the expression of the gibberellin biosynthesis gene GA20-OXIDASE (ga20ox; Hay et al., 2002; Chen et al., 2004) and by activating the cytokinin biosynthesis gene ISOPENTENYL TRANSFERASE7 (IPT7) in Arabidopsis (Yanai et al., 2005). Prior to lateral organ initiation, KNOX gene activity becomes repressed at the initiation sites, and auxin has been proposed as a major factor influencing this process. In the PZ, auxin flux is directed to local sink positions by the action of auxin efflux facilitators such as PINFORMED (PIN), resulting in high local auxin concentrations, pinpointing the location of lateral organ primordium emergence (Reinhardt et al., 2000, 2003; Heisler et al., 2005). In these auxin peak positions, KNOX gene activity is silenced by the converging activities of auxin and a transcriptional repressor complex consisting of the MYB protein ASYMMETRIC LEAVES1 (AS1) and the LOB domain protein AS2 which results in the promotion of leaf development (Hay et al., 2006; Guo et al., 2008). The AS1/AS2 complex may also serve to maintain KNOX silencing during leaf development by recruiting the chromatin-remodelling factor HIRA to the KNOX gene (Phelps-Durr et al., 2005).

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38 Fruit Development and Seed Dispersal (a) HD-ZIPIII ta-siRNA AS

KNOX CUC

ETT/ARF4 KAN ETT/ARF4 YABBY miR165/6

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Figure 2.1 (a) Interactions between transcription factors, small RNAs and hormones for SAM maintenance, lateral organ initiation and establishment of adaxial–abaxial polarity in the leaf. (b) Hormonal and genetic determination of boundaries between SAM and lateral organs. (c) Genetic factors involved in the establishment of distal–proximal polarity of the leaf.

2.2.2

Establishment of boundaries

Next, we will tackle the question how boundaries between the SAM and the newly initiated lateral organ are established? Once the new organ primordium has been initiated, auxin flux in the SAM is redirected to a new position for initiation of the next lateral organ (Heisler et al., 2005). This results in the depletion of auxin and a concomitant change in gene expression in the cell layers immediately adjacent to newly formed primordia, thus creating distinct cell types with reduced cell division activity which forms a morphological boundary that separates the primordia from the rest of the meristem (Fig. 2.1b; Heisler et al., 2005; reviewed by Aida and Tasaka, 2006). Growth repression in the boundary between the SAM and lateral primordia appears to be mediated by the NAC domain transcription factors CUP-SHAPED COTYLEDON (CUC1), CUC2 and CUC3 (Vroemen et al., 2003; Hibara et al., 2006; Sieber et al., 2007) and the JAGGED LATERAL ORGANS (JLO) LBD

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domain protein, which promotes the boundary function by activating KNOX genes and repressing PIN activity (Borghi et al., 2007). 2.2.3 Establishment of the adaxial–abaxial asymmetry Outward growth of the delimited and initially radial leaf primordium is immediately accompanied by the establishment of an adaxial–abaxial (dorsoventral) asymmetry required for the expansion of a flattened lamina. Cells adjacent to the SAM develop adaxial characteristics typical of the photosynthetic upper part of the leaf, whereas cells on the opposite side become abaxialized and form the lower part carrying out gas exchange (for recent reviews see: Golz, 2006; Chitwood et al., 2007; Kidner and Timmermans, 2007; Xu et al., 2007). Establishment of the adaxial–abaxial axis is dependent on the conversion of positional signals provided by the SAM and likely also other surrounding areas, into differential expression of mutually antagonistic transcription factors (e.g. HD-ZIPII and KAN, see below) in the adaxial or abaxial domains, respectively (Fig. 2.1a). Adaxial identity in Arabidopsis leaves is primarily specified by the partially redundant class III homeodomainleucine zipper (HD-ZIPIII) genes PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV) (McConnell and Barton, 1998; McConnell et al., 2001; Emery et al., 2003; Prigge et al., 2005) and exclusion of activity of these genes from the abaxial domain is mediated, at least in part, by miRNA165/166 via posttranscriptional cleavage and/or chromatin modifications (Emery et al., 2003; Kidner and Martienssen, 2003; Tang et al., 2003; Bao et al., 2004a; Mallory et al., 2004; Williams et al., 2005b; Alvarez et al., 2006; Zhou et al., 2007). Adaxial fate also appear to be promoted by AS1 and AS2 via the repression of abaxial identity in the adaxial domain (Lin et al., 2003; Xu et al., 2003; Fu et al., 2007). Abaxial fate, on the other hand, is cooperatively specified by the redundant KANADI (KAN) transcription factors belonging to the GARP family and the auxin response factors ARF3/ETTIN (ETT) and ARF4 (Eshed et al., 2001, 2004; Kerstetter et al., 2001; Pekker et al., 2005). The activity of the two ARF genes is restricted by ta-siRNAs encoded by TAS3 and recent work shows that the ta-siRNAs accumulate on the adaxial side of the leaf primordium where they repress the activity of the ARFs and other abaxial factors (Vazquez et al., 2004; Williams et al., 2005a; Adenot et al., 2006; Fahlgren et al., 2006; Garcia et al., 2006; Hunter et al., 2006; Nogueria et al., 2007). YABBY transcription factor encoding genes that appear to act downstream of both KANs and the ARFs are sufficient to specify abaxial fate and promote leaf lamina expansion in Arabidopsis (Sawa et al., 1999; Siegfried et al., 1999; Eshed et al., 2001, 2004). The mutually antagonistic activity of the KAN and HD-ZIPIII genes is not only required during leaf blade formation but also to establish polarity during embryogenesis and the development of the vasculature (Eshed et al., 2001; Emery et al., 2003; Izhaki and Boman, 2007). How is the polarity information obtained? Microsurgical separation of emerging leaf primordia from the shoot apex results in abaxialized radial

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40 Fruit Development and Seed Dispersal structures that fail to expand laterally (Sussex, 1954; Reinhardt et al., 2005). It has therefore been suggested that a meristem-derived signal (the Sussex signal) specifies adaxial identity. This signal, the nature of which still is unknown, must continuously enter the primordium during the early developmental stages in order to suppress abaxial identity (Reinhardt et al., 2005). The HD-ZIPIII START domain appears capable of lipid/sterol binding and has therefore been suggested to be a putative target of an unknown SAM-derived lipid/sterol signal (McConnell et al., 2001). Recently, the ta-siRNA encoded by TAS3 has also been suggested as a Sussex signal that could repress the activity of abaxial determinants in the adaxial domain (Garcia et al., 2006). In contrast to microRNAs, which appear to be cell autonomous, siRNA-induced gene silencing in Arabidopsis spreads from cell to cell (Tretter et al., 2008). Mobile signals specifying abaxial identity have also been suggested. The involvement of auxin response factors in abaxial cell fate specification introduces auxin as a possible abaxial polarizing signal (Pekker et al., 2005). The auxin influx facilitator AUX1 localizes specifically to the abaxial epidermal layer, suggesting that auxin flows into the abaxial half of the incipient primordium and establishes an auxin gradient across the primordium (Reinhardt et al., 2003). 2.2.4

Patterning of leaf shape and vasculature formation

How is leaf shape controlled? Auxin gradients appear to coordinate processes determining the final shape and size, as well as venation patterning of the developing leaf. It has been proposed that the tip of the developing leaf primorida is established as an auxin sink via directed auxin transport (Reinhardt et al., 2003), thereby generating a distal–proximal auxin gradient with its maximum at the tip of the developing primordium (Benková et al., 2003). This gradient could provide positional information for morphogenesis and has been suggested to be important for midvein development (Mattsson, 1999; Zgurski et al., 2005). As they develop, primordia would become auxin sources, synthesizing auxin first at the leaf tip and then at the symmetrically positioned hydathodes at the leaf margins. This proposed sink–source transition coincides with the lateral growth of primordia and the formation of the secondary veins (Avsian-Kretchmer et al., 2002; Aloni et al., 2003; Sawchuk et al., 2007). Eventually, gradients of cell proliferation and differentiation/expansion become established in the distal–proximal and medial–lateral (from midvein to the margin of the leaf lamina) axes. Cell division rates gradually decline in a gradient from the tip of the leaf to the base as cells begin to differentiate (Donnelly et al., 1999; Nath et al., 2003), whereas cell divisions cease in the mid-region of the leaf slightly ahead of divisions at the margins (Byrne, 2005). Although no comprehensive view of growth regulation in lateral organs has emerged so far, the activity of JAGGED (JAG), ARGOS, AINTEGUMENTA (ANT), ANGUSTIFOLIA3 (AN3), GROWTHREGULATING-FACTOR5 (GRF5), PEAPOD (PPD), LEAFY PETIOLE (LEP) and class I TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) encoding genes have been shown to prolong the period during which proliferative

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cell divisions occur during leaf organogenesis (Fig. 2.1c; Misukami and Fischer, 2000; Van Der Graaff et al., 2000; Hu et al., 2003; Horiguchi et al., 2005; Li et al., 2005; Dinneny et al., 2006; Ingram and Waites, 2006; White, 2006; reviewed in Anastasiou and Lenhard, 2007). The auxin-inducible ARGOS acts through ANT, and the class I TCP proteins bind to and activate the promoters of cyclin and ribosomal protein genes. Class II TCP proteins appear to have an opposite function and promote growth cessation and tissue differentiation from the tip to the leaf base (Fig. 2.1c; Nath et al., 2003; Palatnik et al., 2003; Ori et al., 2007; Efroni et al., 2008). Thus, modulations in activity of these factors may constitute the evolutionary basis for variations in leaf size and shape. Apart from a gradual polarity in cell proliferation, the Arabidopsis leaf also shows a distinct distal–proximal asymmetry. The leaf blade develops in the distal part whereas the very proximal end is occupied by a narrow petiole lacking leaf blade. Recently, two genes specifying petiole identity by the repression of cell proliferation and growth have been identified; BLADE-ON-PETIOLE1 (BOP1) and BOP2, both encoding BTB/POZ domain proteins. These are expressed in the proximal part of the leaf where they promote petiole differentiation by repressing JAG activity as well as, indirectly via AS2/LOB, several KNOX genes (Ha et al., 2003, 2004, 2007; Hepworth et al., 2005; Norberg et al., 2005), resulting in early cellular determination. 2.2.5 Marginal shapes Fine-tuned regulation of cell proliferation and differentiation also determines the shape of the leaf margins (Palatnik et al., 2003; Dinneny et al., 2004). In leaves with smooth edges, auxin responsiveness is evenly distributed along the margins (Aloni et al., 2003) whereas serrations with hydathodes at their tips, both producing and strongly responding to auxin, are formed in a specific pattern during leaf serration. Leaf serration is associated with repression of the meristem and boundary gene CUC2 at the teeth site, and an activity balance of CUC2 and miRNA164A between the teeth determines the depth of the sinus (Nikovics et al., 2006). These data exemplify a common reuse of processes related to meristem function during lateral organ development. In summary, robust and complex genetic networks ensure the maintenance of the meristem–primordia, adaxial–abaxial, proximal–distal and medial–lateral dichotomies (Fig. 2.1). Crosstalk, redundancies and common regulators are characteristics of these pathways, as well as a general coordination provided by the interplay of hormonal balances, with auxin as a major morphogen.

2.3 The Arabidopsis gynoecium The gynoecium in Arabidopsis consists of two carpels congenitally fused that, at maturity form a bilocular chamber with parietal placentation that contains

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Figure 2.2 The Arabidopsis gynoecium. The different axes of polarity and morphological terms are indicated (a) scanning electron micrograph of the mature gynoecium at anthesis. (b) Chloral hydrate cleared anthesis gynoecium to reveal vascular patterns. Primary and secondary bifurcations of the medial veins are indicated with arrows. (c) Cross section of the ovary at anthesis. (For a colour version of this figure, please see Plate 1 of the colour plate section.)

around 50–80 ovules. Gynoecium primordium is first visible at stage 5 of flower development (according to Smyth et al., 1990) as an oval-shaped bulge at the centre of the floral primordium surface. At stage 6, it becomes clearly distinct, as an oval platform that subsequently (stages 7–8) develops a central invagination and grows into an open hollow tube. At later stages, the internal medial regions bulge giving rise to two opposing meristematic ridges, which in turn produce the placentae and ovules laterally and fuse in the centre to form the septum. At stage 9, the epidermis at the open apical end starts to differentiate stigmatic cells and the style becomes distinct. At stage 11, the gynoecial tube closes and different cell types are clearly visible. At anthesis, all tissues required for fertilization have fully developed and those required for fruit maturation and dehiscence are already specified, although they will become functional after fertilization (Bowman et al., 1999; Alvarez and Smyth, 2002; Roeder and Yanofsky, 2005). In Fig. 2.2, the different regions of a mature gynoecium are shown. Along the apical–basal axis: the stigma, composed of a single layer of papillar cells; the style, a solid structure that externally shows characteristic crenellated cells and is rich in stomata; the ovary, comprising most of the length of the gynoecium, which houses the ovules; and the gynophore, a short basal stalk-like structure which connects the ovary to the base of the flower. In a transverse section of the ovary (Fig. 2.2c), the patterning along the medial–lateral axis is shown: the valves correspond to the two carpel walls and are placed in lateral positions; in the valve margins 2–3 columns of smaller cells are visibly forming narrow furrows. The medial positions are formed by the

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fused margins of the carpels: the replum corresponds to the external medial domain; the septum, which forms postgenitally and divides the ovary longitudinally; and the ovules, derived form the placentae. The transmitting tract, a specialized tissue that facilitates pollen tube growth, runs along the entire length of the gynoecium from the stigma and through the centre of the style and the septum. All these medial tissues, including the apical style and stigma, are called marginal tissues, since they all derive from the margins of the two fused carpels. Both lateral and medial tissues show adaxial–abaxial polarity, with different cell types and tissues in the abaxial (external) and the adaxial (internal) positions. The vasculature in the gynoecium at anthesis is formed by four major veins, two lateral and two medial, that longitudinally span the entire gynoecium (Fig. 2.2b). The lateral veins are ontogenetically related to the middle vein in the leaf, and run through the centre of the valves terminating close to their apical end. The medial veins extend through the repla, bifurcating extensively when they reach the style. The mature Arabidopsis fruit is a dehiscent silique, and therefore, specialized tissues develop to facilitate the shattering process and seed dispersal. The valve margins develop the so-called dehiscence zones, comprising a separation layer of small cells, which defines a longitudinal plane of rupture at both sides of the replum, and a patch of adjacent lignified cells. These lignified cells, together with the lignification of the valve internal subepidermal layer, are critical for the dehiscence process, since, when the mature fruit dries the harder lignified regions provide mechanical tensions that facilitate pod opening (Ferrándiz, 2002).

2.4 Genetic and hormonal factors controlling gynoecium development Many mutations that affect gynoecium development in Arabidopsis, and their corresponding genes, have been identified in the last few years, and the list is still growing. As we will see, only one of them causes a complete loss of carpel identity, few others can be related to the development of specific tissues within the gynoecium, and many others contribute to the distribution of territories and the establishment of polarity axes. As expected, most of the genes in this later group also have a role in leaf patterning. Thus, the genetic networks that direct lateral organ development and which we have described previously also work at patterning the two carpels that constitute the Arabidopsis gynoecium. Interestingly, many of the genes with a role in lateral organ development were first identified by the effect that their mutations caused in carpel development, suggesting that these genetic networks may be more robust in leaves than in the more complex and evolutionary younger carpels.

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44 Fruit Development and Seed Dispersal 2.4.1

From the leaf to the carpel: identity genes

Two decades after the original formulation of the ABC model, which explained floral organ identity based on the phenotypes and genetic interaction of floral homeotic mutants (Coen and Meyerowitz, 1991), it has been extensively validated. Intense research carried out in many labs during these years has confirmed the ABC models original postulates and, moreover, it has revealed much of the underlying molecular mechanisms, integrating all this information in the nowadays widely accepted ‘floral quartet model’ for organ identity (Krizek and Fletcher, 2005; Theissen and Melzer, 2007). We have previously introduced how, according to this model, leaves can be transformed into floral organs by expressing the corresponding combination of organ identity genes. In Arabidopsis, to make a carpel out of a leaf, we need to express the C-function gene AGAMOUS (AG) and one of the SEP genes (Honma and Goto, 2001). AG stands alone at the top of the hierarchy of carpel identity genes because ag single mutants are unique in lacking carpels completely: carpels are replaced by a reiteration of the sequence sepals–petals–petals. This is because, in addition to C-identity function, AG is required to prevent floral meristem indeterminacy. Also, as the ABC model predicted, A and C functions are mutually exclusive and in ag mutants, A-function expands to all whorls, causing the homeotic transformation of stamens into petals. Because of its central role in reproductive development, much is currently known about AG regulation, mechanisms of action and targets. AG is a member of the MADS-box gene family of transcription factors, like SEP and all but one of the ABC genes. Both AG and SEP are already expressed at the inception of the carpel primordia, before any morphological sign of differentiation (Yanofsky et al., 1990; Savidge et al., 1995; Mandel and Yanofsky, 1997). AG is activated in the floral meristem by the joint action of the products of the floral meristem identity gene LEAFY (LFY) and the meristem maintenance homeobox gene WUSCHEL (WUS). Once present, AG negatively feeds back on WUS through currently unknown mechanisms, terminating WUS expression and thus, floral meristem activity (Lenhard et al., 2001; Lohmann et al., 2001). While the LFY–WUS pathway appears to play a major role, independent mechanisms for AG activation must exist, as inferred from the phenotypes of lfy mutants, where AG is still expressed and carpelloid organs develop, or the normal stamens found in wus mutants (Weigel and Meyerowitz, 1993; Laux et al., 1996). Little is known about these alternative routes, although it has been shown that in 35S::SEP3 plants, AG is ectopically expressed, suggesting that SEP3, likely in combination with other MADS-box genes, could participate in these LFY-independent pathways (Castillejo et al., 2005). The A-function gene APETALA2 (AP2) prevents AG expression in the two outer whorls of the flower. In addition, many other factors have been described that refine the AG spatial and temporal pattern of expression, both at the transcriptional and posttranscriptional levels (Liu and Meyerowitz, 1995;

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Sieburth and Meyerowitz, 1997; Franks et al., 2002; Cheng et al., 2003; Bao et al., 2004b). The prominent role of AG in establishing carpel identity is not, however, unique in providing carpelloid features to floral organs. This fact was revealed by the phenotypes of some ag mutant combinations like the ap2 ag double mutants, where most carpel tissues still develop in the first whorl organs, even in the absence of AG function (Bowman et al., 1991). This observation led to the proposal of an AG-independent pathway for carpel identity, but in fact, it is better explained by the functional redundancy of AG with two other highly related MADS-box genes, SHATTERPROOF1 and SHATTERPROOF2 (SHP1, SHP2), primarily involved in the specification of valve margin identity (Liljegren et al., 2000). All carpelloid features disappear in the ap2 ag shp1 shp2 quadruple mutant. Moreover, complementation studies have shown that SHP and AG proteins are largely equivalent at the functional level, and that their distinct roles mostly derive from their different expression patterns (Pinyopich et al., 2003). In the wild-type flower, SHP1 and SHP2 are activated in a broad domain at early stages of carpel development, and later they become restricted to placental tissue, valve margins and ovules. SHP expression, as well as these tissues, is absent in ag mutants, and this observation, together with additional molecular studies, places the SHP genes downstream of AG (Savidge et al., 1995). However, this appears to be only partially true since, as seen in ap2 ag mutants, they can be activated independently of AG and are also under negative regulation by the A-function. Filamentous Flower (FIL), YABBY3 (YAB3) and JAG, present in both leaves and floral organs, have been shown to jointly activate SHP in the valve margins (Dinneny et al., 2005; see below), but their possible roles in activating SHP in the first whorl organs of ap2 ag mutants are unexplored at this point. As we will see later, when discussing the development of the different territories, AG seems to be absolutely required for valve identity, but the development of most of the other tissues depend on two factors that appear to act downstream of AG/SHP. SPATULA (SPT) encodes a bHLH transcription factor that is widely expressed in different specific tissues throughout vegetative and reproductive development (Heisler et al., 2001). CRABS CLAW (CRC) belongs to the YABBY family but is specifically expressed in nectaries and carpels (Bowman and Smyth, 1999). spt mutants show defects in the development of most carpel specific tissues (see below), whereas crc gynoecia are shorter and wider than wild type and partially unfused at the top. The crc spt gynoecium develops as two unfused organs with a very reduced amount of ovules and of stigmatic and stylar tissue. Furthermore, loss of SPT and CRC function in the ap2 ag background mimics the phenotype of ap2 ag shp1 shp2 mutants (Alvarez and Smyth, 1999). CRC has been identified as a direct target ´ of AG (Gomez-Mena et al., 2005), and less is known about SPT activation, although both genes are still expressed in the ectopic carpels of ag ap2 mutants (Bowman and Smyth, 1999), suggesting that their expression is driven by SHP. Thus, CRC and SPT appear to mediate AG/SHP specification of many

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46 Fruit Development and Seed Dispersal carpel tissues, although expression of CRC or SPT outside the gynoecium context does not appear to be sufficient to induce ectopic carpelloid features, suggesting that they could require AG/SHP activities to exert their functions. Supporting this idea, CRC and SPT are also involved in other aspects of AG function, such as the control of meristem determinacy (Alvarez and Smyth, 1999). In this sense, it is also noteworthy that in rice, for example the CRC orthologue DROOPING LEAF (DL) has a prominent role in C-function and, while mutants in the rice AG orthologue only have mild floral defects, dl mutant flowers show complete homeotic conversions of carpels into stamens and loss of floral determinacy (Yamaguchi et al., 2004). 2.4.2

Partitioning the gynoecium

At inception (stage 6 of flower development), the gynoecium primordium appears as a small mound of cells. Early patterning events act to partition the growing primordium, first specifying abaxial (outer) versus adaxial (inner) domains and medial versus lateral domains, and only later, as the primordia grows into a tube-shaped organ, to define apical–basal polarity. Adaxial–abaxial patterning basically follows the plan at work in all lateral organs that we have discussed above. Already at stage 6, all types of abaxial factors are expressed in the external domains of the gynoecium: KAN genes (Sessions et al., 1997; Kerstetter et al., 2001; Pekker et al., 2005), the auxin response factors ETT and ARF4 and the YABBY genes like FIL or YAB3 are also expressed abaxially, together with CRC, which is not found in leaves or other floral organs. As previously discussed, genetic networks directing adaxial–abaxial polarity are complex and rely on redundancy to provide robust patterning along this axis. Most single mutants in any of the ‘abaxial’ loci only show weak phenotypes, but mutant combinations can cause strong polarity defects that can turn the gynoecium inside out. Thus, for example crc kan1, kan1 kan2 or ett arf4 double mutants exhibit dramatic phenotypes with ovules and transmit tract tissues developing on the external side of the gynoecium (Eshed et al., 1999, 2001; Pekker et al., 2005). Adaxial HD-ZIP factors in the wild-type gynoecium primordium are expressed in their expected internal domains. Although the effect of HD-ZIP mutations in gynoecium development has not been documented in detail and appears to be less conspicuous than in leaves, it has been described that gain-of-function mutations in PHB or REV cause phenotypes similar to loss of function mutations in the abaxial KAN1 (McConnell and Barton, 1998; Zhong and Ye, 2004; Dinneny et al., 2006). In addition, mutants in the small RNA processing machinery show polarity defects in the carpels as well (Bohmert et al., 1998). All these data indicate that the same antagonistic relationships between KAN/YAB and HD-ZIP found in leaves are maintained in the gynoecium. However, not only a new abaxial player such as CRC is incorporated to the scheme, but also additional factors appear to contribute to specify adaxial fate in the carpels. NUBBIN (NUB) is a C2H2 zinc-finger transcription factor closely related to

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JAG (Dinneny et al., 2006). JAG is expressed in lateral organs, including floral organs, in a non-polar manner, and its role in lateral organ development has been related to promote cell proliferation and differentiation (Dinneny et al., 2004; Ohno et al., 2004). However, NUB expression is restricted to the adaxial domain of leaves, stamens and carpels and the double jag nub mutants display abaxialized stamens and carpels. The expression of polarity genes in jag nub carpels is not significantly affected and thus, NUB/JAG activities have been placed downstream or in parallel to the KAN/YAB/HD-ZIP network. However, it is still unclear whether the role of NUB/JAG in promoting adaxial fate is direct or reflects a requirement for correct organ growth to maintain organ polarity (Dinneny et al., 2006). Medial and lateral domains within the gynoecium are also specified in early stages of development. Since we have been stressing parallelisms between leaves and carpels to understand the common mechanisms that direct their development, we need to point out here their major differences. Medial–lateral symmetry in leaves (Fig. 2.1c) cannot be directly translated into the medial–lateral domains in the gynoecium. As previously described, the Arabidopsis gynoecium develops from two congenitally fused carpels and, in such a structure, medial domains correspond to the fused margins of the two carpels and will give rise to the marginal tissues, while the lateral domains are the ‘blade’ of these organs and later will develop into the valves. Medial domains in the gynoecium differentiate adaxially a new meristem, the medial ridge, which shares the typical three-layered structure of the SAM although instead of dome shaped, develops as an elongated ridge along the gynoecial tube (Hill and Lord, 1989; Azhakanandam et al., 2008). Thus, within the emerging gynoecium primordium, the dichotomy of meristematic versus differentiated lateral domains appears along the medial–lateral axis. Accordingly, several genes involved in SAM maintenance like the KNOX genes SHOOT MERISTEMLESS (STM) or BREVIPEDICELLUS (BP), or other homeobox genes like REPLUMLESS (RPL; a.k.a. PENNYWISE, BELLRINGER and VAAMANA) are expressed in the medial domain, as well as the ‘boundary’ genes CUC1 and CUC2 (Long et al., 1996; Aida et al., 1997; Ori et al., 2000; Pautot et al., 2001; Byrne et al., 2003; Smith and Hake, 2003; Bhatt et al., 2004; Alonso-Cantabrana et al., 2007). In contrast, genes that repress the undifferentiated state in the primordia of lateral organs and promote their development (like AS1/AS2, the YAB genes, the HD-ZIP genes or JAG/NUB) are restricted to lateral domains (Bowman and Smyth, 1999; Siegfried et al., 1999; Dinneny et al., 2006; Alonso-Cantabrana et al., 2007). Since most mutants in meristem maintenance genes do not make flowers, strategies to overcome this problem have been used to determine their specific role in carpel development. Thus, for example flowers produced out of calli regeneration of cuc1 cuc2 mutants fail to fully develop marginal tissues (Ishida et al., 2000). A significant contribution has been recently made by Scofield et al. (2007). Using inducible STM-RNAi, the effect of reduced STM activity on gynoecium development was studied. Phenotypes ranged from total absence of carpel

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48 Fruit Development and Seed Dispersal development to gynoecia that appeared completely unfused and lacked all signs of marginal tissue development. Conversely, overexpression of STM or the related gene KNAT2, was able to induce ectopic carpel formation in a largely AG-dependent manner, indicating a prominent role of KNOX genes in both gynoecium specification and patterning (Pautot et al., 2001; Scofield et al., 2007). As discussed above, genes expressed in emerging leaf primordia that direct adaxial–abaxial polarity in both leaves and floral organs are generally excluded from the medial domains of the gynoecium primordium. Thus, the KAN/YAB/HD-ZIP network appears to work essentially the same to specify the adaxial–abaxial axis in the gynoecium, but, surprisingly, at least the KAN genes are also required to maintain the medial–lateral dichotomy, since in the heavily adaxialized kan1 kan2 double mutants, adaxial marginal tissues such as ovules and transmitting tract develop all over the external side of the gynoecium in a radial manner (Eshed et al., 2001). While these radialized phenotypes could just reflect the requirement of abaxial–adaxial polarity to maintain the correct growth of lateral domains, they could also point to a possible role of KAN genes in the coordination of both adaxial–abaxial and medial–lateral patterning, although the mechanisms involved in such coordination are still unclear. KAN1 and KAN2 expression patterns in the developing gynoecium have not been extensively documented, although it appears that they change dynamically from some compartments to others during development. Likewise, the effect of kan mutations on the expression of gynoecium patterning genes has only been limitedly addressed (Eshed et al., 2001; Kerstetter et al., 2001). The auxin response factors ETT and ARF4, as in leaves and other lateral organs, appear to work together with the KAN genes in the specification of abaxial fate in the gynoecium, as revealed by the almost identical phenotypes of kan1 kan2 and ett arf4 double mutants and the nature of their genetic interactions (Pekker et al., 2005). ETT expression in the young gynoecium primordia is abaxial, and spans the whole perimeter of the gynoecial tube (Sessions et al., 1997) yet, in the ett arf4 double mutant, radialization of the primordium occurs. In addition, in mutants with reduced activity of the YUCCA (YUC) genes, which encode enzymes in the auxin biosynthetic pathway, as in yuc2 yuc4, or mutants in the PINOID (PID) kinase, a regulator of auxin transport, gynoecia develop as a solid radial structure capped by a ring of stigma (Bennett et al., 1995; Cheng et al., 2006). Taking all these data together, it is tempting to speculate that auxin could play a role not only in abaxial–adaxial polarity but also in medial–lateral compartmentation and be part of this hypothetical coordination. 2.4.3

Development of the lateral domains

The lateral domains differentiate into the valves, including the valve margins that eventually will develop into the dehiscence zones. Several factors involved in the specification of carpel identity also play major roles in the

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development of these tissues (reviewed in Ferrándiz, 2002; Dinneny and Yanofsky, 2005; and Chapter 5 of this book). Thus, AG is required for valve identity specification, as deduced from the phenotype of ap2 ag mutants, where most of the gynoecium tissues still form in the first whorl organs but they lack the typical cell layer structure and some specific cell types of the valves (Alvarez and Smyth, 1999). CRC, expressed in the lateral domains at early stages, also influences valve growth and development, and in crc mutants, vascular development in the carpels shows an abnormal pattern of branching resembling that of leaves or sepals and suggesting a partial loss of carpel identity. Conversely, SHP1 and SHP2 are placed at the top of the hierarchy directing valve margin development, as shp1 shp2 double mutants fail to differentiate valve margins and develop siliques unable to open. Downstream additional factors involved in valve margin specification are INDEHISCENT (IND) and ALCATRAZ (ALC), two members of the bHLH family of transcription factors whose mutations also result in indehiscent phenotypes. IND is exclusively expressed in the valve margins, while SHP and ALC are expressed in the margins and in additional domains that, notwithstanding, do not appear to be affected in the corresponding mutants (Liljegren et al., 2000; Rajani and Sundaresan, 2001; Liljegren et al., 2004). SHP, IND and ALC expression is tightly confined to the valve margin by two antagonistic activities, the MADS-box factor FRUITFULL (FUL) and the homeodomain protein RPL, that operate in the valves and the replum, respectively (Gu et al., 1998; Roeder et al., 2003). In ful mutants, SHP, IND and ALC are ectopically expressed in the valves which, as a consequence, develop the small lignified cells typical of dehiscence zones and are unable to elongate and break prematurely. Conversely, SHP, IND and ALC are not expressed in 35S::FUL carpels, which in turn give rise to indehiscent fruits (Ferrándiz et al., 2000; Liljegren et al., 2004). In rpl mutants, the replum is indistinct and the lignified cells of both dehiscence zones appear merged (Roeder et al., 2003). FUL and SHP appear to be the major factors directing cell fate in the lateral domains, namely the valves and the valve margins, respectively. It has been shown that both are under the regulation of some of the ‘lateral factors’ that we have described before (Fig. 2.3). Thus, the cooperative activities of FIL, YAB3 and JAG positively regulate the transcription of FUL and the SHP genes in the valve and the valve margin, respectively. It has been proposed that, while a higher FIL/YAB3/JAG activity in the valves would turn on FUL expression in this domain, SHP genes would only be activated by the weaker FIL/YAB3/JAG activity present in the valve margins (Dinneny et al., 2004). Then, FUL would repress SHP expression, neatly delimiting the two domains of FUL-expressing valve and SHP-expressing valve margin territories (Ferrándiz et al., 2000). In the replum, RPL represses the expression of FIL/YAB3/JAG genes, therefore preventing SHP activation (Roeder et al., 2003; Dinneny et al., 2005). Thus, FUL and RPL restrict SHP expression to a narrow domain spanning 3–4 cell layers between the valve and the replum. In this domain, SHP activates IND and ALC, which are ultimately in charge

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STM BP

AS FIL/YAB3 JAG

RPL SHP IND ALC

FUL

Valve

Replum Margin

Figure 2.3 Model for medial–lateral patterning of the gynoecium. Genetic interactions of the factors directing the development of valves, valve margins and repla. A hypothetical model based on two opposing gradients of valve factors and replum factors explains the positioning of the valve margins at the overlapping domain of both activities.

of directing the differentiation of the tissues in the dehiscence zone (Liljegren et al., 2000, 2004; Rajani and Sundaresan, 2001). Both ful and rpl show extreme phenotypes of ectopic dehiscence zone formation. However, a change in dehiscence zone position, and therefore, of the proportions of valve/replum territories in the fruit can be observed in other genetic backgrounds, generally related to the establishment of the medial–lateral boundaries discussed previously. This supports the idea of medial–lateral dichotomy as equivalent to meristematic-differentiated. Thus, as1 or as2 mutants show enlarged medial regions resulting in expanded repla and narrow valves, as do fruits overexpressing the class I KNOX gene BP (Alonso-Cantabrana et al., 2007). KNAT6, another class I KNOX gene, which in wild-type fruits is expressed in the valve margins, is ectopically expressed in the repla of rpl mutants and this expression appears to be crucial for the rpl phenotypes, as in rpl knat6 double mutants, the replum is restored to a wild-type size (Ragni et al., 2008). YABBY genes have been described to downregulate KNOX genes in lateral organs (Kumaran et al., 2002), thus providing a link between the AS/KNOX antagonism to control replum size and the

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FIL/JAG activities to direct valve regionalization. A hypothetical but attractive model has been proposed to explain how the position of the boundaries between lateral and medial domains, and therefore the dehiscence zones, is established. In this model, a gradient of FIL/YAB3/JAG and AS valve factors with a lateral maximum would coexist with a gradient of KNOX/RPL replum factors with a medial maximum. Both gradients would antagonize each other and only overlap in a narrow domain, where the valve margin would be formed (Fig. 2.3; Alonso-Cantabrana et al., 2007; Mart´ınez-Laborda and Vera (Chapter 5 of this book)).

2.4.4 Marginal tissue development Early during gynoecium development, the meristematic medial ridge develops on the internal, adaxial part of the medial domain, localized at the boundary between the two fused carpels. Genetic data and gene expression patterns strongly suggest the medial ridge to coordinately generate all tissues required for the reproductive competence of the gynoecium; the placenta, ovules, septum and associated transmitting tracts, as well as portions of the style and stigma. The lateral organ boundary genes CUC1 and CUC2 are expressed in the medial ridge from very early stages of development (Fig. 2.4; Ishida et al., 2000; Takada et al., 2001). In cuc1 cuc2 mutant gynoecia, there is an

LUG/SEU/ANT/YAB SPT/HEC

SHI/STY TOP

CUC KNOX HD-ZIP Auxin

ETT

Figure 2.4 Model for apical–basal patterning of the gynoecium. According to the auxin gradient hypothesis (Nemhauser et al., 2000), auxin levels are proposed to be high in the apical regions promoting development of style and stigma whereas intermediate levels determine the ovary and low levels the gynophore. Auxin synthesis in the apical end could be under the control of STY/SHI and NGA genes, while SPT and ETT may be involved in translating the auxin gradient into differentiation programmes.

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52 Fruit Development and Seed Dispersal apical gradient of decreasing medial tissue production, sometimes resulting in an unbroken cylinder of valve tissue (Ishida et al., 2000). It has therefore been suggested that the CUC genes initially act to maintain an undifferentiated state of the medial cells and subsequently promote the expansion and fusion of the medial ridge resulting in septum formation (Alvarez and Smyth, 2002). As discussed previously, class I KNOX meristem maintenance genes are also expressed in the medial ridge from early in gynoecium development (Fig. 2.4) and, in the few stm mutants that flower, carpels exhibit reduced fusion and medial tissue growth (Endrizzi et al., 1996; Long et al., 1996; Pautot et al., 2001). Activity of class I KNOX genes has also been detected in the meristematic placenta, as well as in the ovules and the style suggesting multiple roles of this class of genes during development of marginal tissues (Ori et al., 2000; Venglat et al., 2002; Alonso-Cantabrana et al., 2007; Scofield et al., 2007). As mentioned in an earlier section, expression of adaxial identity genes, such as the HD-ZIP genes, within the adaxial margin has been suggested to be required for development of the placenta and for ovule initiation (Alvarez and Smyth, 2002). As in the SAM, all data so far suggest that the pluripotent medial cells represent a low auxin level/response zone, whereas high auxin responses are detected in some of the tissues subsequently formed by the medial cells. Several genes required for carpel fusion and development of the medial ridge-derived structures have been identified through mutant screening, but it is still unclear if they are involved in determining marginal tissue identity or merely growth of the medial ridge required for further development. Many of these genes share some functional redundancy, and although the contribution of each gene is rather limited, their collective activity is essential for the development of marginal tissues. It has been suggested that LEUNIG (LUG), SEUSS (SEU), ANT and FIL may be part of a multimeric complex important for the development of the medial domain of the gynoecium (Fig. 2.4; Azhakanandam et al., 2008). LUG and SEU encode transcriptional coregulators, with sequence similarity to the Tup1/Groucho and Ssdp/Chip protein families, and to the LIM-domain-binding protein family, respectively (Conner and Liu, 2000; Franks et al., 2002). LUG and SEU act as repressors of AG activity in the first two floral whorls, can interact physically and have been proposed to coregulate common targets throughout numerous stages of plant development (Liu and Meyerowitz, 1995; Franks et al., 2002; Sridhar et al., 2004). The lug mutant gynoecia are unfused at the apical end, and exhibit defects in the septum fusion and ovule development, consistent with its expression pattern (Liu and Meyerowitz, 1995; Chen et al., 2000; Conner and Liu, 2000). Gynoecia of seu mutants are also often split at the apical end whereas mutations in both SEU and LUG completely abolish the development of fourth whorl organs, and the homeotic carpelloid organs that form in whorl one show no trace of marginal tissues (Franks et al., 2002). The Arabidopsis LUG homologue LUH shares redundant functions with LUG, including interactions with SEU, and overlapping functions among SEU-like

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(SLK) genes has also been suggested (Conner and Liu, 2000; Franks et al., 2002; Azhakanandam et al., 2008; Sitaraman et al., 2008). Like lug and seu, ant mutants exhibit partially unfused carpels (Elliot et al., 1996). Additional functional similarities of LUG, SEU and ANT include promotion of leaf growth as well as repression of AG in outer floral whorls (Liu and Meyerowitz, 1995; Chen et al., 2000; Misukami and Fischer, 2000; Franks et al., 2002; Krizek et al., 2000; Cnops et al., 2004). Marginal tissue development is fully dependent on the cooperative activities of ANT and LUG, as revealed by the complete lack of replum, style, septum and placental tissues in the gynoecia of ant lug double mutants (Liu et al., 2000). ANT encodes an AP2/ERF domain DNA-binding transcriptional activator expressed in the medial ridge and medial-derived tissues, as are also several ANT-like (AIL) genes (Krizek, 2003; Nole-Wilson et al., 2005; Krizek and Sulli, 2006). Knock-down of individual AIL genes has no effect on medial tissues, suggesting functional redundancy within the gene family. The defects in medial tissue development in seu ant double mutant gynoecia are also severe. seu ant gynoecia are split in the apical part, fail to initiate ovule primordia, produce reduced amounts of other medial tissues and display a reduced activity of the adaxial identity genes PHB and REV suggesting that ANT and SEU may potentiate the expression of the adaxial fate determinants (Azhakanandam et al., 2008). Apart from ANT, LUG and SEU, some YABBY genes may also be important for medial tissue formation. First, gynoecia of fil lug and fil ant double mutants show similarities to seu ant or even lug ant double mutants (Chen et al., 2001; Nole-Wilson and Krizek, 2006). Second, evidence that SEU and LUG may physically cooperate with FIL comes from analysis of orthologous genes in Antirrhinum majus (Navarro et al., 2004). Finally, LUG, SEU and ANT appear to be required for maintaining abaxial–adaxial polarity by positively regulating FIL expression and/or activity (Franks et al., 2006; Nole-Wilson and Krizek, 2006). Apart from the HD-ZIP genes, additional putative downstream effectors of the LUG/SEU/ANT/YAB proteins in gynoecia have been suggested, which all appear auxin related. The Antirrhinum orthologue of LUG, STYLOSA, was shown to have a role in auxin responses and in Arabidopsis the auxin response genes ARFX15 and ETT/ARF3 were recently identified as potential downstream targets of LUG action (Navarro et al., 2004; Gonzales et al., 2007). Furthermore, SEU physically interacts with ETT/ARF3 and it has been suggested that ETT/ARF3 may recruit SEU, and perhaps thereby also LUG, to auxin response genes to regulate their transcription (Pfluger and Zambryski, 2004). Members of the SHI/STY gene family, encoding zinc-finger transcriptional activators expressed in the apical end of gynoecium primordia, style and ovule primordia, have also been suggested as downstream effectors of LUG because lug mutations are epistatic over sty phenotypes and STY1 expression is reduced in lug mutants (Kuusk et al., 2006). STYLISH1 (STY1) shows only a subtle defect in style development, and mutations in additional members of the family gradually enhance the sty1 phenotype in a redundant and dose-dependent manner. Multiple mutant lines reveal that at least six of

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54 Fruit Development and Seed Dispersal the SHI/STY-related genes contribute to carpel fusion as well as to the formation of all marginal tissues in the apical part of the gynoecium; the style, the stigma and the internal septum with placenta and ovules. These lines also show similar leaf defects as mutants in LUG/SEU/ANT/YAB; highly serrated leaf margins and partial loss of adaxial–abaxial polarity (Fridborg et al., 1999; Kuusk et al., 2002, 2006). One of the direct downstream targets of STY1 is the auxin biosynthesis gene YUC4, suggesting SHI/STY family members positively regulate local auxin biosynthesis (Sohlberg et al., 2006; Eklund et al., 2008). Indeed, exogenous application of auxin on the apical end of young sty1 sty2 double mutant gynoecia induced wild-type-like style and stigma morphogenesis, suggesting high local auxin levels to be required for style development and proliferation. Because ectopic expression of STY1 can rescue the style defects not only of lug and seu, but also of crc, jag and spt mutant gynoecia, STY1 most likely induces a pathway far downstream in the medial tissue programme (St˚aldal et al., 2008). Members of the NGATHA (NGA) gene family encode four partially redundant B3 domain proteins required for apical fusion and marginal tissue development of gynoecia (Weigel et al., 2000; Alvarez et al., 2006. Interestingly, the activity of these genes are necessary for YUC2 and YUC4 expression in the style, and mutations in SHI/STY and NGA genes show synergistic interactions resulting in dramatic reduction in apical fusion and marginal tissue development. It has been suggested that SHI/STY and NGA either act in same pathway or co-operatively (Alvarez et al., 2009; Trigueros et al., 2009). How TOUSLED (TSL) relates to the LUG/SEU/ANT/YAB complex is still unresolved. TSL encodes a protein kinase required for growth and fusion of the apical gynoecial tissue and has been suggested to play a role in the maintenance of transcriptional gene silencing, thereby controlling, for example cell division (Roe et al., 1993, 1997; Ehsan et al., 2004; Wang et al., 2007). SPT is required for transmitting tract development, and for supporting development of the septum, style and stigma. The absence of transmitting tract cells in spt mutants results in poor pollen tube growth and fertilization. SPT encodes a bHLH transcriptional activator and is expressed in all medial tissues, including ovule primordia and funiculi (Alvarez and Smyth, 1999; Heisler et al., 2001; Alvarez and Smyth, 2002; Groszmann et al., 2008). Three other bHLH genes, HECATE (HEC) 1, 2 and 3, are expressed in the SPT domain, and multiple knock-down of all three HEC genes results in an unfused septum and loss of transmitting tract, signifying overlapping function of all four bHLH genes. HEC proteins heterodimerize with SPT in yeast cells, suggesting these proteins cooperate in the control of marginal tissue development (Gremski et al., 2007). Another gene, NO TRANSMITTING TRACT (NTT), appears important for the final stages of transmitting tract development and the two auxin response factors, ARF6 and ARF8, expressed within the stigma, style and transmitting tract, have been suggested to regulate the production of some component necessary for pollen tube germination or growth (Wu et al., 2006; Crawford et al., 2007). SPT is likely to be involved in

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auxin patterning, both as a target of auxin regulation and as a mediator of auxin effects (Nemhauser et al., 2000). Interestingly, when the transcriptional activation capacity of SPT is enhanced via fusion to the VP16 activation domain, ectopic STY2 expression is induced in the valves suggesting SHI/STY family members act downstream of SPT. Although strong synergistic interactions of spt and shi/sty mutations have been observed, spt mutations are epistatic over some shi/sty phenotypes, suggesting that they may, at least in part, act in the same genetic pathways (Kuusk et al., 2002, 2006). However, the STY2 expression in spt is unaffected implicating that STY2 is not a direct target of SPT activation, or alternatively, other factors redundantly regulate its expression (Groszmann et al., 2008). SPT has also been connected to other hormonal processes and was identified as a repressor of a key gene in the GA biosynthetic pathway, GA3ox, during seed dormancy (Penfield et al., 2005). Although a corresponding function in the gynoecium has not yet been established, the data suggest a possible role of SPT in mediating a balance between auxin and gibberellin signalling pathways. Interestingly, ectopic expression of STY/SHI family genes appears to repress GA responses (Fridborg et al., 2001; Sundberg, unpublished). Although mutant analysis has so far not implicated GA in patterning of the gynoecium, we find it tempting to speculate that the antagonistic relationship between auxin and GA in the SAM may also be acting in the ‘meristematic’ medial domain of the gynoecium. It would thus be interesting to investigate if SPT and STY/SHI genes may cooperate in stimulating auxin responses, while simultaneously repressing GA signalling, and if this balance is required for gynoecium medial tissue development. 2.4.5 Apical–basal patterning Several of these genes required for marginal tissue development, for example SPT, STY/SHI and TOP/NGA, may also be considered as major factors promoting the proliferation of the apical end of the gynoecium. Besides the mutations resulting in disturbed development of apical tissues, additional mutants display an altered partitioning of tissues along the apical–basal axis. Mutants defective in polar auxin transport (PAT), such as pin and pid, show severe defects in apical–basal patterning with enlarged apical and basal regions and strongly reduced ovaries (Okada et al., 1991; Bennett et al., 1995), implicating auxin as a major morphogen directing patterning in this axis. Accordingly, mutations in the auxin response factor genes MP/ARF5 and ETT/ARF3 result in similar apical–basal defects (Sessions and Zambryski, 1995; Przemeck et al., 1996; Sessions et al., 1997) and it has been shown that ETT promotes ovary formation largely through the repression of SPT in this domain (Heisler et al., 2001). Based on the majority of these results, Nemhauser et al. (2000) proposed a model for apical–basal patterning of the gynoecium relying on a proposed auxin gradient spanning the gynoecia primordia. This model predicts high auxin levels in the apical region to promote

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56 Fruit Development and Seed Dispersal differentiation and proliferation of the style and stigma, and intermediate and low levels to specify ovary and gynophore, respectively (Fig. 2.4). Reduced PAT results in apical shifts in the boundaries between the different tissues because of high accumulation of auxin in the source tissues, hypothesized to be the most apical parts, and depletion of auxin in the cells normally downstream of the transport (Nemhauser et al., 2000). Further support for the model comes from the recent finding that STY1 and TOP/NGA genes, expressed in the apical end of the gynoecia, promote the activity of the auxin biosynthesis gene YUC4 (Sohlberg et al., 2006; Trigueros et al., 2009). Because several YUC genes are active in the distal gynoecial tip and share partially redundant functions, multiple mutants were required to reveal a common role in patterning of the gynoecia (Cheng et al., 2006). Inhibition of PAT, as well as overexpression of STY1, can restore apical tissue proliferation in various mutant lines affected in apical tissue development, for example lug, seu, ant, sty1, spt, crc and jag, suggesting auxin to act downstream of, or in parallel with, the corresponding apical tissue promoting factors during style/stigma development (Chen et al., 2000; Nemhauser et al., 2000; Sohlberg et al., 2006; St˚aldal et al., 2008). At the other end of the gynoecium, KNUCKLES (KNU) encodes a zinc-finger protein and may restrict development of the gynophore by establishing or maintaining the basal ovary boundary (Payne et al., 2004). Although the auxin gradient model provides a nice framework to explain the apical–basal patterning, the evidence for the auxin gradient model is only based on mutant data and has not yet been supported by auxin level measurements along the length of the gynoecia. We also need to further understand the mechanisms of action of the genes involved. Generally, auxin biosynthesis (yuc1, yuc4) and response mutants (axr1-3, ett/arf3 and tir1) are hyper-responsive to chemically – or genetically inhibited PAT (Nemhauser et al., 2000; Cheng et al., 2007; St˚aldal et al., 2008). So are also gynoecia carrying mutations in any of the medial tissue genes LUG, SEU and STY, suggesting that these genes may support apical–basal patterning by promoting auxin gradient or auxin response pathways (Pfluger and Zambryski, 2004; Sohlberg et al., 2006; St˚aldal et al., 2008). In contrast, spt mutants are less sensitive to decreased PAT (Nemhauser et al., 2000) and it is tempting to speculate that the SPT/HEC bHLH genes normally may act to modulate PAT or to sense parts of the auxin gradient. In summary, we propose a model placing SHI/STY-controlled YUCmediated auxin synthesis at the apical end of the developing gynoecium (Fig. 2.4). The SPT/HEC genes could direct stigma, style and transmitting tract differentiation by mediating the response to high levels of auxin and/or by participating in the formation of the auxin gradient by regulating its polar transport. ETT may establish the size of the ovary by responding to intermediate levels of auxin and by repressing the activity of SPT/HEC from this region. ETT may also restrict stigma and style differentiation to the apical, high auxin level region. However, whether an auxin gradient is established,

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and what is the nature of such a gradient, are important questions to address in the near future.

2.5 Conclusion The impressive progress in elucidation of genetic pathways regulating Arabidopsis gynoecium development has given us a good platform for modelling and has pointed out some clear directions for future work in order to understand how gynoecium patterning is established. Because the present knowledge is still very fragmented, the interrelationship between identified genetic factors must be developed and their downstream targets identified. Although the role of hormones in establishing gynoecium morphogenesis has also become clear, very little is known about the hormone dynamics and localization during gynoecium development, and even less about the regulation of the dynamics. Using modern technology for isolation of small amounts of tissue together with high quality hormone measurements, or immunolocalization methods, we will be able to address these questions in the near future. Establishing the localization of auxin, GA and cytokinin peak sites and the response dynamics of these hormones would greatly improve our understanding of the regulatory pathways required for specific tissues or developmental stages. Another intriguing question is how common or specific are the complex genetic and hormonal pathways directing lateral organ patterning. A large number of parallels between the developmental programmes in leaves and gynoecia have already been discovered, and it remains to be seen if some of the pathways still seemingly specific to gynoecia also are active during leaf development and vice versa.

Acknowledgements We would like to thank Desmond Bradley for critical reading and careful corrections to the manuscript. Our work is supported by research grants ´ y Ciencia to CF and from the BIO206–10358 from Ministerio de Educacion Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning to ES.

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Chapter 3

THE INS AND OUTS OF OVULE DEVELOPMENT Raffaella Battaglia,1 Monica Colombo2 and Martin M. Kater1 1 Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria Milano Italy 2 Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria Milano Italy

Abstract: In the past few years, ovule development has been widely studied in different plant species, both from a morphological and a more molecular point of view. At early stages of flower development, the placenta becomes specified inside the carpel and ovules develop from this tissue as meristematic protuberances. Shortly after, a complicated genetic network regulates ovule patterning controlling the differentiation of three regions named funiculus, chalaza and nucellus. In the past decade, genes playing important roles during ovule development have been identified, and in a few cases, genetic models that could explain the molecular relationship among these genes have been proposed. Here we focus our attention on the molecular genetic mechanisms that stand at the base of ovule development in the model species Arabidopsis thaliana and we report an updated description of the molecular networks controlling both sporophytic and gametophytic tissue development in the ovule. Furthermore, we observe that the mechanisms controlling ovule development seem to be evolutionary conserved, even in a distantly related species such as rice. Keywords: ovule; female gametophyte; embryo sac; haploid generation; ovule identity determination

3.1

Introduction

Ovule development has been addressed in different species since many years, first from a morphological point of view and later from a molecular point of view. The great interest towards the comprehension of the genetic and molecular aspects controlling ovule development relies not only on the fact that ovules can be considered as model organs to study differentiation processes 70


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The Ins and Outs of Ovule Development 71

in plant but also on the fact that they are essential sites for sexual plant reproduction where megagametogenesis, fertilization and embryogenesis take place. Recently, the combination of advanced morphological analysis techniques together with molecular data and mutant characterization allowed a deeper understanding on the genetics at the base of ovule development. As expected, the availability of information concerning ovule development in the model species Arabidopsis thaliana has somehow facilitated the investigation in other model species such as Petunia but also in non-related species such as rice. More data are therefore now published about ovule identity determination and differentiation in different species; not only this is of great interest for applicative projects in cultivated species but also it will help in tracing the evolutionary steps of ovule ontogenesis. Due to the large amount of data concerning ovule development in Arabidopsis, this chapter will focus on this model species in order to offer a complete scenario regarding the molecular control of ovule formation. An intriguing characteristic of this organ is the close relation between sporophytic and gametophytic tissues which suggest the existence of molecular signals regulating the coordinate development of these tissues. Different aspects concerning the molecular control of sporophytic and gametophytic tissue development are reported and a paragraph focused on the molecular signals between these tissues is presented. Moreover, the most recent findings concerning ovule identity determination in Arabidopsis, Petunia and rice are described at the end of the chapter. As it will be illustrated, the characterization of ovule homeotic mutants in different species has been particularly helpful to highlight some aspects concerning the evolutionary conservation at the base of ovule identity determination.

3.2 The origin of ovule Ovules represent the salient feature of gymnosperms and angiosperms; as precursors of seeds, these organs are therefore an integral aspect of the plant life cycle. Different theories have been proposed in order to explain the origin of the ovule. Based on fossil evidence, the telomic theory seemed to be the most plausible. It proposes that the nucellus is derived from a megasporangium that retained a single megaspore. Moreover, according to this theory, the integument(s) are derived from the fusion of sterile branches around the megasporangium (De Haan, 1920). In seed plants, the megaspore is designed as energy transfer-megaspore since the female gametophyte develops and matures with the energy derived from the nucellus over the entire course of megagametophyte development. On the contrary, megaspores in non-seed plants are defined as energy-storage megaspores since the nutrients required for female gametophyte development are accumulated prior the first nuclear division. Many other theories have been proposed and the main difference

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72 Fruit Development and Seed Dispersal relies on the nature of the integument(s) which are proposed to derive from fertile branches (Herr, 1995). More than a century ago, Charles Darwin defined the origin and early evolution of flowering plant as the ‘abominable mystery’ (Charle’s Darwin letter to J.D. Hooker in the year 1879). Indeed, Cenozoic extinctions are responsible for the absence of fossils which could highlight the morphology of ancient non-flowering seed plants and early angiosperms. The identification of ancient lineages of flowering plants are fundamental to understand which are the evolutionary steps that created the extant flowering plants. In order to study the early evolution of the gametic structures, Friedman (2006) reported the developmental analysis of megagametophyte development in Amborella trichopoda, the appearance of which predates the establishment of all the other angiosperms (monocotyledons, eumagnoliids and eudicotyledons). He showed that this ancient female gametophyte is composed of eight cells and nine nuclei; nevertheless, more studies on other ancient species are necessary to understand the evolutionary origins of the Polygonum-type angiosperm embryo sac. Concerning integument(s) evolution in the angiosperm, Impatiens species have been recently studied (McAbee et al., 2005). Most basal angiosperms are bitegmic (i.e. ovule possess two integuments) and the earliest angiosperms were likely bitegmic while many extant angiosperm are unitegmic. Therefore, the idea is that reduction in integument number has occurred several times among different angiosperms.

3.3

Ovule development in Arabidopsis

In Arabidopsis, the morphology of the ovule has been described in detail and the process that leads to the formation of mature ovules has been subdivided into six distinct stages (Modrusan et al., 1994). At stage 8 of flower development (Smyth et al., 1990), ovule primordia arise from the placental tissue as finger-like protrusions with a radial symmetry (Figs. 3.1a and 3.1b). During the subsequent stages (9 and 10), three distinct regions can be distinguished along the proximal–distal (PD) axis: a proximal part, named funiculus, that connects the ovule to the placenta; a medial part, the chalaza, where the inner and outer integuments emerge; and a distal part, the nucellus, that contains the megaspore mother cell. The inner and outer integuments develop from the chalaza at stage 11. The outer integument displays asymmetrical growth through increased cell divisions on the side facing the central septum (Fig. 3.1c). During mid-stage 12, the growth of inner and outer integument continues upwards enclosing the nucellus, and at maturity, the outer integument completely overgrows the inner integument leaving only a small opening, the micropile, through which the pollen tube will pass. The faster growth of the integuments on the abaxial side forces a curvature in the developing ovules.

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Figure 3.1 Ovule development in Arabidopsis thaliana. The upper panel shows the microscopic analysis of ovule development in Arabidopsis while a schematic representation is reported below. (a) At stage 8 of flower development, ovule primordia develop from the placenta. (b) At stage 9, ovule primordia elongate as finger-like structures. The megaspore mother cell is visible in the nucellus. (c) At stage 11, the integuments elongate in the chalaza. The outer integument displays asymmetrical growth. (d) At stage 12, integuments grow to cover the nucellus where the meiotic division takes place. (e) DIC microscopy image of mature ovule. The integuments completely cover the mature embryo sac. In the embryo sac, the egg cell, the two synergids, the central cell and the three antipodal cells are visible. op, ovule primordia; MMC, megaspore mother cell; oi, outer integument; ii, inner integument; f, funiculus; es, embryo sac.

In the nucellus, megasporogenesis is initiated at early stage 11 when the megaspore mother cell (MMC) undergoes a meiotic division that originates a tetrad of haploid megaspores, three of which degenerate (Fig. 3.1d). At stage 12, the distal cell in the tetrad, the functional megaspore, proceeds in megagametogenesis. Three mitotic divisions originate the embryo sac, which is surrounded by the integuments. At the beginning of stage 13, the ovule is ready to be fertilized: the nucellus has degenerated and the medial part of it is occupied by the mature embryo sac, which is composed of eight nuclei and seven cells (Fig. 3.1e). Mature ovules are therefore composed of sporophytic diploid tissues like the funiculus and the integuments while the embryo sac represents the gametophytic haploid tissue.

3.4 Sporophytic tissues 3.4.1 Specification and formation of the placenta Arabidopsis ovules arise from specialized meristematic regions of the internal surface of the carpels, referred to as the placenta (Robinson-Beers et al., 1992), which belong to the carpel medial domain. The Arabidopsis gynoecium is derived from two congenitally fused carpels that appear as a single

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Figure 3.2 Adaxial–abaxial polarity establishment in the developing carpel and ovule. In general, adaxial refers to being situated towards an axis of an anatomical structure while abaxial refers to being situated away from an axis of an anatomical structure. (a) SEM image of a developing carpel. At stage 5, the carpel appears as a open hollow cylinder. If an axis passes through this cylinder, adaxial refers to those structures which are close to the axis while abaxial refers to structures which are located away from the axis. (b) Schematic representation of a transversal section of a carpel at stage 8 of flower development. Ovule primordia arise from the placenta. At this stage, the septum is not completely fused. The adaxial face of the carpel is indicated in black while the abaxial side is grey. (c) Developing ovule at stage 12 of flower development. The faster growing outer integument forces a curvature in the developing ovule. Adaxial sides of the developing integuments are indicated in black and abaxial sides are indicated in grey. cr, carpel; st, stamen; s, septum; p, placenta; op, ovule primordium; ii, inner integument; oi, outer integument; ad, adaxial; ab, abaxial.

primordium in the centre of the flower. Subsequently, a central invagination forms and the primordium elongates as an open hollow cylinder (Fig. 3.2a) (Smyth et al., 1990). During gynoecium development, two opposing internal meristematic outgrowths (the medial ridges) form at medial positions and protrude into the centre of the cylinder where they fuse, giving rise to the septum. Placental tissue differentiates along the length of the septum adjacent to the lateral walls (Fig. 3.2b) (Bowman et al., 1999). Early patterning events divide the gynoecial primordium into distinct domains that will give rise to specific tissues (Bowman et al., 1999; Balanza et al., 2006). Along the apical–basal axis, there are, from top to base, the stigma; a short, solid style; the ovary, which contains the ovules; and basally the gynophore, which attaches the ovary to the flower base. The lateral domains develop into the valves, corresponding to the ovary walls. In medial position, the fused margins of the carpels are found. Several medial tissues (replum, septum, placenta and ovules, transmitting tract) arise from these fused margins. All these medial tissues, together with the apical style and stigma, are collectively termed marginal tissues. Therefore, the gynoecium medial domain is really important since it gives rise to the placenta and the ovules, and several other structures critical for reproductive competence. Despite the importance of the placenta for ovule development, the molecular events that lead to the development of this tissue remain still not well understood. Many mutations that affect the development of the medial domain have been identified (Bowman et al., 1999; Ferrandiz et al., 1999; Sessions, 1999;

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Balanza et al., 2006). However, extensive redundancies between the pathways and genes involved in the formation of the medial tissues have made genetic analysis of this region problematic. So far, no single mutant has been identified that completely blocks marginal tissue formation. However, severe developmental alterations have been reported in a variety of double mutants. Good examples are AINTEGUMENTA (ANT), LEUNIG (LUG), SEUSS (SEU) and FILAMENTOUS FLOWER (FIL), which show to have overlapping and partially redundant functions in marginal tissue development. For instance, the gynoecia of the double mutants lug ant (Liu et al., 2000) and fil ant (NoleWilson and Krizek, 2006) lack almost completely the structures derived from the medial domain, including the placenta and ovules. The double mutant lug seu shows severe developmental phenotypes, including first whorl carpelloid organs that develop as filamentous organs with no traces of marginal tissues (Franks et al., 2002; Sridhar et al., 2004). The seu ant mutant gynoecia fail to initiate ovule primordia and show reduced growth of other medialridge derived tissues (Azhakanandam et al., 2008). A possible model is that these proteins form a multimeric complex, with the SEU/LUG corepressor complex physically interacting with FIL and/or ANT, that is involved in supporting medial domain development and ovule initiation in wild-type gynoecia (Nole-Wilson and Krizek, 2006; Azhakanandam et al., 2008). 3.4.2 Primordium outgrowth In Arabidopsis, approximately 50 ovules develop inside a gynoecium. The outgrowth of ovule primordia requires a signal to direct cells in the placenta to expand and divide out of the plane of the developing septum. Some genes have been identified that seem to play a role in ovule primordium initiation, for instance the CUC genes. Before ovule initiation, indeed, CUC2 is detected in the placenta and, in many cases, the gynoecium of cuc1 cuc2 double mutants form fewer ovules than wild-type, cuc1 or cuc2 (Ishida et al., 2000). Moreover CUC genes may play a role in the location of primordium initiation, as suggested by their expression pattern: CUC2 and CUC3 are expressed at the boundaries of ovule primordia (Ishida et al., 2000; Vroemen et al., 2003). A putative role in ovule primordium initiation seems to be played by ANT. The ANT gene encodes for a transcription factor required for proper initiation and growth of plant lateral organs. It is expressed in carpel primordia and reaches very high levels in the placenta before and during ovule initiation. Moreover, ANT is strongly expressed in the developing ovule primordium and becomes restricted to the chalaza region where the integuments will arise (Elliott et al., 1996). ANT plays important and multiple roles in placenta and ovule formation and development. Concerning the primordium, loss of the ANT function leads to the formation of fewer and more distantly spaced ovule primordia that continue to develop until integument initiation (Elliott et al., 1996; Klucher et al., 1996).

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76 Fruit Development and Seed Dispersal Another gene that seems to be involved in placenta and ovule primordium initiation is CRABS CLAW (CRC). crc mutant carpels are wider and shorter than wild-type and contain fewer ovules. Moreover, early during carpel development, CRC is expressed in a longitudinal strand in the internal region of the carpel, adjacent to where the placenta will develop (Alvarez and Smyth, 1999; Bowman and Smyth, 1999), suggesting a possible role for this gene in ovule primordia formation. Ovule primordium outgrowth seems to be dependent on metabolic energy, necessary to sustain growth and cell division, as demonstrated by the huellenlos (hll) mutant, which exhibit defects in ovule growth and development (Schneitz et al., 1998). In hll mutant ovule primordia, the integuments are highly reduced or absent and the cells in the distal regions often collapse. The HLL gene encodes for a mitochondrial ribosomal protein (Skinner et al., 2001). Mitochondria perform processes essential for the cell; in addition to energy production, they are required for carbon backbone synthesis and the final steps of some amino acid biosynthesis pathways; thus, loss of HLL may slow growth and cause cells to collapse. Double mutant analysis of hll in combination with either short integuments2 (sin2) or ant, both promoters of ovule growth, has shown to result in an even more severe, synergistic reduction in ovule primordium outgrowth (Schneitz et al., 1998; Broadhvest et al., 2000). Thus, the effects of reduction in growth-promoting activity (as in the ant and sin2 mutants) are enhanced by a reduction in metabolic competence, resulting in a dramatic alteration in growth and form. 3.4.3

Patterning the ovule primordium

As already mentioned, ovule primordia appear as finger-like protrusions which show radial symmetry. Along the PD axis, three distinct regions can be recognized: the funiculus, the chalaza and the nucellus. At stage 11 of flower development, the appearance of integument primordia in the chalaza visibly marks the switch from PD polarity towards the adaxial–abaxial (Ad–Ab) polarity. A key regulator of pattern establishment is the NOZZLE (NZZ) gene (Schiefthaler et al., 1999; Yang et al., 1999; Balasubramanian and Schneitz, 2000, 2002; Sieber et al., 2004). nzz mutant ovules show many defects; the funiculus appears longer than wild-type ovules due to a problem in the establishment of the chalaza region. Integuments do not present the typical asymmetrical growth and the nucellus is extremely reduced. Furthermore, the MMC does not differentiate. The molecular characterization of the nzz single mutant together with other ovule defective mutants (Balasubramanian and Schneitz, 2002; Sieber et al., 2004) led to the hypothesis that NZZ plays a pivotal role in the temporal regulation of pattern development. The lack of NZZ activity seems to cause a precocious establishment of the Ad–Ab polarity when the PD axis has not been completed yet (Fig. 3.3a).

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Figure 3.3 Schematic representation of ovule pattern establishment. (a) The NZZ gene plays an important role in the coordination of pattern formation along both the distal–proximal and the adaxial–abaxial axes. nzz mutant ovule displays pleiotropic defects during development. The distal region is reduced or missing and the MMC does not develop. Integuments are variably shortened and the funiculus is longer due to extra cell proliferation. (b) Funiculus development. The MADS-box gene STK regulates funiculus growth. stk single mutant ovules develop a longer and thicker funiculus compared to wild-type ovules. (c) Integuments develop from the chalaza region. Integument initiation is controlled by the ANT and WUS genes. ant and wus mutant ovules show a very similar phenotype since integuments do not develop. During the next stages, the PHB, INO and ATS genes play important roles in the determination of inner and outer integument development. The PHB gene is specifically expressed in the inner integument. Interestingly, the phb mutant shows a phenotype very similar to a weak ino allele since the outer integument growth is blocked. INO loss of function results in the lack of outer integument development. The ATS gene is expressed at the boundary between the outer and inner integuments. The ats mutant ovules are characterized by a single integument which derives from the fusion of the two integuments. (d) Nucellus development. n, nucellus; ch, chalaza; f, funiculus.

3.4.3.1 Funiculus The funiculus connects the ovule to the placenta and it hosts the vascular tissue. One of the genes that has been shown to control funiculus development is the MADS-box gene SEEDSTICK (STK) (Pinyopich et al., 2003). In the stk mutant, defects in the differentiation of the seed abscission zone cause the lack of seed dispersion. Moreover, the funiculus appears thicker and longer than in wild-type ovules suggesting that STK is required to control cell expansion and division in this structure (Fig. 3.3b).

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78 Fruit Development and Seed Dispersal Also in the nzz mutant, the funiculus is longer when compared to wildtype ovules (Balasubramanian and Schneitz, 2000). As already discussed, NZZ plays an important role in the coordination of the pattern formation along both axes. The longer funiculus of the nzz mutant ovules seems to be a consequence of the precocious onset of the Ad–Ab axis instead of a specific role of NZZ in the regulation of funiculus development. The lack of NZZ activity interferes with PD pattern formation in the primordium resulting in the absence of a nucellus and the presence of a longer funiculus (Balasubramanian and Schneitz, 2002). 3.4.3.2 Chalaza 3.4.3.2.1 Integument development: initiation and elongation. The formation of integument primordia in the chalaza is linked to the determination of Ad–Ab polarity (see Fig. 3.2 for adaxial–abaxial definition). Nevertheless, it seems that at a molecular level, the Ad–Ab polarity is established before the asymmetrical initiation of the outer integument (Sieber et al., 2004). A key regulator of adaxial cell fate in Arabidopsis is the transcriptional regulator PHABULOSA (PHB) (McConnell and Barton, 1998; McConnell et al., 2001). In the developing ovules, PHB mRNA is detected at very early stages of development and it is restricted to the adaxial side of the ovule. Later, at the time of integument development, PHB expression is restricted to the distal chalaza where the inner integument develops (Sieber et al., 2004). Outer integument formation occurs immediately after the polar expression of INNER NO OUTER (INO) on the abaxial side of the proximal chalaza (Villanueva et al., 1999; Balasubramanian and Schneitz, 2000; Meister et al., 2002). INO belongs to the YABBY family and it is necessary for outer integument initiation since ino mutant ovules are characterized by the absence of the outer integument (Villanueva et al., 1999). The presence of PHB and INO mRNAs can therefore be considered as molecular markers for inner and outer integument formation, respectively. Moreover, it was reported that these genes do not regulate each other during ovule development (Sieber et al., 2004). The isolation and characterization of the aberrant testa shape (ats) mutant led us to better understand how adaxial and abaxial identities are established both in the inner and outer integuments. The ATS gene belongs to the KANADI (KAN) gene family of putative transcription factors. In ovules, ATS is specifically expressed at the boundary between the inner and outer integuments (McAbee et al., 2006). The lack of ATS activity results in the formation of a single integument which derives from the fusion of inner and outer integuments which did not separate. McAbee et al. (2006) proposed that, like in the leaf, ovule integuments, adaxial and abaxial identities and growth direction are controlled by the expression domain of adaxial and abaxial identity factors. In both these organs, the expression of the PHB gene is correlated to adaxial identity while the expression of KAN and YABBY members (like ATS and INO, respectively) is linked to the determination of abaxial domain. The

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direction of growth is strongly correlated to the juxtaposition of adaxial and abaxial factors (Fig. 3.3c). The ANT gene plays a fundamental role during integument development since ant mutant ovules completely lack integuments (Elliott et al., 1996; Klucher et al., 1996). A few years after the characterization of ant ovules, another mutant that fails to form integuments was described. It was shown that ovules that lack WUSCHEL (WUS) activity do not develop integuments (Fig. 3.3c) (Gross-Hardt et al., 2002). In the shoot apical meristem, WUS is part of a feedback loop with the CLAVATA3 (CLV3) gene to regulate size homeostasis of the stem-cell population (Laux et al., 1996; Brand et al., 2000; Schoof et al., 2000). A similar feedback loop occurs at the end of flower development between WUS and AGAMOUS (AG). AG is required to terminate WUS expression and therefore floral meristem activity (Lenhard et al., 2001; Lohmann et al., 2001). In wild-type ovules, the WUS homeobox gene is specifically expressed in the nucellus and it is sufficient to promote integument initiation in the neighbouring chalaza in a non-cell autonomously way (Gross-Hardt et al., 2002). In situ hybridization experiments demonstrated that PHB mRNA is not detected neither in the chalaza of the ant mutant nor in the chalaza of wus mutant ovules, indicating that both ANT and WUS act upstream of PHB during inner integument formation (Sieber et al., 2004). Furthermore, INO expression is temporally delayed in the ant mutant background and is not sufficient to induce outer integument development (Sieber et al., 2004). Information about the different roles that WUS and ANT play during integument development came from the expression analysis of INO in wus mutant ovules and from the molecular characterization of ANT::WUS ovules (Gross-Hardt et al., 2002; Sieber et al., 2004). In situ hybridization experiments showed that INO mRNA is still present in wus mutant ovules but it is spatially deregulated since it can be detected in the distal chalaza instead of the proximal chalaza as observed in wild-type ovules. Moreover, when WUS is ectopically expressed in the chalaza under the regulation of the ANT promoter, it is sufficient to induce the formation of ectopic integuments (Gross-Hardt et al., 2002). In situ hybridization experiments performed on these ectopic integuments showed that PHB is expressed in the distal integument while INO is expressed in all the ectopic integuments that develop proximally but its expression cannot be maintained (Sieber et al., 2004). These data suggest that WUS can be considered a patterning gene which plays an important role in the establishment of the chalaza since it is necessary for the proper PHB expression in the distal chalaza and for the restriction of INO expression in the proximal chalaza. Moreover, WUS seems not to be sufficient for the maintenance of INO expression and outer integument development suggesting that integument development requires additional region-specific factors. Sieber et al. (2004) suggested that ANT can be considered as such a factor promoting primordia growth and cell proliferation (Elliott et al., 1996; Mizukami and Fischer, 2000).

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80 Fruit Development and Seed Dispersal INO expression is deregulated also in the nzz mutant background, since nzz mutant ovules show early initiation of the outer integument suggesting that NZZ behaves as a negative temporal regulator of INO expression. The early INO expression in nzz mutants interferes with PD development. Ovules of nzz mutants develop a smaller nucellus, but form a hyperplasic funiculus. Therefore, the temporal control of NZZ prevents INO from being active too early, leaving enough time for the completion of PD axis development. NZZ seems to act redundantly with ATS to spatially restrict INO expression in the adaxial chalaza (Balasubramanian and Schneitz, 2000). Moreover, NZZ is also required to restrict PHABULOSA (PHB) expression to the distal chalaza, from where the inner integument initiates, since in nzz ino double mutant ovules, PHB is ectopically expressed throughout the nucellus. In situ hybridization experiments performed in the nzz mutant background showed that WUS expression was strongly reduced suggesting that NZZ might also play as a positive regulator of WUS in the nucellus. Taken together, these results indicate that INO, PHB, and WUS all act downstream of NZZ during the switch from PD to Ad–Ab development (Sieber et al., 2004). Another regulator of INO is the zinc finger transcription factor SUPERMAN (SUP) (Sakai et al., 1995). SUP acts as a spatial repressor of INO since it interferes with the maintenance of INO on the adaxial side of the ovule primordium (Balasubramanian and Schneitz, 2002; Meister et al., 2002). INO therefore extends in the chalaza of sup mutant ovules resulting in the equal growth of the outer integument on the adaxial and abaxial side causing the absence of the typical curvature that is observed in wild-type ovules (Gaiser et al., 1995; Balasubramanian and Schneitz, 2002; Meister et al., 2002). 3.4.3.2.2 Integument elongation. Despite the fact that inner and outer integument initiation seems to be regulated independently, the subsequent elongation of both the integuments is somehow correlated. Different genes involved in the regulation of cell division and/or expansion participate in the coordinated regulation of ovule integument extension. The SIN2 gene encodes for a mitochondrial GTPase which is necessary for cell division specifically in the ovule integuments (Hill et al., 2006). sin2 mutant ovules are in fact characterized by the presence of short integuments which do not cover the nucellus (Broadhvest et al., 2000; Hill et al., 2006). More often, mutations in cell division controlling genes result in pleiotropic effects. For instance, the SEU and LUG genes encode for components of a putative transcriptional regulatory complex which is necessary for the regulation of floral homeotic genes. Both in seu and lug single mutants, a reduction in the ovule outer integument growth and an over-proliferation of the inner integument, which is supposed to be a consequence of the reduced outer integument growth, were observed (Franks et al., 2002). The specific role of SEU and LUG during cell expansion has recently been studied in petals. SEU and LUG seem to regulate petal polarity along the Ad–AB axis and they are supposed to enforce

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the key polarity genes PHB (Franks et al., 2006). The same mode of action can therefore be supposed during ovule development. Cell proliferation in developing ovules is impaired in the sin1 mutant which is female sterile due to the uncoordinated growth of the integuments and over-proliferation of the chalazal nucellus (Robinson-Beers et al., 1992; Lang et al., 1994). The mutant phenotype is not restricted to ovule integument growth since SIN1 also plays a role in the maternal sporophytic control of embryo pattern formation (Ray et al., 1996). Cloning the SIN1 gene showed that it encodes a RNA III/RNA helicase important for the translational regulation of developmental genes (Golden et al., 2002). The emerging scenario seems to indicate that there are only few specific integument growth regulators. Whereas the majority of genes that control integument growth seem to be general regulators that also control the growth in other tissues. 3.4.3.3 Nucellus The nucellus is derived from the distal part of the ovule primordium and hosts the MMC. In the nzz mutant, nucellus development is impaired since the MMC does not differentiate and the nucellus appears shorter than in wild-type (Yang et al., 1999; Balasubramanian and Schneitz, 2000). Recent work supports the hypothesis that the lack of MMC formation and the reduced size of the nucellus in the nzz mutant is a consequence of the temporal deregulation of pattern establishment instead of a specific role for NZZ in the control of MMC differentiation and nucellus formation. This hypothesis comes from the observation that the WUS gene is still expressed in the nucellus of the nzz mutant indicating that nucellus determination is independent from MMC differentiation. Moreover, in the nzz ino double mutant, the MMC is visible in the nucellus leading to the conclusion that the mutant phenotype of nzz can be mostly ascribed to the lack of temporal regulation of INO by NZZ (Balasubramanian and Schneitz, 2002; Sieber et al., 2004). As already mentioned, the meristem maintenance gene WUS is specifically expressed in the nucellus of developing ovules. wus mutant ovules are characterized by the absence of both integuments indicating that in the ovule WUS expression is a marker for nucellus determination while the WUS protein acts non-cell autonomously to regulate integuments growth (Gross-Hardt et al., 2002).

3.5 Gametophytic tissue 3.5.1 Development of the female gametophyte Female gametophyte development occurs over two phases referred to as megasporogenesis and megagametogenesis. More than 15 different patterns of female gametophyte development have been described, which arise mainly

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82 Fruit Development and Seed Dispersal from variations in cytokinesis during meiosis, number and pattern of mitotic divisions, and pattern of cellularization. The developmental pattern exhibited by most species is the Polygonum type, which was first described in Polygonum divaricatum (Drews and Yadegari, 2002). The Polygonum-type female gametophyte is found in more than 70% of flowering plants and is the pattern found in many economically and biologically important groups, including Brassicaceae (e.g. Arabidopsis, Capsella, Brassica), Gramineae (e.g. maize, rice, wheat), Malvaceae (e.g. cotton), Leguminoseae (e.g. beans, soybean) and Solanaceae (e.g. pepper, tobacco, tomato, potato, petunia). Polygonum-type female gametophyte development has been well described in Arabidopsis by Schneitz et al. (1995) (Fig. 3.4). In the nucellus, shortly after ovule initiation, a single subepidermal cell, directly below the nucellus apex, enlarges and differentiates into the MMC. During megasporogenesis, the diploid MMC undergoes meiosis to produce four haploid megaspores (Fig. 3.4, stage 2). The megaspore closest to the chalaza survives and enlarges. The other three non-functional megaspores undergo cell death, degenerate and are eventually crushed by the expanding functional megaspore (Fig. 3.4, stage 3-I). The female gametophyte is generated from the functional megaspore via a process termed megagametogenesis (described in Schneitz et al. (1995), Christensen et al. (1997)). The megaspore undergoes three sequential mitotic nuclear divisions to form a coenocytic embryo sac with eight haploid nuclei. After the first mitosis, the two nuclei migrate to opposite poles and the smaller vacuoles coalesce into a large central vacuole (Fig. 3.4, stage 3-III). Each of the two nuclei then divides two more times, resulting in an eight-celled coenocytic megagametophyte (Fig. 3.4, stage 3-V). Two nuclei (the polar nuclei), one from each pole, then migrate towards the centre of the cell. In Arabidopsis, the polar nuclei fuse, forming the secondary endosperm nucleus. During polar nuclei migration, the embryo sac cellularizes to form a seven-celled structure consisting of the egg cell (the future zygotic embryo after fertilization) closely associated with two supporting cells called synergids at the micropylar end, a large central cell with a diploid secondary nucleus (the future endosperm after fertilization), and at the chalazal end three cells of undetermined function, the antipodals that disintegrate prior to fertilization (Fig. 3.4, stage 3-VI). In some species, this is the final form: the Polygonum female gametophyte is typically a seven-celled structure at maturity. However, this structure may be modified by cell death or cell proliferation events in various species. For example, in Arabidopsis, the antipodal cells undergo cell death immediately before fertilization, whereas in grasses (e.g. maize), the antipodal cells proliferate to form a cluster of up to 40 cells (Kiesselbach, 1949). 3.5.2

Regulation of female gametophyte development

In the pathway leading to female gametophyte development, the first step is the differentiation of a single subepidermal cell at the tip of the ovule

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Figure 3.4 Schematic representation of wild-type female gametophyte development in Arabidopsis thaliana. Stages according to Schneitz et al., 1995. The grey areas represent cytoplasm, the white areas represent vacuoles and the black points are the nuclei. ac, antipodal cells; cc, central cell; ec, egg cell; m, megaspore; sc, synergid cell; sn, secondary nucleus.

primordium as the archesporial cell. In Arabidopsis, the archesporial cell directly functions as the megasporocyte or MMC (Reiser and Fischer, 1993). So far, little is known about the molecular mechanisms underlying archesporial cell fate determination. In maize, the multiple archesporial cells (mac1) mutation results in the formation of multiple archesporial cells in ovules

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84 Fruit Development and Seed Dispersal and anthers produce excessive numbers of sporogenous cells (Sheridan et al., 1996, 1999). The rice gene MSP1 (MULTIPLE SPOROCYTES1) also controls early sporogenic development by restricting the number of cells entering into male and female sporogenesis (Nonomura et al., 2003). MSP1 encodes a leucine-rich repeat receptor kinase, which is orthologous to EXCESS MICROSPOROCYTES1 (EMS1)/EXTRASPOROGENOUS CELLS (EXS) in Arabidopsis (Canales et al., 2002; Sorensen et al., 2002; Zhao et al., 2002). Like mac1 and msp1 mutants, exs/ems1 mutants produce excess microsporocytes in the anther instead of a tapetum, causing male sterility, whereas no effect has been observed on the female side. The exs/ems1 phenotype is also observed in mutants of TAPETUM DETERMINANT1 (TPD1) (Yang et al., 2003), which encodes a small protein that serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction (Jia et al., 2008). The control of meiosis is a key step in the transition from the sporophytic to the gametophytic phase in the plant life cycle. Several meiotic mutants affecting key stages in meiosis (chromosome cohesion, recombination, synapsis, chromosome segregation, and cell cycle regulation) have been identified in Arabidopsis using a combination of forward genetic (screening for plants exhibiting reduced fertility or sterility) and, more recently, using a reverse genetics approach (reviewed in Bhatt et al., 2001; Mercier et al., 2001; Caryl et al., 2003; Hamant et al., 2006). The use of reverse genetics has proven to be very powerful and led to the characterization in Arabidopsis of many homologues of yeast genes, implying that several of the basic mechanisms underlying meiotic functions are conserved between higher plants and fungi (Caryl et al., 2003). For instance, the Arabidopsis dmc1 mutant has been shown to be defective in bivalent formation (Couteau et al., 1999). A mutation in an Arabidopsis homologue of the SPO11 gene, which encodes a type II topoisomerase responsible for generating double-strand breaks in meiosis in yeast, has been shown to reduce meiotic recombination and bivalent formation (Grelon et al., 2001). Functioning downstream of AtSPO11-1, the Arabidopsis homologue of the RAD51 gene is essential for chromosome pairing and synapsis at early stages in meiosis (Li et al., 2004). The Arabidopsis asynaptic (asy1) mutant is defective in chromosome synapsis. This phenotype is due to a mutation in an Arabidopsis homologue of the yeast HOP1 gene which is required for homologous pairing (Caryl et al., 2000). Gametophytic mutations affect those aspects of female gametophyte development and function after meiosis, including megagametogenesis, fertilization and seed development. Embryo sac-expressed genes therefore play an important role in the control of embryo sac development as well as seed development. Although extensively studied at the morphological and cytological level, relatively little was known about the genes and the pathways involved in gametophytic development (Schneitz et al., 1995; Christensen et al., 1997). However, this is changing since in the last few years several

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studies have been carried out to identify genes that control female gametophyte development. Gametophytic mutants, typically, are identified using two criteria: reduced seed set and segregation distortion. Reduced seed set in a plant heterozygous for a female gametophyte mutation is due to the fact that half of the female gametophytes are mutant and non-functional and therefore they fail to undergo normal seed development. The segregation distortion that is observed in female gametophyte mutants is due to the fact that these mutations affect the haploid gametophyte phase of the plant life cycle, and therefore, in the case of female gametophyte mutants, the mutant allele is only transmitted through the male gametes. As a consequence, they are transmitted to subsequent generations at reduced frequency and exhibit non-Mendelian segregation patterns. The consequence is that these mutations can only be transmitted from generation to generation as heterozygotes (Drews et al., 1998; Drews and Yadegari, 2002). Many genes required for female gametophyte development and function have been identified and analysed using reverse genetic approach; among them are cell cycle genes (e.g. NOMEGA (Kwee and Sundaresan, 2003) and RETINOBLASTOMARELATED1 (Ebel et al., 2004)), transcription factors (e.g. MYB98 (Kasahara et al., 2005), AGL80 (Portereiko et al., 2006a) and AGL23 (Colombo et al., 2008)) and several others (including FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) (Ohad et al., 1996), MEDEA (MEA) (Chaudhury et al., 1997; Grossniklaus et al., 1998), FERTILIZATION-INDEPENDENTSEED2 (FIS2) (Chaudhury et al., 1997), GFA2 (Christensen et al., 2002), DEMETER (Choi et al., 2002), ARABINOGALACTAN PROTEIN18 (Acosta-Garcia and Vielle-Calzada, 2004), SLOW WALKER1 (Shi et al., 2005), CHROMATIN REMODELLING PROTEIN11 (Huanca-Mamani et al., 2005) and LPAT2 (Kim et al., 2005)). The screening of Arabidopsis T-DNA or transposon insertion lines has led to the identification of mutants defective in almost all stages of female gametophyte development (e.g. Christensen et al., 1998; Pagnussat et al., 2005). Drews and Yadegari (2002) identified five phenotypic mutant categories corresponding to key developmental events involved in the formation of a mature female gametophyte. Category 1 mutants are affected at the earliest possible step and fail to progress beyond the one-nucleate stage, suggesting that expression of the haploid genome is required very early in megagametogenesis. Category 2 mutants have defects during the nuclear division phase of megagametogenesis and fail to cellularize. These mutants have defects in nuclear number and/or positions, and developmental arrest at stages 3-III to 3-V (Fig. 3.4). Category 3 mutants become cellularized but have defects in cellular morphology, including abnormal nuclear positions within cells, misshapen cells, and unusual cell features. Category 4 mutants have defects in fusion of the polar nuclei. They migrate properly, come to lie side by side, but fail to fuse. Category 5 mutants have phenotypically wild-type female gametophytes at the terminal developmental stage (stage 3-V; Fig. 3.4), which suggests that

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86 Fruit Development and Seed Dispersal megagametogenesis is not affected. However, they show alteration at the level of female gametophyte’s reproductive functions, like pollen tube guidance, fertilization, induction of seed development and maternal control of seed development. Pagnussat and co-workers (2005) reported the results of a large-scale mutant screen of Ds transposon insertion lines. They identified 130 Arabidopsis mutants with defects in female gametophyte development and function and a wide variety of mutant phenotypes were observed, ranging from defects in different stages of early embryo sac development to mutants with apparently normal embryo sacs, but exhibiting defects in processes such as pollen tube guidance, fertilization or early embryo development. The successive sequence identification of the genes disrupted in these mutants revealed genes involved in several different processes, that is secondary metabolism, protein degradation, cell death, signal transduction and transcriptional regulation, therefore providing a wide-ranging assessment of the genes and functions required for embryo sac development, fertilization and early embryogenesis. Despite the progresses made with the identification of female gametophytic mutants, the total set of genes expressed in the embryo sac was poorly defined due to the fact that the embryo sac is embedded within the sporophytic tissues of the ovule, making it difficult to directly isolate embryo sac tissue for gene expression analysis. In the last few years, different attempts have been made to identify female gametophyte-expressed genes. Hennig and co-workers (2004) analysed the expression profiles of thousands of genes present on the Arabidopsis ATH1 microarray, focusing on developmental stages of the female gametophyte. They found that more than 2300 genes are specifically regulated during these developmental transitions or are expressed preferentially in the tested samples. Furthermore, they observed that several members of the YABBY, MADS-box and Myb transcription factor families are significantly over-represented, suggesting an important role for these families during reproduction. Interestingly, among the specifically expressed MADS-box genes, several of them belong to the recently discovered type I family (Alvarez-Buylla et al., 2000). Recently more information has become available through the comparison of expression profiles of ovules with and without an embryo sac, which led to the isolation of several gametophytically expressed genes. Yu et al. (2005) performed a screen for genes with reduced expression in sporocyteless/nozzle mutant ovules in Arabidopsis and identified a set of 249 genes. As a first step towards dissecting the gene regulatory networks of the female gametophyte, Steffen et al. (2007) identified, using determinant infertile1 (dif1) ovules, a large collection of genes expressed in specific cells of the Arabidopsis female gametophyte. Johnston et al. (2007) identified hundreds of genes expressed or enriched in the embryo sac, using as approach a genetic subtraction and microarray-based

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comparative profiling between the wild-type and two sporophytic mutants lacking a functional embryo sac, coatlique (coa) and sporocyteless (spl). They also identified a set of genes that are over-expressed in the maternal sporophyte in the absence of a functional embryo sac, suggesting that a substantial portion of the sporophytic transcriptome involved in carpel and ovule development is, unexpectedly, under the indirect influence of the embryo sac. Jones-Rhoades et al. (2007) used two recently developed technologies, whole-genome tiling microarrays and high-throughput cDNA sequencing, to identify hundreds of genes expressed in embryo sacs of Arabidopsis. Most of these embryo sac dependent genes have unknown function, and include entire families of related genes that are only expressed in the embryo sac. The fact that so many paralogous genes have overlapping domains of expression in the embryo sac suggests that there will be a high degree of functional redundancy between embryo sac genes and may explain why genes from these families have not been identified previously in mutant screens. Moreover, many of these embryo sac dependent genes are not expressed at high levels in tissues other than ovules, suggesting that they may be specialized for roles in female reproductive development and function. Furthermore, most embryo sac dependent genes encode small proteins that are potentially secreted from their cells of origin, suggesting that they may act as intracellular signals or to modify the extracellular matrix during fertilization or embryo sac development. 3.5.3 Function and patterning of the female gametophyte The four different cell types that form the gametophyte are distinct with respect to morphological and molecular attributes and perform unique functions that are essential for the reproductive process. The two gametic cells, the egg cell and the central cell, are fertilized by one sperm cell each to form the embryo and the endosperm, respectively. These two gametic cells are flanked by accessory cells, the synergids and the antipodal cells, that aid in the fertilization process. Soon after pollen is transferred from anther to stigma, the male gametophyte forms a pollen tube that grows through the carpel to reach the female gametophyte. Pollen tube guidance is mediated by multiple signals emitted by both sporophytic female cells and the embryo sac which, using short-range attractants, guides the final stage of tube growth (reviewed in Higashiyama and Hamamura, 2008). Several lines of evidence indicate that the synergid cells are the source of one or more pollen tube attractants (reviewed in Punwani and Drews, 2008). Laser ablation of the synergid cell in T. fourieri abolished the pollen tube guidance process (Higashiyama et al., 2001, 2003). Moreover, in Arabidopsis, it has been shown that loss of function of synergid-expressed genes also abolished the ovule ability to attract pollen tubes. For instance, in the Arabidopsis myb98 mutant, pollen

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88 Fruit Development and Seed Dispersal tube guidance and the development of the filiform apparatus are affected. The filiform apparatus is an elaborated cell wall present at the synergid micropylar pole that likely plays a role in pollen tube guidance and reception (Kasahara et al., 2005). Punwani et al. (2007) demonstrated that MYB98 functions as a transcriptional regulator within the synergid cells and that many of the downstream genes encode proteins that are secreted into the filiform apparatus, suggesting that they play a role in the formation or function of this structure. In maize (Zea mays), RNA interference knockdown of the ZmEA1 gene, initially isolated from egg cells and expressed in the synergid cells, abolished micropylar pollen tube guidance (Marton et al., 2005). The expression of ZmEA1 in egg cells suggested that in addition to the synergids, other cells of the female gametophyte may be involved in pollen tube guidance. Recently, this hypothesis was strengthened by Chen et al. (2007) who demonstrated that the central cell also plays a critical role in this process. They identified the central cell guidance (ccg) mutant, which is defective in micropylar pollen tube guidance. The CCG gene is expressed in the central cell of the female gametophyte and its specific expression in the central cell alone is sufficient to restore the pollen tube guidance defect in the mutant. Many other Arabidopsis mutants with synergid cell defects function have been identified, for example feronia (fer), which is allelic to sirene (srn) (Huck et al., 2003; Rotman et al., 2003; Escobar-Restrepo et al., 2007) and gametophytic factor2 (gfa2) (Christensen et al., 2002). In wild-type plants, when the pollen tube arrives to the female gametophyte, it enters into one of the synergid cells, ceases to grow and bursts to release its two sperm cells. The synergid cell that interacts with the pollen tube typically undergoes cell death. Degeneration appears to occur only upon contact with the pollen tube but before tube discharge (Sandaklie-Nikolova et al., 2007). In the feronia (fer) mutant, when a wild-type pollen tube enters the receptive synergid of a mutant female gametophyte, it fails to arrest its growth and does not rupture to release the sperm cells, so it continues to grow inside the female gametophyte. Thus, the fer mutation disrupts the female control of pollen tube reception. FER encodes a synergid-expressed, plasma membrane localized receptor-like kinase that might interact with a ligand from the pollen tube (Escobar-Restrepo et al., 2007). gametophytic factor2 (gfa2) female gametophytes fail to undergo synergid cell death following pollination (Christensen et al., 2002). GFA2 encodes a J domain containing protein required for mitochondrial function, suggesting a role for mitochondria in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. Finally, the two sperm cells migrate to the egg cell and central cell, and the gametes fuse to accomplish double fertilization (Lord and Russell, 2002). In the cdka;1 (also called cdc2a) mutant, double fertilization does not occur because the central nuclei are never fertilized. Indeed in cdka;1 mutant pollen only one sperm cell, instead of two, is produced and it fertilizes exclusively

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the egg cell. This shows that egg and central cell are not equally likely to be fertilized. However, it is not clear whether this preferential fertilization is due to the egg cell closeness to the sperm cell, to an active signalling or to predetermination (Iwakawa et al., 2006; Nowack et al., 2006). A series of mutants affecting central cell development and function are known. Several of them show defects in fusion of the polar nuclei (e.g. Pagnussat et al., 2005) and, interestingly, among them, several of the mutated genes encode mitochondrial proteins or predicted mitochondrial proteins, such as GFA2 (Christensen et al., 2002), RPL21M (Portereiko et al., 2006b) and SDH1-1 (Leon et al., 2007), suggesting that the karyogamy defect is a consequence of an impairment in basic mitochondrial functions. On the contrary, in the lachesis (lis) mutant, defects in polar nuclei fusion appear to be a secondary consequence of the fact that the central cell differentiates egg cell attributes at the expenses of central cell fate (Gross-Hardt et al., 2007). In the agl80 mutant, polar nuclei fuse but central cell development is compromised; nucleolus and vacuole fail to mature properly and, after fertilization, endosperm is not formed (Portereiko et al., 2006a). Within the ovule and seed, AGL80 is expressed exclusively in the central cell and during early endosperm development, suggesting that this transcription factor is required for late steps of central cell differentiation. Moreover, central cell-expressed gene products control the activation of endosperm development (Grossniklaus et al., 1998; Luo et al., 1999; Ohad et al., 1999). Until now, no clear function has been assigned to antipodal cells. Christensen et al. (1998) identified some mutants where antipodal cells fail to degenerate, but these mutants also showed other defects in female gametophyte development. Gross-Hardt et al. (2007) showed that in lis-1 mutant, antipodal cells can adopt a central cell fate and eventually disintegrate their membrane allowing nuclei fusion. It is therefore possible that the antipodal cells might function as a backup in case of gametic failure. Despite the large number of mutants showing defects during megagametogenesis isolated in Arabidopsis, the mechanisms underlying the establishment of cell identity in the female gametophyte are not known. Mutant analysis suggests that the differentiation of the distinct cell fates is tightly controlled and appears to follow regional cues, with a pivotal role for cell–cell communication during the patterning process. Important aspects of gametophyte development are gametophytically regulated. Moreover, the female gametophyte seems to be a flexible structure with enormous respecification potential suggesting that the status of distinct cell fates is constantly monitored. In the lis-1 mutant, synergids and central cells adopt egg cell fate, whereas antipodal cells behave like central cells (Gross-Hardt et al., 2007). Therefore, accessory cells of lis-1 mutant gametophytes frequently adopt a gametic cell fate, suggesting that all the cells in the female gametophyte are competent to differentiate gametic cell fate and that this competence is repressed in accessory cells of wild-type gametophytes. Intriguingly, shortly after

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90 Fruit Development and Seed Dispersal cellularization, expression of LIS, which is homologous to the yeast splicing factor PRP4, is highly up-regulated in gametic and down-regulated in accessory cells. A lateral inhibition model has been proposed based on these results: the gametic cells upon differentiation generate a LIS-dependent signalling molecule that is transmitted to the adjacent accessory cells to inhibit their gametic cell competence, thereby not only preventing excess gametic cell formation but also assuring that the accessory cells can act as a backup if required. Moreover, the central cell in lis-1 gametophytes additionally adopted egg cell fate, suggesting that a further, LIS-dependent mechanism suppresses egg cell fate in the central cell. Thus, the lis-1 mutant phenotype reveals two levels of cell fate regulation, one between gametic and accessory cells, and the other between egg and central cell. Pagnussat et al. (2007) observed that, although this mechanism of lateral inhibition can explain the maintenance of only one egg cell in the embryo sac after the initial specification of cell fate, the mechanisms involved in the early establishment of different cell fates in the embryo sac are not addressed by the model. They proposed that a positional mechanism would explain how cell fate is specified in early megagametophyte development. The migration and position of nuclei during megagametogenesis in Arabidopsis have been shown to be highly regular (Webb and Gunning, 1990; Webb and Gunning, 1994), and previous studies suggested that cytoplasmic domains may determine the fate of cells during cell partitioning (Brown and Lemmon, 1991, 1992). According to this positional model, the nuclei that move towards the micropylar or chalazal poles acquire accessory cell fates and, upon cellularization and egg cell formation, the lateral inhibition mechanism maintains their cell fates. In this way, superimposition of a gametic module onto existing micropylar and chalazal domains can generate four distinct cell types (Kagi and GrossHardt, 2007; Pagnussat et al., 2007). The idea of a positional mechanism is also supported by other studies. In the maize indeterminate gametophyte1 (ig1) mutant, the female gametophyte has a prolonged phase of free nuclear divisions, which results in a variety of embryo sac abnormalities, including the presence of extra egg cells, extra polar nuclei, and extra synergids. The phenotypes of the ig1 mutant embryo sacs suggest a position-based determination of cellular identity. The ability of the extra cells and nuclei to function as egg cells or polar nuclei, for example, appears to depend on their position in the embryo sac (Guo et al., 2004; Evans, 2007). Another mutant supporting the idea that a positional mechanism might be directing establishment of cell fates in early megagametophyte development is the eostre mutant which shows a synergid to egg cell fate switch (Pagnussat et al., 2007). The eostre-1 mutant embryo sac appears to contain two functional egg cells and only one synergid. As eostre-1 embryo sacs also show nuclear migration abnormalities, it has been proposed that the cell fate switch might be due to the unusual position of the nuclei within the embryo sac. This makes them exposed to different cytoplasmic domains compared to what happens in wild-type embryo sac.

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3.6 Interaction between the female gametophyte and the maternal sporophyte A feature of ovule morphology is that diploid sporophytic tissues such as the funiculus and the integuments and the haploid tissue of the embryo sac develop very close to each other. Despite the increasing number of information about the molecular events that control ovule development in Arabidopsis, an intriguing aspect that is still not completely understood at the molecular level is the relation between the sporophytic and gametophytic tissues. It is still not clear which are the molecular signals that coordinate the development of both these tissues. More than 10 years ago, Schneitz et al. (1997) classified the ovule defective mutants in three different classes. Mutants that show defects in both sporophytic and gametophytic tissues are named sporophytic and megagametogenesis defective mutants (smd). When the defects are restricted to the development of the gametes, these mutants are named megasporogenesis defective (msd) and embryo sac defective mutants (emd). The characterization of several smd mutants provides information about the interdependency between sporophytic and gametophytic tissues during ovule development. A strong smd mutant is the nozzle (nzz) mutant. In fact, when NZZ activity is missing, both sporophytic and gametophytic tissues are impaired (Schiefthaler et al., 1999; Yang et al., 1999). Regarding many other smd mutants characterized until now, a common feature seems to be the fact that the meiotic division proceeds normally while a block at different stages of megagametogenesis occurs later. Based on this observation, it was proposed that genes which regulate megasporogenesis act independently from those that regulate sporophyte development, while megagametogenesis is strongly influenced by the ovular context. Until now, many smd mutants have been isolated. The bel1 single mutant and the stk shp1 shp2 triple mutant have been classified as smd mutants (Schneitz et al., 1997; Battaglia et al., 2008). Aniline blue staining performed on these mutant backgrounds marked the cells resulting from meiosis meaning that megasporogenesis is not affected; later in development, the homeotic conversion of integuments into carpelloid tissue is accompanied by a block during megagametogenesis. Together with the bel1 single mutant and the stk shp1 shp2 triple mutant, the majority of integument-defective mutants can also be classified as smd mutants. Among them, a few examples are ino (Baker et al., 1997; Schneitz et al., 1997), sup (Sakai et al., 1995) and most of the ant alleles (Klucher et al., 1996). Since smd mutant defects are to a large extent region-specific, it is likely that funiculus, chalaza and nucellus develop independently for a certain period. Later, the sporophytic tissue influences embryo sac ontogenesis. In particular, it seems that megasporogenesis is independent from integument primordia

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92 Fruit Development and Seed Dispersal development in the chalaza while megagametogenesis is strongly influenced by the morphogenesis of the inner and outer integuments. The characterization of the bel1 stk shp1 shp2 quadruple mutant allowed not only to better understand the molecular regulation of ovule identity determination but this mutant also represents an example of an integumentdefective mutant which is blocked during megasporogenesis (Brambilla et al., 2007; Battaglia et al., 2008). Despite the fact that at early stages the bell stk shp1 shp2 quadruple mutant resembles the bel1 ovules in the chalaza, surprisingly, in the nucellus, differences with the wild-type ovules appear more precociously when compared with the stk shp1 shp2 triple mutant and the bel1 single mutant. DIC microscopy analysis using aniline blue staining showed that in this quadruple mutant, the MMC never goes into meiotic division. Furthermore, callose accumulation indicates that cells in this region are degenerating (Battaglia et al., 2008). Until now, it is still not clear if the ovule identity factors STK, SHP1 and SHP2 together with BEL1 directly regulate target genes involved in the control of megasporogenesis. Another possibility is that a cross talk between nucellus and chalaza coordinates ovule ontogenesis during the early stages of development. So far, the characterization of megasporogenesis-affected mutants led to the identification of genes that regulate meiosis in both the female and male megaspore/microspore mother cells or exclusively during female or male meiotic divisions. For instance, the sterile apetala (sap) mutant is both male and female sterile due to a missing meiotic division (Byzova et al., 1999). A different example is the dyad mutation which affects exclusively the female germ line while pollen development and male fertility are normal (Siddiqi et al., 2000). Interestingly, in both the sap and dyad mutants, the remainder of the sporophyte is normal, indicating that the activity of these genes is independent from the neighbouring tissues. In the attempt to identify embryo sac specific transcripts, Johnston et al. (2007) used a genetic subtraction and microarray-based comparative profiling approach. The authors compared the transcriptome of wild-type pistils with those of the spl (nzz) and cotilique (coa) mutant pistils (Vielle-Calzada, Moore and Grossniklaus, unpublished data). These mutants are characterized by the absence of the embryo sac and they can therefore be used as a powerful tool for the identification of genes which regulate the female gametes development. The results obtained from the microarray analysis allowed them to identify two groups of genes: (i) those that are specifically expressed during female gametogenesis and (ii) those that appear to be over-expressed in both the spl and coa mutants. These genes are deregulated in the maternal sporophyte probably due to the absence of the embryo sac meaning that not only the diploid sporophytic tissues influence megagametogenesis but also the presence of a mature embryo sac controls the sporophytic transcriptome. Among this group of genes, transcriptional regulators such as MYB genes,

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homeobox genes (including SHOOT MERISTEMLESS (STM)) and zinc-finger genes (including SUP) were identified and signalling factor encoding genes were deregulated (Johnston et al., 2007). The emerging scenario indicates that the development of the female gametophyte is strictly controlled by the development of the neighbouring maternal sporophytic tissues and vice versa. Nevertheless, the molecular nature of this interaction is still unknown.

3.7 Ovule identity determination 3.7.1 Arabidopsis Information about ovule identity determination came available through the characterization of mutants which showed transformations of ovules into carpelloid tissues. A good example is the bell1 (bel1) mutant reported by Robinson-Beers et al. (1992). The loss of the BEL1 function results in the formation of a single integument-like structure which in the strong bel1-4 allele develops into carpelloid-tissue bearing ectopic ovules (Robinson-Beers et al., 1992; Ray et al., 1994). It was suggested that this change in identity was due to the ectopic expression of the AGAMOUS (AG) gene in the developing integuments. AG belongs to the well-characterized Arabidopsis MADS-box family of transcription factors and it controls stamen and carpel identity (Bowman et al., 1989; Yanofsky et al., 1990). A similar conversion was later described by Pinyopich et al. (2003) in the stk shp1 shp2 triple mutant. A more recent detailed morphological analysis of this mutant showed that ovule development proceeds like in wild-type plants until stage 12 of flower development. Subsequently, in most of the ovules, integuments grow abnormally and do not cover the nucellus, with some of them converted into carpel-like tissue (Brambilla et al., 2007). SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2 also encode for MADS-box transcription factors and in a protein maximum likelihood tree, they all group together with AGAMOUS (Parenicova et al., 2003). The fact that AG also plays a role in ovule development was demonstrated by the comparison of the apetala2 (ap2) single mutant with the ap2ag double mutant. In the ap2 single mutant, petals are mostly absent and sepals are converted into carpel structures on which ectopic ovules develop, of which some have been converted into carpelloid structures. Interestingly, the first whorl organs of the ap2ag double mutant still had carpel identity; however, the number of ovules that lost their identity and were converted into carpel structures was significantly increased indicating that AG activity also contributes to ovule identity (Western and Haughn, 1999; Pinyopich et al., 2003). Protein interaction studies allowed a better understanding of the mode of action of these factors. Yeast two-hybrid experiments have shown that STK,

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94 Fruit Development and Seed Dispersal SHP1, SHP2 and AG cannot directly interact but higher order protein complexes can be formed when the SEPALLATA SEP3 MADS-box factor is added in yeast three-hybrid experiments (Favaro et al., 2003). Interestingly, AG is not only able to interact with SEP alone to regulate carpel identity but it can also participate in the formation of the ovule identity complex (Favaro et al., 2003). The genetic relationship between the ovule identity genes BEL1, AG, STK, SHP1 and SHP2 has been very recently elucidated with the characterization of the bel1 stk shp1 shp2 quadruple mutant (Brambilla et al., 2007). This mutant shows an enhancement of the bel1 phenotype since the single integument is transformed into carpel-like structure. Furthermore, on this structure and on the funiculus, new ectopic ovules develop. In situ hybridization experiments allowed to study the identity of the developing tissues in the ovules of this mutant. The loss of integument identity in the bel1 stk shp1 shp2 mutant was demonstrated using the ovule identity marker STK and the carpel identity marker CRC as probes. This analysis showed that until stage 12 of flower development, integument identity is maintained. After this stage, STK expression disappeared while CRC starts to be expressed in the mutant chalaza clearly showing the change from integument to carpel identity (Brambilla et al., 2007). BEL1, STK, SHP1 and SHP2 loss of function is also responsible for WUS deregulation in the developing ovules. In fact, starting from stage 10, WUS transcripts were not only detected in the distal part of the ovule primordia but also in the chalaza, in structures developing from the chalaza region and in the funiculus. Moreover, WUS expression was still clearly detectable in mutant ovules at late stage 12 and stage 13, while in wild-type ovules, its expression weakens from stage 11 to become undetectable at later stages (Gross-Hardt et al., 2002). These results suggest that the cell proliferations observed from the chalaza and the ectopic ovule-like structure formation from the funiculus, as observed in the quadruple mutant, might be due to a combination of ectopic WUS expression and the absence of integument identity gene activity. Interestingly, deregulation of WUS was only observed when the BEL1 gene was inactive, since in the stk shp1 shp2 triple mutant, WUS expression was not changed relative to wild-type plants. Protein interaction experiments allowed the authors to interpret the genetics results (mutant ovule phenotypes) and the molecular data obtained through in situ hybridization analysis and to suggest a model for integument identity determination (Fig. 3.5). Brambilla et al. (2007) demonstrated that BEL1 strongly interacts with the AG–SEP dimer. Since in the bel1 mutant ovules, integuments are converted into carpel tissue, it means that the interaction between BEL1 and the AG–SEP dimer is necessary to prevent AG carpel identity activity during ovule integument development. Whenever the BEL1 protein is missing, the free carpel identity AG–SEP dimer induces carpel development in the chalazal region (Fig. 3.5). Moreover, the WUS gene is ectopically expressed in the chalaza of the bel1 mutant indicating that the AG–SEP–BEL1 complex also plays a role in the restriction of the WUS expression domain.

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BEL1

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Figure 3.5 Schematic representation of integument identity determination in Arabidopsis. (a) In wild-type plants, the BEL1 factor interacts with the carpel identity complex. The ovule identity complex seems to stabilize the CIC–BEL1 complex. (b) In the bel1 single mutant, the lack of BEL1 protein results in an increasing amount of free carpel identity complex which induces carpel development in the chalaza. (c) In the stk shp1 shp2 triple mutant, the absence of the ovule identity complex probably causes the destabilization of the BEL–CIC interaction which results in an increased carpel identity activity in the chalaza region. (d) In the case that the amount of AG protein is reduced, like in the stk shp1 shp2 ag3/+ mutant, integument development is impaired but differently from the stk shp1 shp2 triple mutant, they do not longer develop in a carpel structure. (e) In the bel1 stk shp1 shp2 quadruple mutant, the presence of only the carpel identity complex strongly induces carpel tissue instead of integuments. CIC, carpel identity complex (AG-SEP); OIC, ovule identity complex; ii, inner integument; oi, outer integument; f, funiculus; cls, carpel-like structure.

In the stk shp1 shp2 triple mutant, ovule integuments are transformed into carpel tissue. This phenotype suggests that the MADS-box ovule identity complex somehow stabilizes the AG–SEP–BEL1 protein complex. The lack of STK, SHP1 and SHP2 activity results once again in the ectopic carpel activity of the AG–SEP dimer (Fig. 3.5). The role of the AG–SEP dimer in promoting carpel development in the stk shp1 shp2 mutant background has been further confirmed by the phenotype of the stk shp1 shp2 ag3/+ quadruple mutant which is characterized by a less extension of the carpel-like structures that developed instead of integuments (Fig. 3.5). The enhanced ovule phenotype of the bel1 stk shp1 shp2 quadruple mutant is due to the fact that only the AG–SEP dimer is available, so that it strongly induces carpel development in the chalaza of this quadruple mutant (Fig. 3.5).

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96 Fruit Development and Seed Dispersal 3.7.2

Petunia and rice

The first results towards the understanding of the molecular mechanisms controlling ovule identity determination came from studies performed in Petunia (Angenent et al., 1995; Colombo et al., 1995). In Petunia plants in which two putative STK orthologues, FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, were cosuppressed, carpel structures developed directly from the placenta replacing the ovules. The carpel identity of these structures was confirmed molecularly by showing that the carpel marker genes pMADS3 and FBP6 were active in these structures. Furthermore, FBP7 and FBP11 also interact with SEP-like proteins, which are FBP2 and FBP5 (Ferrario et al., 2003; Immink et al., 2003). These SEP-like proteins are, like in Arabidopsis, essential for correct ovule development since in the fbp2 fbp5 double mutant instead of ovules, leaf-like structures are directly formed from the placenta (Vandenbussche et al., 2003), suggesting that the ovule identity complex cannot be formed without SEP-like proteins and therefore the ovule identity pathway cannot be initiated. Another example that shows that correct ovule identity determination is dependent on the same basic principle and evolutionary well-conserved factors comes from the more distantly related species rice. In rice, it has been shown that OsMADS13, which is orthologous to STK, determines ovule identity (Dreni et al., 2007). In the osmads13 mutant, ovules are converted into carpelloid structures. Furthermore, in this mutant, a reiterated set of carpels develops in the place of the ovule (Dreni et al., 2007). Interestingly, the complexes that control ovule identity also seem to be conserved between grasses and core eudicot plants since OsMADS13 is also able to interact with the SEP-like rice proteins OsMADS24 and OsMADS45 (Favaro et al., 2002). The conservation of the interactions of the Arabidopsis, Petunia and rice ovule identity proteins is further illustrated by the fact that OsMADS13 interacts with the SEP proteins of Arabidopsis and Petunia, and STK and FBP7 with OsMADS24 and OsMADS45. Although this suggests that OsMADS24 and OsMADS45 are also involved in ovule identity determination, functional analysis of these genes has still to be done to prove this hypothesis. Phylogenetic analysis of the AGAMOUS-subfamily revealed that in the angiosperm lineage, an ancient gene duplication occurred that produced the ovule specific group to which STK, FBP7, FBP11 and OsMADS13 belong (Kramer et al., 2004; Yamaguchi and Hirano, 2006). This group further contains a large variety of angiosperm species and it will be interesting to analyse if all these genes are still controlling ovule development. One of the differences that can be observed in ovule identity defective mutants in Arabidopsis, Petunia and rice is that while in the Arabidopsis stk shp1 shp2 triple mutant, ovule integuments are converted into carpelloid tissue, in Petunia and in rice, carpel structures develop instead of complete ovules. Another important difference is the origin of the placenta. In Petunia and rice, the floral meristem is maintained after carpel primordia development, and

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placenta and ovules subsequently arise directly from the inner part of the floral meristem. By contrast, in Arabidopsis, both the placenta and the ovules differentiate from the inner ovary wall and the floral meristem is already completely ‘consumed’ by the carpel primordia. Related to this difference in the origin of the placenta is the regulation of floral meristem activity through the action of stem-cell maintenance genes such as WUS (Lenhard et al., 2001; Lohmann et al., 2001). In Arabidopsis, appearance of the carpel primordia is accompanied by the repression of WUS by the carpel identity gene AG (Sablowski, 2007). In rice and Petunia, where floral meristem identity is maintained after the development of the carpel primordia, the ovule identity genes seem to be involved in the determinacy of the floral meristem (Ferrario et al., 2006; Dreni et al., 2007). However, in rice, indeterminacy due to the loss of OsMADS13 activity is different in respect to Arabidopsis since it results in a reiteration of carpels and not in a complete reiteration of the floral meristem, as observed in the Arabidopsis ag mutant (Yanofsky et al., 1990). It will be interesting to study the rice orthologue of WUS and investigate whether the ovule identity gene OsMADS13 controls this stem-cell maintenance gene. In Petunia, simultaneous ectopic expression of FBP2 (which encodes a SEP orthologue in Petunia) and the ovule identity gene FBP11 caused an early arrest in development at the cotyledon stage. Molecular analysis of these transgenic plants revealed a possible combined action of FBP2 and FBP11 in the repression of the Petunia WUS homologue named TERMINATOR (TER) (Ferrario et al., 2006). The role of SEP orthologues in floral meristem determinacy in Petunia was already suggested previously by the observation that in the fbp2 mutant, determinacy is lost in the centre of the flower (Angenent et al., 1994; Vandenbussche et al., 2003). However, cosuppression of both FBP7 and FBP11 does not result in the loss of meristem determinacy. This could be explained by the presence of a third still-unidentified ovule identity gene that is not silenced in the cosuppressed plants. In fact, in the cosuppression plants, some of the ovules develop normally. It will be interesting to investigate whether the meristem determinacy function is maintained for this class of ovule identity genes in all plants that develop the placenta directly from the floral meristem.

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104 Fruit Development and Seed Dispersal Portereiko, M.F., Sandaklie-Nikolova, L., Lloyd, A., Dever, C.A., Otsuga, D. and Drews, G.N. (2006b) NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization. Plant Physiology 141, 957–965. Punwani, J.A., Rabiger, D.S. and Drews, G.N. (2007) MYB98 positively regulates a battery of synergid-expressed genes encoding filiform apparatus localized proteins. Plant Cell 19, 2557–2568. Punwani, J.A. and Drews, G.N. (2008) Development and function of the synergid cell. Sexual Plant Reproduction 21, 7–15. Ray, A., Robinson-Beers, K., Ray, S., Baker, S.C., Lang, J.D., Preuss, D., Milligan, S.B. and Gasser, C.S. (1994) Arabidopsis floral homeotic gene BELL (BEL1) controls ovule development through negative regulation of AGAMOUS gene (AG). Proceedings of the National Academy of Sciences of the United States of America 91, 5761–5765. Ray, S., Golden, T. and Ray, A. (1996) Maternal effects of the short integument mutation on embryo development in Arabidopsis. Developmental Biology 180, 365– 369. Reiser, L. and Fischer, R.L. (1993) The ovule and the embryo sac. Plant Cell 5, 1291–1301. Robinson-Beers, K., Pruitt, R.E. and Gasser, C.S. (1992) Ovule development in wildtype Arabidopsis and two female-sterile mutants. Plant Cell 4, 1237–1249. Rotman, N., Rozier, F., Boavida, L., Dumas, C., Berger, F. and Faure, J.E. (2003) Female control of male gamete delivery during fertilization in Arabidopsis thaliana. Current Biology 13, 432–436. Sablowski, R. (2007) Flowering and determinacy in Arabidopsis. Journal of Experimental Botany 58, 899–907. Sakai, H., Medrano, L.J. and Meyerowitz, E.M. (1995) Role of SUPERMAN in maintaining Arabidopsis floral whorl boundaries. Nature 378, 199–203. Sandaklie-Nikolova, L., Palanivelu, R., King, E.J., Copenhaver, G.P. and Drews, G.N. (2007) Synergid cell death in Arabidopsis is triggered following direct interaction with the pollen tube. Plant Physiology 144, 1753–1762. Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier, D., Wisman, E. and Schneitz, K. (1999) Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 96, 11664–11669. ¨ Schneitz, K., Hulskamp, M. and Pruitt, R.E. (1995) Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. The Plant Journal 7, 731–749. Schneitz, K., Hulskamp, M., Kopczak, S.D. and Pruitt, R.E. (1997) Dissection of sexual organ ontogenesis: a genetic analysis of ovule development in Arabidopsis thaliana. Development 124, 1367–1376. Schneitz, K., Baker, S.C., Gasser, C.S. and Redweik, A. (1998) Pattern formation and growth during floral organogenesis: HUELLENLOS and AINTEGUMENTA are required for the formation of the proximal region of the ovule primordium in Arabidopsis thaliana. Development 125, 2555–2563. Schoof, H., Lenhard, M., Haecker, A., Mayer, K.F., Jurgens, G. and Laux, T. (2000) The stem cell population of Arabidopsis shoot meristems in maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100, 635–644. Sessions, A. (1999) Piecing together the Arabidopsis gynoecium. Trends in Plant Science 4, 296–297.

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106 Fruit Development and Seed Dispersal Yamaguchi, T. and Hirano, H.Y. (2006) Function and diversification of MADS-box genes in rice. TheScientificWorldJournal 6, 1923–1932. Yang, W.C., Ye, D., Xu, J. and Sundaresan, V. (1999) The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes and Development 13, 2108–2117. Yang, S.L., Xie, L.F., Mao, H.Z., Puah, C.S., Yang, W.C., Jiang, L., Sundaresan, V. and Ye, D. (2003) Tapetum determinant1 is required for cell specialization in the Arabidopsis anther. Plant Cell 15, 2792–2804. Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldmann, K.A. and Meyerowitz, E.M. (1990) The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346, 35–39. Yu, H.J., Hogan, P. and Sundaresan, V. (2005) Analysis of the female gametophyte transcriptome of Arabidopsis by comparative expression profiling. Plant Physiology 139, 1853–1869. Zhao, D.Z., Wang, G.F., Speal, B. and Ma, H. (2002) The excess microsporocytes1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Genes and Development 16, 2021–2031.

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Chapter 4

FERTILIZATION AND FRUIT INITIATION Sara Fuentes1 and Adam Vivian-Smith2 1

Department of Crop Genetics, John Innes Centre, Norwich, United Kingdom Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg, Leiden, The Netherlands

2

Abstract: Angiosperms have evolved the unique processes of double fertilization and fruit development as key steps of their survival and dispersal strategies. In this chapter, we will examine fertilization and fruit initiation as central restriction points to fruit and seed development. Pollination and fertilization appear essential for fruit initiation, since angiosperm flowers universally enter terminal senescence and abscission phases if pollination is prevented. We review key developmental processes, pathways and genes that were recruited to control and restrict the reproductive growth of the carpel and ovule until fertilization is achieved. Ever since the discovery that exogenous application of phytohormones results in the development of seedless fruit without fertilization (termed parthenocarpy), most research has concentrated on the role of endogenous phytohormones as triggers for fruit initiation after fertilization. We will highlight how uncoupling of fruit initiation from fertilization through mutational studies can further contribute to the understanding of these complex processes. Initial analysis shows that strict local control of auxin signalling, through a transcription factor network, forms one of the decisive and primary events that leads to the hierarchical control over gibberellin metabolism and perception. Testing and challenging these assumptions will provide further knowledge indispensable for controlling fruit set and yield in agriculture. Keywords: double fertilization; ovule; female receptivity; fruit initiation; vascular development; phytohormonal signalling cascades

4.1 Introduction From a purely biological standpoint, the success of higher plants hinges on reproduction strategies and the dissemination of viable progeny into


107

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108 Fruit Development and Seed Dispersal habitable environmental niches. Fertilization and fruit initiation are essential processes in angiosperm plant reproduction and play a part in maximizing the success and variation. This is evident since the conquest and rise of the angiosperm plant lineage in the dominance of land environments can be directly attributed to the developmental innovations of the flower, ovule, carpel and fruit as vital components of propagation and dispersion. Selection pressure has lead to large variation in fruit development; however, the control on fruit initiation appears to be a universally conserved mechanism amongst all angiosperms. Without pollination and successful fertilization, the ovary and sometimes the accessory tissues, cease to grow and the flower begins a terminal phase of senescence that ends in floral abscission. Alternatively, when pollination and fertilization take place, a cascade of events is triggered, leading to growth and development of the fruit and seed. Speculation about the evolutionary origin of the fruit has largely been focused on the role of the carpel in initiating and providing protection around the developing ovules and seeds. The development of the carpel and integuments presumably also played a role together with the stigma and style in the selective discrimination of male gametes during pollination and, thus, constitutes an important mechanism in the control of outbreeding (Mulcahy, 1977, 1979). The development of the fleshy fruit most likely coevolved with extinct megafauna and avifauna where the function was to enable vectorial dispersion. The whole seed would be swallowed at the end of fruit development, and subsequent excretion would ensure a fertile environment for seeds to germinate and colonize. Various mechanisms arose to strictly manage and restrain carbon partitioning to optimal levels in developing flowers and fruits in order to match fitness with success of the zygote in the environment. From an economical standpoint, fruit initiation and fruit set are essential processes in many horticultural and agricultural cropping systems. Shortly after fruit initiation commences, large diversions in plant resources often occur and the fruit actively recruits photoassimilates and nutrients into the reproductive tissues. Plant breeders, both past and present, have sought to maintain and stabilize high yields and prevent premature fruit drop, while on the other hand, they concentrated selection on plant varieties which maximize their resource allocation into fruits and seeds to provide larger and more various fruit forms (Paran and Van der Knaap, 2007). The economic relevance of pollination and fertilization is clear if we consider the economic costs associated with their potential loss. In 1998, reduced pollination of crops and harvest loss, examined in a combination of 30 crops, comprised US $54.6 billion, a total loss of 46% in harvest yield (Kenmore and Krell, 1998). Broader estimates in 2005, placed the worldwide economic value of pollination alone at €153 billion, although no loss in harvest yield was calculated (Gallai et al., 2008). More recent studies did not support a hypothesis that pollinator decline has yet affected crop yield at a global scale (Ghazoul, 2005; Aizen

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Fertilization and Fruit Initiation 109

et al., 2008); it has been proposed that the increase in pollinator-dependent crops may result in important ecological and economic consequences if pollinator decline is to further continue (Aizen et al., 2008). On the other hand, fruit initiation and fruit development are also strong determinants of plant weed invasiveness which has detrimental effects on cropping systems and the natural environment. In Australia, the economic cost of plant weed invasiveness, facilitated by fruit development and seed dispersal, was estimated at an annual cost of AUD $ 4.039 billion (Sinden et al., 2004; Keller et al., 2007). Thus, it is easy to envisage how mechanisms of altering fruit initiation and retention may offer a way to stabilize and increase yields in crops or, alternatively, become an Achilles’ heel for the manipulation and control of invasive plant species. Several different pillars of research characterize the published knowledge about fruit initiation and how it relates to fertilization. Early research focused on induced parthenocarpy (Noll, 1902), whereby fruit was artificially stimulated independent of fertilization by application of plant-growth regulators (PGRs; Gustafson, 1936) or by various pollination treatments that restricted fertilization or compatibility (Noll, 1902; Yasuda, 1930, 1935). As success with artificial growth regulators gained momentum (Gustafson, 1939a,1939b, 1942; Nitsch, 1952), another area of research was directed at quantifying and localizing phytohormones in specific tissues of the fruit based on the hypothesis that fruit development was initiated and sustained by the developing seeds (Talon et al., 1990a, 1992; Kim et al., 1992; van Huizen et al., 1995; Fos et al., 2000, 2001). Both areas of research now cover a vast number of agricultural and horticultural crops (Schwabe and Mills, 1981), but often the relationship remained obscure between phytohormonal activity and fruit initiation. In contrast, heritable parthenocarpy that occurs naturally or through induced genetic lesions has provided valuable breakthroughs in crops and several genetic loci involved in fruit initiation are now known (Lin, 1984; Rotino et al., 1997; Vivian-Smith et al., 2001; Yao et al., 2001; Bassel et al., 2008; Marti et al., 2008). Recent advances in understanding fruit initiation and the intrinsic linkage to fertilization are now being completed by genetic analysis and transcriptome profiling. In this chapter, we examine the role of female receptivity in fruit set and the key pathways and genes that control fruit initiation together with their complex relationship with fertilization, and with flower maturation. We present data that reinforce the idea that fruit initiation occurs in a very short period of time, characterized by hours and minutes, and not necessarily days. The contribution of various phytohormones such as auxin and gibberellins is also examined, as is the molecular genetic study of parthenocarpy as a tool to interrogate the early and immediate steps in fruit initiation. Through the course of understanding the molecular basis of fruit initiation, the evolution of the angiosperm fruit structure is also addressed, since extensive conservation of candidate regulatory genes exists.

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4.2 4.2.1

Pollination Pollen-stigma recognition and interaction

The landing of pollen on a compatible stigma marks the beginning of pollination. The pollen will then adhere, hydrate and germinate to produce the pollen tube, a structure specialized in the delivery of the sperm cells to the ovule (Fig. 4.1). Stigmas can be divided into two broad types, wet and dry, depending on the amount of exudate present (Heslop-Harrison and Shivanna, 1977). It has been suggested that in plants with dry stigmas (e.g. crucifers), the pollen coat plays a more active role in the adhesion and hydration of the pollen grain (Heslop-Harrison and Shivanna, 1977; Heslop-Harrison, 1992 ). The role of the pollen coat in hydration has been further elucidated thanks to the grp17-1 Arabidopsis mutant, where the loss of a single oleosin protein from the coat resulted in a significant delay in pollen hydration (Mayfield and Preuss, 2000). Pollen coat substances are also involved in early pollenstigma recognition events such as the Brassicaceae-type self-incompatibility system (for more detailed reviews on this subject see Lord and Russell, 2002; Takayama and Isogai, 2005). In the stigma surface, aquaporin-like proteins have been suggested to play a major role in the control of pollen hydration (Tyerman et al., 2002) as well as in pollen acceptance (Lord and Russell, 2002). Additionally, the female determinants of various self-incompatibility systems have also been identified on the stigma surface (Takayama and Isogai, 2005). Pollen-stigma recognition is an active process which subsequently leads to pollen-tube germination (Fig. 4.1). Nevertheless, there is no evidence suggesting that recognition alone is sufficient to trigger fruit initiation (Zhang and O’Neill, 1993). The time period between pollen landing and pollen-tube germination varies greatly among plants. In Phalaenopsis orchids, pollen germinates 4 days after landing (Duncan and Curtis, 1942) providing a unique system for the study of the effect of pollen landing on fruit initiation. Orchids are also unusual among flowering plants in that the ovary and ovules of many orchid species mature after pollination (Withner, 1974). Zhang and O’Neill (1993) showed that physical contact of pollen alone is sufficient to trigger ovary maturation in Phalaenopsis orchids. Nevertheless, this interaction failed to induce fruit initiation (Zhang and O’Neill, 1993). 4.2.2

Pollen germination and pollen-tube growth

After pollen hydration, germination occurs which results in the emergence of the pollen tube (Fig. 4.1). The pollen tube is formed by a generative cell which contains the two sperm cells and the vegetative nucleus. Both pollen germination and pollen-tube growth are subjected to gibberellic acid (GA)mediated control. It has recently been shown in rice that de novo synthesis of

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Fertilization and Fruit Initiation 111 1 2

3

4

(a)

X

(b)

(c)

p g

p

sp

f

m

(d)

rv

p

g sp

fv

e

s

z pn

cr

cc oi

a

en

ii

Figure 4.1 Pollination and fertilization in Arabidopsis and Brassicaceae. (a) Flower at anthesis stage (left) and during pollination (right) showing several rows of ovules within the pistil. During pollination, the stages of adhesion and hydration (1), recognition and pollen tube emergence (2), tube growth (3) and guidance (4) are shown (right). When anthers dehisce, the inhibitory stimulus for fruit development is removed (see the cross symbol). (b) A pollen tube containing two sperm cells is guided to the ovule micropyle by signals emanating from a fertile female gametophyte and the surrounding sporophytic tissue. (c) The tube tip enters the micropyle of the ovule and unites with a synergid cell that degenerates upon fusion. Two sperm cells migrate to combine with the egg cell and polar nuclei of the central cell, respectively. (d) Synergid cells degenerate and the diploid zygote and triploid endosperm begin development. The outer and inner integuments undergo cell expansion and division to form the seed testa. a, antipodal cells; cc, central cell; cr, chalazal region; e, egg cell; en, endosperm; f, funiculus; fv, funiculus vascular tissue; g, generative cell; ii, inner integument; m, micropyle; p, pollen tube; pn, polar nucleus; oi, outer integument; rv, replum vascular tissues; s, synergid cell; sp, sperm cells; z, zygote.

GA in the pollen grains is required for pollen germination (Chhun et al., 2007). Similarly, gibberellins are also necessary for pollen-tube growth across different plant species (Singh et al., 2002; Cox and Swain, 2006; Chhun et al., 2007). The pollen tube grows by tip growth and periodic callose deposition (for a detailed review on this subject see Krichevsky et al., 2007). Directional tube

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112 Fruit Development and Seed Dispersal growth towards the egg cell is guided by several mutual signalling processes in the pistil, style, funiculus, ovules and female gametophyte (Fig. 4.1). Several studies have focused on the role played by the pollen tube in fruit initiation. It was first observed by Hildebrand (1865) that in certain orchids, where self-incompatible pollinations arose, pollen tubes grew only a small amount but this appeared to stimulate the development of parthenocarpic fruit. Fruit initiation has also been related to the degree of penetration into the style by the pollen tube. It was concluded that to stimulate fruit initiation in cucumbers and eggplants, the base of the style had to be reached by the pollen tube (Yasuda, 1936). These early observations led to the hypothesis that pollen-tube growth resulted in the transfer of growth-promoting substances from the pollen to the ovary and ‘stimulated’ the growth of the fruit (Gustafson, 1939b). Later measurements of growth-promoting hormones (namely auxin) in pollen grains and pollen tubes ruled out these as the possible sources of growth hormones. However, it was suggested that pollen tubes may secrete an enzyme responsible for the activation of auxin precursors in the style and ovary (van Overbeek et al., 1941; Muir, 1942). Recently, Schijlen et al. (2007) provided further evidence of the possible contribution of pollen-tube growth on fruit initiation. In this case, the downregulation of the flavonoid biosynthesis pathway genes, CHALCONE SYNTHASE 1 and 2 (CHS1/CHS2), by RNA interference led to parthenocarpic tomato fruit development (Schijlen et al., 2007). Downregulation of the flavonoid pathway arrested pollen-tube development in self-pollinated gynoecia. Although the pollen tubes failed to reach and fertilize ovules, their initial growth appeared to be sufficient in triggering fruit set and produce stimulatory parthenocarpy (Schijlen et al., 2007). The role of the flavonoid pathway in the gynoecium and the stimulatory effect of pollen-tube growth in tomato fruit initiation require further examination since several interactions between polar auxin transport and flavonoids have been identified. Most notably the loss of flavonoid biosynthesis in Arabidopsis led to increased polar auxin transport (PAT; Murphy et al., 2000; Brown et al., 2001; Lazar and Goodman, 2006; Santelia et al., 2008), leading to the hypothesis that PAT could stimulate fruit initiation. Roles for PAT in fruit initiation are considered in sections below. The use of ionizing radiation has also provided data about the role of the pollen tube in fruit initiation. Fruit initiation can occur even when pollen samples are treated with high doses of ionizing radiation (Denissen and Den Nijs, 1987; Knox et al., 1987; Sniezko and Visser, 1987; Polito, 1999; Peixe et al., 2000). This technique, commonly known as ‘prickle pollination’, stimulates parthenocarpic fruit growth and has been documented in various crops including Cacao, Cotton, Pistacio, Capsicum and other Solanaceaous species. High-irradiation treatments do not always impair pollen-tube growth but prevent fertilization or steps immediately after fertilization (Speranza et al., 1982; Denissen and Den Nijs, 1987; Peixe et al., 2000). Although the degree and precise stage of impairment requires clarification, arrest is characteristically earlier and different from the late post-zygotic arrest observed in

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stenospermocarpic seedless grapes (Cain et al., 1983; Emershad and Ramming, 1984; Hanania et al., 2007) or in Capsicum fruit development triggered by interspecific crosses (Tong and Bosland, 2003). The results from prickle pollination suggest that pollen-tube growth and/or processes immediately after fertilization but prior to zygotic development are able to trigger fruit initiation. Pollen-tube guidance towards the ovules depends on a complex signalling network which involves signals from the female gametophyte and components of the style and ovary (Higashiyama et al., 1998; Higashiyama, 2002; Dresselhaus, 2006; Palanivelu and Preuss, 2006; Escobar-Restrepo et al., 2007; Rotman et al., 2008). Disruption to this complex signalling network can lead to the disorientation of pollen tubes and, thus, prevent fertilization. For example, specific fruit initiation mutants can affect pollen-tube polarity and reduce seed set. This is the case in the parthenocarpic pat-2 tomato mutant where the distorted pollen-tube growth has been related to the high proportion of defective ovules present in the pistil (Mazzucato et al., 2003) and associated with increased levels of endogenous gibberellins (Olimpieri et al., 2007). Altered seed set patterns as a result of defective pollen-tube growth are also found in Arabidopsis plants overexpressing the GA2ox2 gene (Singh et al., 2002; Cox and Swain, 2006). In the light of previous observations, it is tempting to suggest that the impaired pollen-tube growth could also play a part in enhancing parthenocarpic fruit development in the pat-2 background. On the other hand, even though stimulatory parthenocarpy has been observed in a few crops, it has not been detected yet in Arabidopsis mutants where pollen or ovules have subtle defects disrupting fertilization (Vivian-Smith, 2001; Vivian-Smith et al., unpublished data).

4.3 Female receptivity and the cessation of gynoecial growth Often in horticulture, two or more varieties are planted together in a specific planting or orchard design to maximize cross-pollination for hybrid production or yield. The female receptive period is an important final component of the floral maturation process and has a direct bearing on fruit set and initiation, since viable female and male components must both exist in space and time, while the plant sufficiently conserves essential resources. The receptive period has also been referred to as the effective pollination period (EPP) and is the mutual or partial sum of the longevity for the stigma, style and ovule, while taking into account the time taken for the pollen tube to grow and fertilize the ovule (Williams, 1966; reviewed Sanzol and Herrero, 2001; Page et al., 2006). During the maturation and receptive periods, specific molecular pathways restrict the growth of the pistil and accessory tissues and, thus, stop them from developing into fruit.

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114 Fruit Development and Seed Dispersal Despite the wealth of data in crop species and extensive analysis of female gametophyte development, female receptivity has not been the subject of any significant genetic analysis. Female receptivity can be quantified by emasculating flowers before anthesis and allowing a sample of flowers to be pollinated on successive days post-anthesis (Williams, 1966). Final seed set reflects the period when pistils were most receptive to pollination. As a rule, pollen-tube growth on floral tissues is temperature dependent and different results would be expected if pistils were incubated at sub-optimal temperatures for stigmatic receptivity, pollen-tube development or ovule longevity (Sanzol and Herrero, 2001). Often the optimum for pollen-tube elongation and the optimum for support on the female component do not coincide, and the optima are usually higher for pollen-tube growth (Hedhly et al., 2005a,b). In Arabidopsis, female receptivity, as assessed by seed set, lasts up to 3 days post-anthesis and effectively correlates with the integrity of the female gametophyte which deteriorates shortly thereafter (Christensen et al., 1998; VivianSmith and Koltunow, 1999; Vivian-Smith and Offringa, unpublished). There are marked differences in female receptivity duration between the ecotypes Landsberg and Columbia (Vivian-Smith and Koltunow, 1999). Nevertheless, the receptivity periods in Arabidopsis ovules are significantly shorter than the period the pistil remains receptive to exposures of 10 nmol GA3 that stimulate fruit development (Vivian-Smith and Koltunow, 1999). A longer period of gibberellin perception suggests that the viability of the gametophyte and ovule is completely independent to the perception and signalling of a GA3 mediated growth in the pistil and that the gibberellin-mediated restriction maybe directly occurring in the carpel. Mutations in the Auxin Response Factor 8 gene (ARF8), which lead to parthenocarpic fruit initiation, dramatically shorten the duration of female receptivity and lead to reduced seed set (Vivian-Smith et al., 2001). ARF8 mutants also initiate fruit development precociously and the pistil protrudes far enough to prevent proper contact between the stigma and anthers to effect proper self-pollination (Vivian-Smith et al., 2001). Taken alone, however, the arf8 mutant data may suggest an indirect link with female receptivity. On the contrary, mutations in ARF8 together with the related gene ARF6 lead to complete sterility and dramatically prevent flower maturation in numerous aspects (Nagpal et al., 2005; Wu et al., 2006). This data suggest a global role for both genes in flower maturation, female receptivity and pollen-tube growth. Distinct genetic pathways halt further development of the egg cell and the central cell at maturity and this has been demonstrated with the use of Arabidopsis gametophytic and sporophytic mutants (see sections below). Evidence that the female gametophyte reciprocally exerts control over the developing sporophyte comes from transcriptional profiling studies where mutants lack a viable gametophyte (Johnston et al., 2007). Significant modulation of the sporophytic genes has been observed for SUPERMAN (SUP), Small Auxin Upregulated RNA (SAUR), C3HC4-type RING finger proteins, the homeobox gene SHOOT MERISTEMLESS (STM) and the STYLISH2 (STY2)

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transcription factor. However, these genes are just a few examples amongst 527 genes identified (Johnston et al., 2007). Many of these genes could be candidates linking female receptivity, the female gametophyte and the regulation of fruit initiation. Other studies have also implicated the phytohormone cytokinin in gametophyte development and maintenance of receptivity. Pischke et al. (2002) and Hejatko et al. (2003) demonstrated that CKI is expressed in the female gametophyte until fertilization and it is essential for gametophyte viability. Previous research showed that overexpression of CKI results in cytokinin independency in somatic tissues (Kakimoto, 1996; Glover et al., 2008). If CKI functions in a similar manner in the female gametophyte, it may play a significant role in maintenance of gametophyte viability via a cytokinin-related pathway. Female receptivity can also be positively influenced by the application of nitrogen fertilizer (Williams, 1965; Tromp et al., 1994), and by stigmatic secretions induced by pollination, that can help release carbohydrates from the transmitting tissue and prolong embryo sac viability (Herrero, 1992). While the beginning of female receptivity is demarcated by the period when pollen tubes can grow on the stigma, style and transmitting tissue (Kandasamy et al., 1994), the end of female receptivity is onset by an irreversible initiation of floral senescence (O’Neill, 1997; O’Neill and Nadeau, 1997; Lewis et al., 2006).

4.4 Additional restraints on flower development and fruit initiation Prior to pollination, the floral whorls surrounding the pistil may play a role in repressing or slowing ovary growth (Vivian-Smith, 2001; Vivian-Smith et al., 2001; Fig. 4.1). Accordingly, the specific removal of stamens in Arabidopsis thaliana has been shown to promote pistil growth slightly in wild-type plants, but moreover, the effect is significantly pronounced in genetic backgrounds that display parthenocarpy (fwf/arf8) or fertilization-independent seed development (fis2-2; Vivian-Smith, 2001). Combinations of these mutants with the conditional male sterile pop1/cer6-1 mutant do not alleviate the retardation in silique growth and emasculation of pop1/cer6-1 flowers is still required to achieve full comparative silique elongation (Vivian-Smith, 2001; Vivian-smith et al., 2001). From these experiments, stamens and pollen have been pinpointed as being fully responsible for the retardation in fruit initiation (Fig. 4.1; Vivian-Smith and Offringa, unpublished). The basis of both the FWF/ARF8 and anther dehiscence pathways is to ensure that wildtype plants are successfully synchronized in dehiscence, self-pollination and fruit initiation, but taken separately, the anther acts independently to prevent precocious pistil growth.

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116 Fruit Development and Seed Dispersal The restraint on ovary growth during female receptivity could be mediated in part by anthers producing high concentrations of free auxins (Aloni et al., 2006), since auxin flow in anther filaments and high auxin levels in the tapetal tissues are critical for pollen development (Feng et al., 2006; Cecchetti et al., 2008). High auxin levels appear to be mediated in part by the auxin biosynthesis genes YUCCA6 and YUCCA2 (Feng et al., 2006; Cecchetti et al., 2008) which are regulated by the SPOROCYTLESS/NOZZLE gene (SPL/NZZ) that controls gametophyte development (Li et al., 2008). Before the female receptive period, a strong auxin maximum is formed in stamens as judged by the auxin transcriptional response reporter, DR5::GUS (Aloni et al., 2006; Feng et al., 2006; Cecchetti et al., 2008; Li et al., 2008). Prior to wild-type pollen dehiscence, the auxin maximum declines and is absent upon dehiscence, providing a natural mechanism to decrease the growth of the anther filaments through reduced PAT (Aloni et al., 2006; Cecchetti et al., 2008). The strongest evidence for pathways facilitating the restraint of Arabidopsis fruit growth from the anther comes from double mutant analysis where several genes have been isolated (Vivian-Smith and Offringa, unpublished). Serendipitously, one was found during the map-based cloning of the fwf-1/arf8-4 mutant (VivianSmith, 2001; Vivian-Smith et al., 2001). Mutations in the aberrant testa shape-1 (ats-1) mutant, also known as kanadi4-1 (kan4-1; McAbee et al., 2006), were observed to enhance silique development in the pop1/cer6-1 ats-1/kan4-1 fwf1/arf8-4 background independently of anther emasculation (Vivian-Smith, 2001; Vivian-Smith et al., 2001). Defects in ATS/KAN4 cause incomplete separation and growth of the ovule integuments. ats-1/kan4-1 mutant ovules consist of three cell layers that have a shared unitegmic identity, as opposed to two outer and three inner integuments in wild type (Léon-Kloosterziel et al., 1994; McAbee et al., 2006). Importantly, total mesocarp cell counts from fully developed siliques of pop1/cer6-1 ats-1/kan4-1 fwf-1/arf8-4 and wild-typepollinated siliques were the same (Vivian-Smith et al., 2001) suggesting that together ATS/KAN4 and FWF/ARF8 control a large portion of the fruit initiation pathway. The identity or reduced integumentary cell layers in ats1/kan4-1 appear to disrupt a key parallel signalling pathway that does not alone trigger fruit initiation but does link signalling with the restriction of fruit growth facilitated by the anther (Vivian-Smith et al., 2001). The study of MADS box gene mutants has further contributed to the understanding of the restraint imposed by the other floral whorls in ovary growth. For example, loss of function mutation in the MdPI (apple PISTILLATA homologue) causes parthenocarpic fruit development in apple (Yao et al., 2001) which could also be attributed in part to the disappearance of the restraint imposed by the third whorl organs (namely stamens) and to the replacement of ovule identity. Similarly, the parthenocarpic fruit development observed in tomato transgenic plants with low expression levels of TM29 (tomato SEPALLATA homologue) could also be linked to the disruption of petal and stamen identity (Ampomah-Dwamena et al., 2002). However, this phenotype can also be attributed to the altered expression levels of TM29 in the ovaries

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(Ampomah-Dwamena et al., 2002) and, thus, the correlation between outer floral whorls disruption and the observed ovary growth remains to be further clarified in these transgenic plants. In Arabidopsis, carpelloid identity replaces ovule primordia in alleles of the bel1-1 mutants (Reiser et al., 1995; Western and Haughn, 1999; Brambilla et al., 2007) and in the knuckles mutant (Payne et al., 2004). Both mutants develop fruit independent of fertilization but the length is completely correlated with the total number of carpelloid ovule structures produced in the carpel (Vivian-Smith, 2001). Similar occurrences are observed in Capsicum and tomato species (Gray-Mitsumune et al., 2006; Tiwari et al., 2006).

4.5 Fertilization In the vast majority of angiosperms, the mature female gametophyte consists of a seven-cell, eight-nucleate ‘Polygonum-type’ structure, bounded by a membrane that lacks a plant cell wall (Fig. 4.1; Christensen et al., 1998; Yadegari and Drews, 2004). This type of gametophyte has two synergid cells and an egg cell located at the micropylar pole, thus comprising the three-celled egg apparatus (Fig. 4.1, note that one synergid is hidden behind the other). Three antipodal cells are positioned at the chalazal pole of the ovule. Two nuclei of the central cell form the polar nuclei that locate adjacent to the egg cell (Fig. 4.1b; Yadegari and Drews, 2004). Considerable variation exists on the general architecture of the female gametophyte, however, the basal angiosperm Amborella has a similar structure to higher angiosperms and consists of an eight-celled, nine-nucleate female gametophyte (Friedman, 2006). In Amborella, an egg cell is derived from a division of one of the three synergid cells to form a four-celled egg apparatus, unlike the Arabidopsis female gametophyte where the egg cell is specified from a designated nucleus and remains in association with the two synergids. Other basal angiosperms frequently contain a four-celled ‘Nuphar/Schisandra-type’ gametophyte that contains an egg cell, two synergids and a uninucleate central cell at maturity (Friedman and Williams, 2003; Williams and Friedman, 2002; Friedman, 2008). However, the majority of higher angiosperms presents a seven-cell polygonum-type gametophyte and, thus, this type of gametophyte is used as a reference point for the remainder of this review. Further information on gametophyte development is extensively covered elsewhere (Drews and Yadegari, 2002; Punwani and Drews, 2008). 4.5.1 Signal transduction before fertilization The delivery of two sperm cells to the mature female gametophyte by the ¨ pollen tube relies on a robust mutual communication (Fig. 4.1; Hulskamp et al., 1995). Palanivelu et al. (2003) showed that pollen-tube growth in stigma,

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118 Fruit Development and Seed Dispersal style and ovule is guided by gradients in ␥ -aminobutryic acid (GABA). Pollen tubes utilize a GABA-transaminase, encoded by the POP2 gene, to provide pollen directionality by degrading the GABA stimulant. Guidance towards the female gametophyte occurs since the GABA gradient peaks at the micropylar integument cells. Degradation of GABA is probably involved in ensuring that only a single tube enters the micropyle (Fig. 4.1). As such, GABA maybe a contact-mediated guidance mechanism (Palanivelu and Preuss, 2006). Palanivelu and Preuss (2006) also defined two other processes regulating pollen-tube guidance. These include diffusible ovule-derived attractants from unfertilized ovules and repellents from fertilized ovules. The male gametophytic tepitzin1 mutant indicates a requirement for the auxininducible homeobox gene WOX5 for Arabidopsis pollen-tube growth (Gonzali et al., 2005; Dorantes-Acosta and Vielle-Calzada, 2006). Auxin, together with calcium produced in synergids, had long been hypothesized as pollen-tube chemotropic attractants (Van Went and Willemse, 1984; Chaubal and Reger, 1990; Raghavan, 2003). The synergids and the central cell also play a role in the guidance of the pollen tube prior to fertilization (Higashiyama et al., 2001; Kasahara et al., 2005; Chen et al., 2007; Rotman et al., 2008). In contrast to the GABA gradient, these appear to be short-range recognition and developmentally regulated (Palanivelu and Preuss, 2006). For instance, the plasma membrane-associated GEX3 protein is expressed in the female gametophyte and required for micropylar pollen-tube guidance (Alandete-Saez et al., 2008). Boisson-Dernier et al. (2008) also show that the AMC gene, that encodes peroxisomal protein, functions at short range in both female and male gametophytes through potential diffusible signals. Mutations in the MYB98 gene specifically prevent proper differentiation of the synergid cells which fail to differentiate the structural filiform apparatus that facilitates the reception of pollen tubes (Kasahara et al., 2005). As a consequence, most myb98 ovules fail to attract pollen tubes, suggesting that MYB98 plays a role in the transcriptional activation of the network of genes involved in signalling or in the structural differentiation required for signalling (Kasahara et al., 2005). Transcriptional profiling has validated transcriptional networks regulated by MYB98 and identified small secreted peptides/proteins as MYB98 targets (Jones-Rhoades et al., 2007; Punwani et al., 2007). Our reanalysis of supplementary data from myb98 (Jones-Rhoades et al., 2007) and female gametophyte transcriptional profiles (Yu et al., 2005) shows that the cytochrome P450 CYP78A9 gene is upregulated in myb98 female gametophytes. This is of potential interest since overexpression of CYP78A9 with the 35S promoter provides strong sterility and fruit initiation (Ito and Meyerowitz, 2000). However, the fact that viable myb98 homozygotes are generated indicates double fertilization per se is not defective (Kasahara et al., 2005). In the context of pollen-tube signalling and fruit initiation, Arabidopsis contrasts with reports of stimulatory parthenocarpy in horticultural crops.

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From Arabidopsis research alone, one could be convinced that pollen-tube development does not produce a stimulus for fruit initiation. Further analysis of the many Arabidopsis mutants that avoid fertilization (Pagnussat et al., 2005) should illuminate this aspect.

4.5.2 Double fertilization Double fertilization, first described by Guignard in 1899, involves one sperm cell uniting with the egg cell, while the second sperm cell fuses with the central cell and undergoes karyogamy with the polar nuclei (Fig. 4.1; Weterings and Russell, 2004; Yadegari and Drews, 2004). It has been extensively reviewed (Raghavan, 2003; 2006; Weterings and Russell, 2004; Dresselhaus, 2006) and recently the technical limitations of live visualization have been resolved by confocal laser scanning microscopy (CLSM; Ingouff et al., 2007; Fig. 4.2; Vivian-Smith and Offringa, unpublished data). Double fertilization begins upon fusion of the tube tip with a synergid cell (Fig. 4.1c; Higashiyama et al., 2000; Weterings and Russell, 2004; Sandaklie-Nikolova et al., 2007). At this point, the pollen tube stops growing and discharges the two sperm cells. In some species, synergid degeneration occurs well before pollen tube arrival (Raghavan, 2003) while in Arabidopsis it has been reported that synergid cell death occurs upon pollen tube contact (Sandaklie-Nikolova et al., 2007). Bidirectional communication can however increase cell permeability well before contact, since preferential propidium iodide (PI) staining occurs in the selected synergid before the pollen tube has arrived (Vivian-Smith and Offringa, unpublished data). This is also the case in ∼15% of ovules from the pop1/cer6-1 male sterile mutant, suggesting that a long-range pollen-synergid signalling triggers events prior to synergid cell death and double fertilization. Upon discharge of the sperm cells into the degenerating synergid, migration of sperm cells to the egg and central cells occurs (Faure et al., 2002; Weterings and Russell, 2004; Ingouff et al., 2007). Movement towards their respective nuclei is facilitated by remnant F-actin coronas and microtubles (Ye et al., 2002; Raghavan, 2003). Following double fertilization, development of a diploid zygote and triploid endosperm is initiated (Fig. 4.1; Faure et al., 2002). The remaining synergid eventually deteriorates (Kasahara et al., 2005) and the integuments expand and divide to accommodate the developing embryo and endosperm. In Arabidopsis and other angiosperms, the integuments differentiate post-fertilization to form the seed coat or testa that protects the seed and facilitates the transfer of nutrients and photoassimilates to the seeds (Fig. 4.1; Bowman, 1993; Wittich, 1998). Many steps are required to enable gamete fusion and karyogamy (Jensen, 1964; Faure et al., 2002), but in Arabidopsis this occurs within 2–3 h after the arrival of the pollen tube (Berger et al., 2008; Fig. 4.1c).

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120 Fruit Development and Seed Dispersal (a)

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s

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(b) cs

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Figure 4.2 Confocal laser scanning microscopy (CSLM) images of unfertilized and fertilized ovules of Arabidopsis expressing the synthetic auxin-responsive reporter gene DR5rev::eGFP. (a) Unfertilized anthesis ovule with minimal GFP expression. (b) Post-fertilized ovule at 5 h. The first nuclear endosperm division has occurred and GFP expression is observed in the endothelium, the chalazal domain and adjacent to the funiculus vascular strand. (c) Ovule after the third endosperm division (9 h post-fertilization) with eight endosperm nuclei and an elongated zygote. Strong GFP expression occurs in the endothelium, the chalaza and funiculus. Weaker expression is observed in the outer integument. (d) Treatment of detached pistils with NAA (50 µM) for 1 h, with subsequent washing for 7 h, induces strong GFP activation in the funiculus and chalaza, and moderate activation in the inner integument and weaker expression in the outer integument. a, antipodal cells; cc, central cell; cr, chalazal region; e, egg cell; en, endosperm; f, funiculus; fv, funiculus vascular tissue; g, generative cell; ii, inner integument; m, micropyle; p, pollen tube; pn, polar nucleus; oi, outer integument; rv, replum vascular tissues; s, synergid cell; sp, sperm cells; t, endothelium; z, zygote. (For a colour version of this figure, please see Plate 2 of the colour plate section.)

4.5.3

Signal transduction during fertilization

After compatible fertilization, rapid changes in membrane-bound calcium occur and the female gametophyte changes cellular polarity, forming the zygote and endosperm (Russell, 1993). A complex signalling network is involved in the coordination of double fertilization (for detailed reviews see Dumas and

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Gaude, 2006; Berger et al., 2008). An early signalling event is the generation of a calcium influx following the fusion of the sperm and egg cell (Antoine et al., 2000). This Ca2+ influx will subsequently spread throughout the egg cell and it is believed to contribute, at least partially, to the transient elevation of cytosolic Ca2+ observed shortly after (Antoine et al., 2001). The release of high concentrations of Ca2+ during the synergid cell degeneration (Chaubal and Reger, 1990, 1992a,b) is also likely to contribute to this process (Digonnet et al., 1997). The increase in cytosolic Ca2+ concentration has been proposed to trigger egg cell activation (Digonnet et al., 1997; Antoine et al., 2000). Kranz (1999) also reported that isolated gametes that were fused using in vitro fertilization techniques required pulses of auxin (25–40 mg L–1 2,4-D) to initiate cell division in the newly formed zygote. Characterization of the Arabidopsis aca9 mutant has further contributed to the understanding of the role played by calcium signalling during fertilization. ACA9 encodes a Ca2+ pump that is primarily expressed in pollen (Schiott et al., 2004). Mutant aca9 pollens not only display reduced pollen-tube growth but are also defective in sperm cell release (Schiott et al., 2004). Disruption in sperm cell release was also previously reported in the sirene (Rotman et al., 2003) and feronia (Huck et al., 2003) female gametophyte mutants. In both siréne/feronia and aca9 mutants, pollen tubes fail to initiate the release of sperm cells into the synergid and, consequently, pollen tubes continue to grow inside the female gametophyte (Huck et al., 2003; Rotman et al., 2003; Schiott et al., 2004; Escobar-Restrepo et al., 2007). However, while sirene/feronia are female gametophyte mutants (see section below), aca9 mutation affects the male gametophyte (Huck et al., 2003; Rotman et al., 2003; Schiott et al., 2004). More recently, a similar phenotype to that observed in siréne/feronia and aca9 mutants was also described in the Arabidopsis amc mutant (Boisson-Dernier et al., 2008). In this mutant, sperm cell release is only impaired when an amc pollen tube reaches an amc female gametophyte, resulting in the pollen-tube outgrowth previously described (Boisson-Dernier et al., 2008). AMC functions as a peroxin in reproductive tissues and it has been postulated that mutations in this gene may result in the loss of a molecule originating from the peroxisome required for female and male gametophyte communication (Boisson-Dernier et al., 2008). Undoubtedly, coordination of an intrinsically complex and costly process such as double fertilization must rely on a robust regulatory network, and miscommunication during female gametophyte development can lead to fertilization-independent fruit initiation. 4.5.3.1 Roles of the egg cell and central cell in fruit initiation The role of the endosperm and egg cell in the control of seed development has been extensively reviewed (Pien and Grossniklaus, 2007) and several mutants have been isolated that uncouple fruit initiation from fertilization. Autonomous endosperm development in the absence of fertilization was first observed in the FERTILIZATION-INDEPENDENT ENDOSPERM (FIE)

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122 Fruit Development and Seed Dispersal mutant (Ohad et al., 1996) and in the FERTILIZATION-INDEPENDENT SEED (FIS) mutant class (Chaudhury et al., 1997). FIE, FIS2 and MEDEA/FIS1 (MEA; Grossniklaus et al., 1998) are all part of the FIS mutant class (Ohad et al., 1996; Grossniklaus et al., 1998; Luo et al., 1999) and derived from a genetic screen that searched for silique elongation in a male sterile background (Chaudhury and Peacock, 1994). The screen additionally produced the fwf mutant (Vivian-Smith et al., 2001). The fis class mutants encode members of the Polycomb group (PcG) proteins and together with MSI1 and RBR1 form various multiprotein complexes involved in transcriptional regulation through chromatin remodelling (Spillane et al., 2000; Sørensen et al., 2001; Kohler et al., 2003; Guitton et al., 2004; Ingouff et al., 2005; Jullien et al., 2008). Mutations in FIS1/MEA, FIS2, FIE, MSI1 or RBR1 in the female gametophyte result in fertilization-independent endosperm development which triggers fertilization-independent silique elongation (Ohad et al., 1996; Grossniklaus et al., 1998; Luo et al., 1999; Kohler et al., 2003; Jullien et al., 2008). Additionally in fis and msi mutants, autonomous endosperm development can also give rise in some cases to seed-like structures containing aborted embryos arrested at an early stage (Chaudhury et al., 1997; Guitton and Berger, 2005), suggesting that the central cell plays an important role in the control of both fruit and embryo initiation. Recently, autonomous endosperm development was also observed in sirène (srn) and scylla (syl) mutants (Rotman et al., 2008). ` receptor-like kinase is expressed in the two synergids The FERONIA/SIRENE and it is involved in the control of the release of the sperm cells (Huck et al., 2003; Rotman et al., 2003; Escobar-Restrepo et al., 2007). In fis loss of function mutants, endosperm proliferation in the absence of fertilization is caused by the relief of the restraint imposed by FIS genes in the central cell; however, the origin of endosperm proliferation in srn and syl mutants remains to be clarified (Rotman et al., 2008). The study of cdka:1/cdc2a mutants has also provided useful insights into autonomous endosperm and fruit initiation (Nowack et al., 2006). In cdc2a mutant pollen, a single sperm cell is produced and is able to fertilize the egg cell (Nowack et al., 2006). Although selfing of heterozygous mutant plants as well as reciprocal crosses with wild-type plants showed that cdc2a pollen caused seed abortion (Nowack et al., 2006), initial egg cell fertilization was able to promote autonomous endosperm proliferation (Nowack et al., 2006). It is widely accepted that upon fertilization auxin originating from the seed is generated (Fig. 4.2). CLSM images have shown that initial endosperm division is enough to trigger the auxin signal (Fig. 4.2b). It would be interesting to investigate whether fertilization of the egg cell by cdc2a pollen can indeed trigger auxin responsiveness to the same extent that double fertilization does. 4.5.3.2 Roles of the integuments in fruit initiation Apart from selection of male gametes arriving at the ovule and providing nutritive support to the developing zygote and endosperm, the integuments have a role in signal transduction that directly stimulates fruit initiation.

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Analysis of the cell cycle marker PCycB1;2 :GUS in integuments indicates that cells remain mitotically active throughout the female receptive period (Ingouff et al., 2006). However, soon after fertilization, the integument cells begin expansion and the mitotic index further increases (Ingouff et al., 2006). Significantly, the expression of auxin-responsive DR5rev::eGFP marker also begins in the integuments around 5 h post-fertilization (Fig. 4.2b; VivianSmith and Offringa, unpublished data). Considering that it takes 2–3 h for green fluorescent protein (GFP) expression from the DR5rev::eGFP reporter to become visible by CLSM (Sauer et al., 2006), auxin responsiveness must at least occur 2–3 h post-fertilization. Another indication that integument development can trigger fruit initiation independent of fertilization comes from two other sources. The knockout of the MET1 DNA methyltransferase enzyme, in the met1-3 mutant, stimulated both the differentiation of the seed coat testa and also that of fruit development without fertilization (FitzGerald et al., 2008). These results imply control of DNA methylation in integument morphogenesis. In another case, the genetic analysis of the bel1-1 mutant also uncouples fruit initiation from fertilization and ovule development suggesting that in wild type, the control of integument development, possibly together with the nucellar identity, is linked to the control fruit initiation (Western and Haughn, 1999; Vivian-Smith, 2001). Associated pseudo-integument development has also been reported in parthenocarpic tomatoes (Mazzucato et al., 2003; Goetz et al., 2007; De Jong et al., 2008) and in the Arabidopsis fwf/arf8 mutants, albeit much weaker than in tomato (Vivian-Smith et al., 2001).

4.6 Hormonal cues during fruit initiation In 1936, Gustafson discovered that application of synthetic auxins to emasculated flowers of several different plant species resulted in parthenocarpic fruit development and, thus, established the initial linkage between fruit initiation and plant-growth regulators (Gustafson, 1936). At present, three main types of plant-growth regulators are recognized as having phytohormonal properties that can potentially induce fruit setting and fruit development (Gillaspy et al., 1993). Application of auxin, gibberellins or cytokinin, either alone or in combination, has been shown to trigger parthenocarpy across a wide variety of plant species (Gustafson, 1936; King, 1947; Srinivasan and Morgan, 1996; Vivian-Smith and Koltunow, 1999; Ozga et al., 2002, 2003). Application of optimal combinations of plant-growth regulators to emasculated pistils can often promote elongation to the extent observed in fully seeded fruits (Vivian-Smith et al., 2001). These results have led to a long standing belief that fruit initiation is sustained by phytohormone biosynthesis occurring during the stages of seed development, although often this assertion remains unchallenged. Understanding the genetics and molecular genetics behind natural parthenocarpic mutants permits further investigation of the phytohormonal

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124 Fruit Development and Seed Dispersal signalling and the relationship to pollination, fertilization and fruit initiation. Uncoupling fruit initiation from fertilization, with parthenocarpy, offers a unique method to examine these relationships. For example, a role for the ovule in fruit initiation during parthenocarpy has been uncovered (VivianSmith, 2001; Koltunow et al., 2002). However, many questions remain to be answered. To what degree does the ovule contribute in signalling? How are phytohormonal responses initiated and then propagated through the entire flower? Which are the sites of biosynthesis and signal transduction? 4.6.1

Auxin

Auxin plays a crucial role in plant development by directing basic processes such as division, elongation, phyllotaxy, organ primordia differentiation, apical dominance, tropic responses and response to shading (Sauer et al., 2006; for a more detailed review see Benjamins and Scheres, 2008). Auxin also appears to have a primary role during fruit initiation since the genetic analysis of wild-type Arabidopsis fruit initiation with gibberellin biosynthesis and perception mutants shows that auxin-mediated differentiation underlies other signalling pathways (Vivian-Smith and Koltunow, 1999; Vivian-Smith et al., 2001). Furthermore, transcriptional profiling during fruit initiation also shows directionality in phytohormonal responses with auxin preceding gibberellin responses at 12–14 h period post-fertilization (Vriezen et al., 2007). The use of the transgenic DEFH::iaaM construct in a broad range of species (Rotino et al., 1997; Ficcadenti et al., 1999; Mezzetti et al., 2004; Yin et al., 2006) additionally suggests a universal role for auxin in triggering fruit set. Accordingly, auxin-mediated signalling is an early response in the Arabidopsis ovule (Fig. 4.2). Auxin-responsive reporters show transcriptional activation 2–3 h post-fertilization expression, when the nuclear endosperm has undergone only one division (Fig. 4.2). Auxin responses continue to increase after the third endosperm nuclear division, primarily in the integument tips, the endothelium and the chalazal region. Activation at the base of the funiculus is observed less than 12 h after fertilization (not shown). Apart from expression data, portrayed in Fig. 4.2, parthenocarpic mutants and quantitative trait loci (QTLs) have also been characterized and these clearly support roles for auxin as a primary fruit initiation cue (Vivian-Smith et al., 2001; Wang et al., 2004; De Jong et al., 2008; Gorguet et al., 2008). 4.6.1.1 Auxin-mediated transcriptional activation Several genetic lesions in the auxin pathway conferring autonomous fruit initiation have been isolated. Each of these mutants appears to work within the auxin-mediated transcriptional network. The Arabidopsis genome encodes 22 functional auxin response transcription factors (ARFs) and 29 Aux/IAA interacting proteins, and each gene appears to have strong sequence conservation in other plant genomes (Remington et al., 2004; Okushima et al., 2005).

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The ARFs are a family of Aux/IAA interacting proteins that contain a DNA-binding domain (DBD) which recognizes auxin response elements (AuxREs) in DNA sequences (Figs. 4.3 and 4.4; Kim et al., 1997; Ulmasov et al., 1999a,b). Information about the role of ARF8 in fruit initiation came from the map-based cloning of the FWF locus (Vivian-Smith, 2001). All nullomorphic lesions in the Arabidopsis FWF/ARF8 gene induce parthenocarpy, but surprisingly the homeologous expression of the arf8-4 gDNA allele can also provide parthenocarpy in tomato and Arabidopsis (Vivian-Smith, 2001; Vivian-Smith et al., 2001; Goetz et al., 2006, 2007). FWF/ARF8 transcripts are naturally downregulated within 24 h post-pollination (Goetz et al., 2006, 2007), unambiguously similar to the nullomorphic phenotype, suggesting that the removal of the ARF8 mRNA induces fruit initiation. QTL mapping and microsynteny between Arabidopsis and tomato has also uncovered a potential link between natural variations in parthenocarpic tendency and the SlARF8 locus located on chromosome 4 (Gorguet et al., 2008). Recently, the related SlARF7 locus has also been implicated in fruit initiation (Vriezen et al., 2007; De Jong et al., 2008). SlARF7 was initially identified through cDNA AFLPs technology used for transcriptional profiling. This methodology was applied to wild type and GA3 -induced fruit initiation and revealed that SlARF9 and the Aux/IAA proteins SlIAA2 and SlIAA14 are upregulated within 24 h post-fertilization, but that the SlARF7 transcript was downregulated within 24 h in ovule and placental tissues (Vriezen et al., 2007). On the basis that SlARF7 may function as a repressor, like AtARF8, De Jong et al. (2008) specifically silenced SlARF7 by RNAi. Indeed SlARF7 silenced lines produced fruit initiation without fertilization, although the morphology was not entirely similar to pollinated fruit. RNAi has also been used to silence the tomato Aux/IAA protein SlIAA9 (Wang et al., 2005). Silencing SlIAA9 also caused parthenocarpy, but it also resulted in the expression of simple leaves and altered leaf vascular differentiation (Wang et al., 2005). Single base deletions in SlIAA9 recapitulated the RNAi phenotype confirming the specificity of the SlIAA9 RNAi phenotype (Zhang et al., 2007). While the expression of SlIAA9 is not known, the expression of SlARF7 in the placenta and ovules (Vriezen et al., 2007) and the expression of AtARF8 in the endothelium, the female gametophyte, the funiculus and the chalaza (Goetz et al., 2006) clearly indicate a strong involvement with the ovule. The expression of the synthetic auxin-responsive reporter gene shown in Fig. 4.2 correlates precisely with ARF8 reporters in the unfertilized Arabidopsis ovule (as observed in Goetz et al., 2006). Initial expression occurs in the chalaza at

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