HORTICULTURAL REVIEWSVolume 47

Editorial Board, Volume 47A. Ross FergusonRobert E. Paull

Horticultural Reviews is sponsored by:

American Society for Horticultural ScienceInternational Society for Horticultural Science

HORTICULTURAL REVIEWSVolume 47

Edited by

Ian WarringtonMassey University

New Zealand

This edition first published 2020© 2020 John Wiley & Sons, Inc.

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.

The right of Ian Warrington to be identified as the author of this editorial material in this work has been asserted in accordance with law.

Registered Office(s)John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USAJohn Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK

Editorial OfficeThe Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK

For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com.

Wiley also publishes its books in a variety of electronic formats and by print‐on‐demand. Some content that appears in standard print versions of this book may not be available in other formats.

Limit of Liability/Disclaimer of WarrantyWhile the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

Library of Congress Cataloging‐in‐Publication data applied for

ISBN: 9781119625339 (Hardback)

Cover Design: Wiley Cover Image: Image courtesy of Jules Janick

Set in 10/12pt Melior by SPi Global, Pondicherry, India

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

v

Contents

Contributors ixDedication: Theodore DeJong xi

Ian Warrington

1. Molecular Physiology of Fruit Growth in Apple 1Anish Malladi

I. Introduction 2 II. Morphology and Anatomy of the Apple Fruit 2 III. Flower Growth Before Bloom 5 IV. Fruit Set 7 V. Fruit Growth 9 VI. Conclusions 31

Literature Cited 33

2. Mechanosensing of Plants 43Marc‐André Sparke and Jens‐Norbert Wünsche

I. Introduction 44 II. Thigmomorphogenesis 47 III. Natural and Artificial Induction of Thigmo Responses 48 IV. Morphological Plant Responses 50 V. Physiological Plant Responses – Cellular Signaling 57 VI. Molecular Aspects 69 VII. Application Strategies in Horticulture 70 VIII. Conclusions 72

Literature Cited 73

3. Microgreens: Definitions, Product Types, and Production Practices 85Sven Verlinden

I. Introduction 86 II. History of Immature Leafy Vegetables 92

vi CONTENTS

III. Seedling Development in Other Crops – Growth and Development of Seedlings 94

IV. Production Strategies 96 V. Nutritional Value 104 VI. Microbiological Safety and Postharvest Biology

and Technology 114 VII. Sensory Attributes and Qualities 117 VIII. Health Effects 117 IX. Future of Microgreens 118

Literature Cited 119

4. The Durian: Botany, Horticulture, and Utilization 125Saichol Ketsa, Apinya Wisutiamonkul, Yossapol Palapol, and Robert E. Paull

I. Introduction 127 II. Botany 140 III. Cultural Practices 149 IV. Chemical Composition and Nutritional Value 173 V. Postharvest Physiology 177 VI. Harvesting and Postharvest Handling 184 VII. Utilization 192 VIII. Conclusions 195

Literature Cited 195

5. The genus Cupressus L.: Mythology to Biotechnology with Emphasis on Mediterranean Cypress (Cupressus sempervirens L.) 213Homayoun Farahmand

I. Introduction 215 II. Cupressaceae (Geographical Distribution

and Horticultural Importance) 215 III. The Genus Cupressus 216 IV. The Role of Mediterranean Cypress in Persian Gardens 249 V. Medicinal Values 252 VI. Breeding and Genetic Improvement 254 VII. Abiotic and Biotic Challenges 256 VIII. Conservation of Genetic Resources 261 IX. Conventional Propagation and Micropropagation 263 X. Biotechnology 265 XI. Conclusions 267

Literature Cited 268

CONTENTS vii

6. Taxonomy and Botany of the Caricaceae 289V.M. Badillo and Freddy Leal

I. Introduction 290 II. History of the Papaya and Other Caricaceae 291 III. Taxonomic History 291 IV. New Proposals for the Taxonomy of Caricaceae 295 V. Botany of the Family and the Genera 297 VI. Concluding Comments 319

Literature Cited 320

7. Entomopathogens: Potential to Control Thrips in Avocado, with Special Reference to Beauveria bassiana 325Gracian T. Bara and Mark D. Laing

I. Introduction 326 II. Commercial Production in South Africa 328 III. Requirements for Export and Local Quality 329 IV. Economics of Avocado Production 329 V. Pests and Diseases of Avocado 330 VI. Thrips of Avocado 330 VII. Management of Thrips 333 VIII. Entomopathogens 336 IX. Conclusions 356

Literature Cited 357

Subject Index 369Cumulative Subject Index 372Cumulative Contributor Index 406

ix

Contributors

V.M. Badillo,† Facultad de Agronomía, Universidad Central de Venezuela, Maracay, Aragua, Venezuela

Gracian T. Bara, School of Agricultural, Earth and Environmental Sciences, University of KwaZulu-Natal, Scottsville, Pietermaritzburg, South Africa

Homayoun Farahmand, Department of Horticultural Science, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

Saichol Ketsa, Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok, and Thailand and Academy of Science, The Royal Society of Thailand, Dusit, Bangkok, Thailand

Mark D. Laing, School of Agricultural, Earth and Environmental Sciences, University of KwaZulu-Natal, Scottsville, Pietermaritzburg, South Africa

Freddy Leal, Facultad de Agronomía, Universidad Central de Venezuela, Maracay, Aragua, Venezuela.

Anish Malladi, Department of Horticulture, University of Georgia, Athens, GA, USA

Yossapol Palapol, Division of Agricultural Technology, Faculty of Science and Arts, Burapha University, Chanthaburi Campus, Thamai, Chanthaburi, Thailand

Robert E. Paull, Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa, HI, USA

Marc‐André Sparke, Department of Crop Science, Institute of Crop Physiology of Specialty Crops, University of Hohenheim, Stuttgart, Germany

Sven Verlinden, Department of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia, USA

Apinya Wisutiamonkul, Expert Centre of Innovative Agriculture, Thailand Institute of Scientific and Technological Research (TISTR), Khlong Luang, Pathum Thani, Thailand

Jens‐Norbert Wünsche, Department of Crop Science, Institute of Crop Physiology of Specialty Crops, University of Hohenheim, Stuttgart, Germany

†deceased

Theodore DeJong

Dedication: Theodore DeJong

xi

Professor Theodore (Ted) DeJong has had a long and distinguished career in the broad area of whole tree physiology, with a particular emphasis on stone fruit species (peach, plum, and nectarine) that are relevant to California but widely grown in many areas of the world. This research has led to an enhanced understanding of tree growth and development, especially in areas relating to carbon balance in the tree, tree architecture, and the growth of the vegetative canopy, fruit, and roots. He has focused considerable effort on modeling these various processes. In addition, he has been a leader in an effective stone fruit breeding program.

Ted grew up in Ripon, CA and spent a good deal of time working on peach and almond farms in the area – he had loved farming ever since he was in grammar school. Ted attended Ripon Christian Schools and then Calvin College in Grand Rapids, Michigan. While in college he became interested in ecology and, because of his farming experience, was mainly interested in plant ecology. After college, in 1968, he mar-ried his wife Rose, and was scheduled to be drafted into the army and so he volunteered for admission to the Army Officer Candidate School. He graduated from OCS in late 1969, was commissioned, spent most of the next year in Fort Riley, Kansas, and went to Vietnam on September 11, 1970.

In September, 1971 he enrolled in an M.S. program at Fullerton State University in Plant Ecology. His mentor there was Dr Ted Haines and he was probably most influential in Ted subsequently choosing an academic career. In Jan, 1974 he enrolled in the Botany Ph.D. program at the University of California, Davis to continue studying Plant Ecology with Prof. Mike Barbour. His research was on the physiological ecology of Californian beach and dune species.

In January, 1978 he began a one‐year post‐doctoral fellowship at the Smithsonian Institution and did research on the physiological ecology of tidal marsh species. One year later, Ted returned to UC Davis on a Post‐Doctoral Fellowship in the Agronomy and Range Science Department

xii DEDICATION: THEODORE DEJONG

with Prof. Don Phillips working on carbon and nitrogen assimilation interactions in legumes. At this time, he realized that he could have a rewarding career working in “applied environmental physiology of crop plants,” otherwise known as “crop physiology.”

He was appointed as a Lecturer/Pomologist/Co‐op Extension Spe-cialist at the Kearney Agricultural Experiment Station and Extension Center (KAC) in the Pomology Department in April 1981. Thanks to good field staff support, he remained at Davis but conducted virtually all of his research and extension work at KAC for the first 25 years of his career. He was assigned to teach practical pomology courses at Davis and also co‐taught graduate level courses in plant/crop physiology. He enjoyed teaching and by the end of his career he was the main instruc-tor for the pomology/tree crop physiology courses at UC Davis, even though he had never taken a formal pomology or horticulture course in his life.

His teaching had a lot of influence on his research because, early in his career, as he was teaching some of the pomological “dogma” to his students, he realized that some of it made little physiological/eco-logical sense. Furthermore, many of the current horticultural practices were often not backed up by sound scientific study, so some of these “dogmas” formed ideas for research. Such ideas led to investigating: the importance of leaf photosynthetic capacity in determining crop yield; fruit effects on photosynthesis; causes of the double sigmoid growth of stone fruit; carbohydrate and nitrogen allocation in fruit trees; car-bohydrate storage in trees; causes of alternate bearing in fruit trees; physiological mechanisms involved in size‐controlling rootstocks; factors driving shoot growth in fruit trees; and interactions between fruit, shoot, and root growth in fruit trees.

Ted’s challenges to much of this “dogma” within the teaching frame-work have also been widely acknowledged internationally. New Zealand pomologist Dr Stuart Tustin states that “Ted’s incisive consideration of the science presented within the ISHS Fruit Section has always been highly valued by symposia participants, as has the friendly pugilism often arising in such discussions with Ted in such fora. He can always be relied upon to challenge concepts and interpretations and in such ways, contribute greatly to the scientific thinking and advances in fruit crop pomology and physiology.”

When he was a graduate student at UCD, he attended Prof. Robert Loomis’s crop ecology lectures for two years in a row and was fascinated with his crop modeling work. When Ted had his first sabbatical oppor-tunity in 1987, he went to Wageningen University in the Netherlands to learn more about developments in that field of research. This was a

DEDICATION: THEODORE DEJONG xiii

watershed experience for his career. While there, he realized that plants can best be viewed as composite organisms made up of semi‐autono-mous organs and the key to understanding/modeling whole plants was in understanding what drives the growth of those individual organs. This also led to development of a new model for explaining stone fruit growth patterns. After this sabbatical, much of his conceptually‐based research involved various aspects of crop modeling and the culmina-tion of this work resulted in development of Functional–Structural Plant Models of peach and almond tree growth and physiology. The L‐Peach and L‐Almond models are still the most detailed and advanced virtual computer simulation models of fruit trees in existence today. They permitted the testing of, and/or demonstrated concepts behind, numerous fruit tree management practices that are commonly used in commercial fruit production.

Underpinning Ted’s work in crop modeling at the Department of Pomology (later Department of Plant Sciences) at UC Davis, was a range of research that focused on understanding tree physiological and orchard management factors that control the carbon balance/budgets of fruit and nut trees. His initial work focused on understanding the func-tioning and photosynthetic efficiencies of tree leaves and on under-standing factors governing the horticultural efficiencies of orchard can-opies. As he gained experience and understanding of factors controlling the “supply side” of the carbon balance equation, later studies focused on the “demand side” of the equation and the integration of both aspects into a functional understanding the how tree carbon budgets work. This “demand side” work focused on characterization and understanding factors governing flowering and fruit set, fruit growth, vegetative (leaf and shoot) growth, and root growth, and eventually involved numerous studies characterizing how rootstocks control shoot growth (see Horti-cultural Reviews 46:39–97 for this latter topic). Much of his intellectual stimulation for conducting the various aspects of this research came from an overall goal of developing an integrated understanding of fruit tree carbon budgets and growth through crop modeling. As indicated above, this led to the development of very sophisticated and complex functional–structural tree simulation models that are not only carbon budget models but also include integrated understanding of the archi-tectural development of fruit trees.

French scientist, Dr Evelyn Costes, notes that: “with an an open‐minded vision Ted has combined skills in plant physiology, classical horticulture and fruit tree cultivation with new technologies involving computer programming and modelling. He thus has made an excep-tional research contribution.”

xiv DEDICATION: THEODORE DEJONG

Italian colleague, Dr Paolo Inglese, states that: “personally I gained a lot of inspiration from his papers on fruit growth and development and carbon partitioning in peach trees. I really learned a lot and, most interesting, it became easy to move from physiology to orchard management, understanding the basic factors of tree behavior. From this point of view, it is clear how strongly Ted DeJong influenced a large number of students, and younger scientists worldwide, with an impres-sive benefit for horticultural development, in terms of knowledge and, most importantly, field practices and orchard management. Indeed, it is worth noting that Ted’s research was always related to real problems experienced by stone fruit growers, particularly peach and almond growers. He is strongly dedicated to solving real problems through a clear scientific standpoint and this deserves our admiration. I have seen Ted giving lectures several times, but I have also seen him talking to growers in the field ready to learn and to share his knowledge as well as to understand the basic facts behind any particular horticul-tural technique.”

Pomology Farm Advisor, Rachel Elkins, summarizes Ted’s research achievements as follows: “Ted’s successful career reflects his thought process: creativity melded with logic and practicality. His upbring-ing on an almond farm in the Central Valley provided the necessary ‘grounding’ needed to ensure his research and extension contributions would impact commercial agriculture. His training in basic biology and ecology have provided the unique holistic perspective enabling him to think ‘out of the box’ about fundamental perennial crop physiology concepts. Those of us fortunate enough to take classes from him, study under him, and work with him have benefitted enormously. Indeed, the concept Ted has developed and demonstrated, that tree vigor and bearing at any given time of the year and life stage are fundamentally and primarily related to carbohydrate partitioning and balance has permanently influenced my own thought processes dealing with tree health issues in the field and in developing my own applied research. I am not alone; California tree crop advisors who have studied under, or worked with Ted, are well‐trained and confident in their understanding of fruit and nut crop physiology.”

In addition to this physiological research, he has also been the prin-ciple investigator on a prune breeding project since 1985. The Cali-fornian prune industry is currently dependent on a single cultivar. The goal of this project is the development of new prune/dried plum cultivars for the Californian industry that will increase orchard and processing efficiencies, spread the harvest season, and maintain or increase dried product quality. Ted holds 11 plant patents that cover

DEDICATION: THEODORE DEJONG xv

the cultivars that have been developed in that program as well as col-laborative development of size‐controlling rootstocks for peach and nectarine production.

In addition to his extensive research activities, Prof. DeJong also held a number of administrative positions at UC Davis, several of which coincided with challenging financial times and consequent organiza-tional restructuring. He served on the College Research Committee and later the College Executive Committee, being the Chair during a College reorganization. In that role he also served on the Campus Senate Exec-utive Council and on the Committee on Academic Planning and Bud-get Review for eight years, during several budget crises and strategic planning projects. From these experiences, he gained an understanding of the politics involved in campus and university decision making and became somewhat disillusioned in the process given the continuing decline in academic values and principles.

He was chair of the Pomology Department for eight years and was also a Vice‐Chair in the Plant Sciences Department for three years. He additionally chaired numerous other committees including the Pomology/Plant Sciences Department’s Field Facilities Committee for nearly 30 years, the College Air Shuttle Committee for 15 years and the Foundation Plant Services (FPS) Strawberry Advisory Committee for 15 years. He also served as a Senate representative on the Athletics Administrative Advisory Committee for more than six years as well as the Administrative Committee for the Transition of UC Davis athletics from Division II to Division I. He regards his main accomplishment in this latter service was to try to maintain the UC Davis vision of “stu-dent‐athlete” as the university moved to higher levels of competition.

Prof. DeJong’s scientific output has been prodigious over the past four decades. He has published over 200 manuscripts in refereed scientific journals. In addition, he has presented numerous talks at grower meet-ings and scientific conferences. He has also maintained a regular teaching program and mentored more than 50 Masters, Ph.D., and post‐doctoral students from the US and several foreign countries.

Fellow Pomologist, Prof. Greg Reighard, Clemson University, states: “His publication record is unmatched by his peers, while his research has been productive, relevant, well received and widely implemented by his clientele. In my opinion, he is the foremost authority on stone fruit whole tree physiology in the world. His advice is frequently so-licited by his peers for his insight into problems related with fruit pro-duction. His prolific publication record and esteemed standing with research journals and professional societies are a testament to his abilities as a scholar and mentor.”

xvi DEDICATION: THEODORE DEJONG

Prof. DeJong has been an active member of professional horticultural science societies for many years, contributing widely and consistently to their scientific programs during that time. In particular, he has support-ed many ISHS symposia and has, consequently, published in a number of Acta Horticulturae volumes (70 manuscripts in 23 Acta Horticulturae volumes). He has also been the convener of two ISHS symposia and is currently (2014–22) the Chair of the Section Pome and Stone Fruits.

His achievements have been recognized by a number of honors and awards, including: Smithsonian Post‐doctoral Fellow, 1979; Nether-lands International Agriculture Center Fellowship, January–July, 1987; NATO Senior Guest Fellowship to Italy, July–August 1987; Fellow, American Society for Horticultural Science, Class of 2002; The National Peach Council Outstanding Peach Researcher Award, 2002; UC Davis Distinguished Professor, 2013; ISHS Lifetime Achievement Award for Outstanding Contributions to Research and Education in Fruit Crop Physiology, 2014; and ISHS Fellow, 2018.

Prof. DeJong observes that he was extremely fortunate to have had his career in a time that he and many of his colleagues, who were in the Pomology Department during this period, call the “golden age of pomology.” It was a time when there was ample financial and personnel support for research. He started with a department‐paid career staff research associate (SRA) and enough San Joaquin Valley/industry support to support another young SRA to plant and main-tain tree crop research plots and to conduct numerous “exploratory” research projects to gain new perspectives on how fruit trees work. In addition, the university believed in, and supported, field research. Research was valued for the help that it provided growers rather than just how much funding it brought into the university. He observes that too often research is increasingly valued now more for the fund-ing that it generates than the actual societal benefits derived from it. Field stations are run now more as “profit centers” than as research support enterprises.

He is deeply concerned that future pomology researchers will not be as successful as he was able to be, not because (he claims) he was espe-cially talented, but because it will be impossible to access the practical, integrated experience that he was able to gain because of the support systems that were in place when he began his career. Because of ear-ly experiences, he was able to sustain funding mechanisms with long term horizons. It will be nearly impossible for young researchers to do the same now, when they are obligated to continually develop new projects and garner new funding for projects lasting only two to three years when working on complex, perennial crops.

DEDICATION: THEODORE DEJONG xvii

Prof. DeJong and his wife have a family of three sons and ten grand-children. Their eldest, Jason, is Professor of Geotechnical Engineering at UCD and the youngest, Matthew, is Professor of Structural Engineering at UC Berkeley. Their middle son, Michael, is a Fire‐fighter/Paramedic, also based in California. Although formally retired, Ted continues to do research and to contribute to the programs at UCD.

“I would like to close on a personal note and state that no one cur-rently working in the field of stone fruit pomology is at Ted’s level as to what he has accomplished. There are some outstanding pomologists working throughout the world on stone fruit physiology, but I think Ted stands as tall or taller (no pun intended) than all of them at this stage of his career” – Dr Greg Reighard.

Ian WarrIngton

Emeritus ProfessorMassey UniversityPalmerston North

New Zealand

Horticultural Reviews, Volume 47, First Edition. Edited by Ian Warrington. © 2020 John Wiley & Sons, Inc. Published 2020 by John Wiley & Sons, Inc.

1

1

ABSTRACT

Fruit growth and development are processes of primary biological importance and of considerable commercial significance. In apple, the fleshy fruit is derived largely from non‐ovarian tissue. Regulation of fruit growth in apple is therefore likely distinct from that in other model fleshy fruit species. Fruit growth is an integration of multiple processes that are regulated through developmental factors, phytohormones, and availability of metabolic resources. These factors differentially influence growth during diverse stages of development, and across different tissues within the fruit. In recent years, substantial progress has been made in identifying some of the major molecular components and mech-anisms involved in the regulation of apple fruit growth. This review presents a comprehensive analysis of our current knowledge of the molecular physiology of fruit growth in apple and identifies gaps where future research is needed to expand our knowledge of the regulation of this trait.

KEYWORDS: cell division; cell expansion; fruit development; fruit size; organ growth

Molecular Physiology of Fruit Growth in Apple

Anish MalladiDepartment of Horticulture, University of Georgia, Athens, GA, USA

I. INTRODUCTIONII. MORPHOLOGY AND ANATOMY OF THE APPLE FRUIT

III. FLOWER GROWTH BEFORE BLOOMIV. FRUIT SETV. FRUIT GROWTH

A. Components of Fruit Growth: Cell Production, Expansion, and Void SpacesB. Fruit Growth and its Regulation

2 ANISH MALLADI

I. INTRODUCTION

Apple (Malus × domestica) is one of the most widely grown temperate fruit crops in the world. Fruit growth and development are not only of botanical significance but are also of vast economic significance in apple production. In this review, growth is defined as the increase in size of the organ, while development is defined as the progression of the organ through various phenological stages. The main emphasis of this review is on the processes and factors mediating fruit growth. However, often, growth of an organ and the processes that mediate it are intimately associated with its development. Hence, where applicable, these inter‐relationships will also be discussed.

II. MORPHOLOGY AND ANATOMY OF THE APPLE FRUIT

The apple fruit is botanically a “pome.” Fruits of this class are charac-terized by the presence of fleshy exocarp and mesocarp tissues and a cartilaginous endocarp. The majority of the fruit tissue is comprised of accessory tissue (Pratt 1988). The central region of an apple fruit is typ-ically constituted by five locules that are derived from five carpels from a syncarpous ovary. Each of these carpels may contain up to four ovules which upon fertilization can yield one to four seeds (Pratt 1988). The seeds are surrounded by the cartilaginous endocarp tissue at maturity. A ring of five sepal and five petal vascular traces occurs towards the periphery of the locules and is often referred to as being the core‐line. Tissue outside of this core‐line develops into the major fleshy and eco-nomically significant part of the apple fruit (Figure 1.1). At maturity, this tissue may constitute over 80% of the fruit volume (Tukey and Young 1942; Goffinet et al. 1995).

Ontogeny of the fleshy region of the fruit outside of the core‐line and the precise localization of ovarian tissue inside of it have been debated extensively and reviewed previously (Pratt 1988). Briefly, two conflicting hypotheses have been proposed to explain the ontogeny of fruit tissues. According to MacDaniels (1940), the receptacular hypothesis indicates

C. Cell Production Related Genes and Regulation of Fruit GrowthD. Organ Size Related Genes and Regulation of Fruit GrowthE. Floral Homeotic Genes and Regulation of Fruit GrowthF. Cell Wall Modifying Genes and Regulation of Fruit GrowthG. Metabolism and Regulation of Fruit GrowthH. Phytohormones and the Regulation of Fruit GrowthI. A Note on the Measurement of Growth

VI. CONCLUSIONSLITERATURE CITED

1. MOLECULAR PHYSIOLOGY OF FRUIT GROWTH IN APPLE 3

that the major fleshy region of the fruit is derived from axial tissues. The central region of the fruit extends from the pedicel into the fruit and the outer fleshy tissue represents an extension of the cortical region peripheral to the vascular tissue within the stem. Hence, this tissue is referred to as the cortex (Figure 1.1). As an extension of this terminol-ogy, tissue inside of the core‐line (vascular tissue) is referred to as the pith. The location of the ovarian tissues is within the pith region and restricted to cell layers immediately surrounding the locules (Figure 1.1). Further, the true fruit is composed of five drupe‐like structures charac-terized by a cartilaginous endocarp. As the cell layers constituted by the exocarp and mesocarp tissues are few, most of the pith is comprised of parenchymatous cells of non‐ovarian origin. The alternative appen-dicular hypothesis presents a divergent view. In this context, the tissue peripheral to the core‐line originates from the fusion of the basal tis-sues of appendages: multiple floral organs including the petals, sepals, and stamens (MacDaniels 1940). Hence, this tissue is often described as a floral tube or a hypanthium derived from fused basal regions of floral appendages. Tissue inside of the core‐line is considered of ovar-ian origin, such that the innermost layer of this tissue is the endocarp while the rest is constituted by fleshy exocarp and mesocarp tissues. The core‐line is regarded as the line of fusion between the floral tube

Fleshy pericarp*Dorsal carpellary tracePith

Core-lineLocule

Sepal vascular trace

Petal vascular trace

Fruit skin

Cortex

Figure 1.1 Transverse section of the apple fruit displaying the primary tissues. *: The fleshy pericarp here is shown to indicate interpretation of the fruit morphology according to the receptacular hypothesis. According to this interpretation, tissue imme-diately surrounding the locule constitutes the pericarp. The appendicular hypothesis considers tissue inside of the core‐line to be of ovarian origin as described in the text.

4 ANISH MALLADI

and the ovary. The relative merits of each of these theories have been evaluated with many recent authors preferring the appendicular hypo-thesis (Pratt 1988), largely owing to interpretations from comparative vascular anatomies across Rosaceae family fruits, as elegantly described by MacDaniels (1940), as well as data from cytochimeras summarized by Pratt (1988). Very few studies since the 1950s have addressed the ori-gin of these tissues, despite its botanical significance.

An effective way to determine the origin of these tissues in the apple fruit is through the application of specific tissue‐based markers for development. As the fruit and some of its constituent parts are derived from specific floral organs, identification of markers defining these floral organs in the fruit tissues, especially during early fruit development, can provide clues to the origin of these tissues. The ABC model describes development of floral organ identity (Bowman et  al. 1991; Coen and Meyerowitz 1991) and has been extensively validated across many plant systems (Bowman et  al. 2012; Irish 2017). According to this model, members of the A class of gene products determine sepal identity, and in interaction with those of the B class gene products, the identity of petals. Interaction of the B and C class gene products influences sta-men development, while the C class gene products regulate gynoecium development (Irish 2017). Putative homologs of these classes of genes, many of which are MADS box transcription factors, have been identi-fied in apple. The apple APETALA2 (AP2) is a putative A class gene, the transcripts for which were shown to be abundant in sepal tissues (Kotoda et al. 2000) and in the cortex/floral tube region during early fruit development (Yao et al. 1999). This suggested that the fleshy region of the apple fruit was likely derived at least from sepal tissues. Further, fac-ultatively parthenocarpic spontaneous mutants of apple, ‘Rae Ime’ and ‘Spencer Seedless’ were identified to be defective in one type of B class genes, PISTILLATA (PI; Yao et al. 2001). Transcript accumulation of the PI gene was abundant within the petals but could not be observed in the cortex/floral tube region of the developing apple fruit at four weeks after bloom (Yao et al. 2001, 2018). These data suggested that petal and stamen tissues did not likely contribute substantially to development of the cortex/floral tube region of the fruit. The A class gene defining sepal identity, AP2, is regulated post‐ transcriptionally by microRNA 172 (miR172). In apple, miR172 has been associated recently with regulation of fruit growth and final size (Yao et al. 2015). Higher levels of miR172 were associated with a reduction in fruit growth while the opposite was true under lower levels of miR172. Overexpression of miR172p (one of several active miR172) in transgenic ‘Royal Gala’ plants resulted in a dramatic reduction in fruit size (Yao et al. 2015). The authors proposed

1. MOLECULAR PHYSIOLOGY OF FRUIT GROWTH IN APPLE 5

that the cortex/floral tube was largely derived from the base of the sepals and post‐transcriptional alteration of the A class gene product, AP2, in these transgenic plants leads to altered growth of tissue derived from this floral organ. Together, these data strongly support the appendicular the-ory of apple fruit development and suggest that the basal regions of the floral organs, particularly the sepals, contribute greatly to development of the major fleshy tissue of the apple fruit. There are, however, a few limitations to the above approaches. Many of the genes described above have been identified and described in apple as floral organ identity genes that have clearly defined roles during flower development, but their roles in post‐flowering fruit development are not as well character-ized (Yao et al. 2016). While their transcript and protein accumulation in specific parts of the flower would be clearly indicative of organ iden-tity, the significance of such accumulation at later stages and during fruit growth may need to be interpreted with caution. Further, in case of miR172, substantial growth and development of the fruit cortex/floral tube tissue was still noted as the hypanthium in transgenic lines, where it was overexpressed, was reduced by only about 25% during early fruit development (Yao et al. 2015). Presumably, basal regions of the petals and stamens may still contribute to the growth and development of this tissue. The current availability of additional floral tissue identity genes and newer approaches that aid in isolating specific tissues of the devel-oping flower/fruit, such as laser capture micro‐dissection, need to be applied to clearly identify the origin of this tissue.

III. FLOWER GROWTH BEFORE BLOOM

Apple flower buds are induced and initiated in the previous season and display substantial growth and development prior to dormancy (Buban and Faust 1982). Eight stages of progression in apical meristem morphol-ogy prior to winter dormancy have been described (Foster et al. 2003). Broadening of the apex of the meristem was identified as a key morpho-logical feature signaling commitment to floral induction. This transition to floral induction peaked around 53 days after full bloom (DAFB) but continued until 127 DAFB. Similarly, broadening of the apex was also considered as a signal committing the meristem to inflorescence initi-ation (Pratt et al. 1959; Pratt 1988). Doming of the apex was rapid and occurred largely between 96 and 109 DAFB. Lateral floral meristem ini-tiation and terminal floral meristem initiation followed this induction. By winter dormancy (~280 DAFB), floral meristems have clearly dis-cernable initiation of sepals. At least in the terminal flower, further differentiation of floral organs and their development is also evident

6 ANISH MALLADI

before endo‐dormancy (Pratt 1988). During the period of endo‐dor-mancy and subsequent eco‐dormancy, little growth and development are observed. Further flower development including microsporogenesis and macrosporogenesis is completed following bud break (Pratt 1988).

The period from bud break to anthesis/bloom is associated with sub-stantial growth of the floral tube (Smith 1950; Malladi and Johnson 2011): a substantial increase in floral tube diameter from around three weeks before, until full bloom was reported (Smith 1950; Malladi and Johnson 2011). However, growth during this period was not uniform and involved rapid growth during the early part of this period followed by a cessation in growth before bloom (Malladi and Johnson 2011). A similar temporary reduction in growth around bloom was noted previ-ously in several cultivars (Smith 1950), indicating that this may be an important feature of pre‐bloom growth in apple. Reductions in growth prior to pollination and fertilization have also been reported previously in tomato (Vriezen et al. 2008; de Jong et al. 2009). Growth during the period from bud‐break to bloom was largely associated with an increase in cell number, indicating that the majority of this growth was support-ed by an increase in the extent of cell production, although cell size also increased slightly during this period. The cessation in growth prior to bloom was associated with a quiescence in cell production (Malladi and Johnson 2011). Transcript accumulation of multiple positive regula-tors of cell production, such as the CYCLIN DEPENDENT KINASE B1;2 (CDKB1;2), was reduced by greater than twofold during this period. Con-versely, transcript accumulation of two KIP RELATED PROTEINS (KRP4 and KRP5), which function as negative regulators of cell production by inhibiting progression of the cell cycle, were enhanced by over four-fold. Together, these data suggest coordinated transcriptional regulation to decrease cell production prior to bloom. Such reduction in growth may serve to restrict further nutrient investment in this organ until after successful pollination and fertilization. Alternatively, this reduction in growth may be reflective of extensive competition for limited resources during this period. Early growth, including flower growth after bud‐break and before bloom, is largely supported by carbohydrate and nutri-ent resources remobilized from stored reserves (Hansen 1971; Titus and Kang 1982). Flower growth and development likely competes with veg-etative growth for these resources, including that of spur leaves during the pre‐bloom period. It may be hypothesized that such competition for resources leads to reduced allocation of these reserves for further growth of the flower before bloom. However, growth cessation during this period is temporary and resumes after successful pollination and fertilization, indicating that internal factors beyond the availability of resources may also temporarily limit growth during the pre‐bloom

1. MOLECULAR PHYSIOLOGY OF FRUIT GROWTH IN APPLE 7

period. There is a general paucity of information regarding the role(s) of such internal factors prior to bloom. It is possible that changes in phy-tohormone content, transport, or signaling play important roles in regu-lating growth during this period. In unpollinated ovaries of garden pea, abscisic acid (ABA) concentration was found to be generally higher (Ro-drigo and Garcia‐Martinez 1998). Similarly, transcripts of genes associ-ated with ABA and ethylene biosynthesis and signaling were generally upregulated in unpollinated tomato ovaries (Vriezen et al. 2008), sug-gesting these phytohormones, particularly ABA, may serve as negative regulators of cell production to limit growth during this period. Inter-estingly, KRPs are known to be positively regulated by ABA (Wang et al. 1998; Vergara et al. 2017). It may be speculated that similar regulation may occur in apple flowers before bloom to alter cell cycle progression and allow for quiescence in cell production. However, this needs to be experimentally verified. Measurement of phytohormone concentrations during the pre‐bloom and bloom stages would be insightful in this con-text. Further, analysis of transcriptome changes during the pre‐bloom period and in relation to cessation of growth is likely to provide further insights into its regulation.

IV. FRUIT SET

The term “fruit set” has different implications in apple botany and pro-duction. Botanically, fruit set refers to the transition from a flower to fruit upon successful pollination and fertilization. The alternative, and a common use of the term in apple literature, is in reference to the total amount of fruit retained on the tree after bloom (“initial set;” Lakso and Goffinet 2017) or after subsequent events of young fruit abscission (“final set”). In this review, the term is used to refer to the botanical interpretation of fruit formation associated with seed formation.

Pollination and fertilization lead to seed set. In other fruits such as tomato, seed set is thought to alter phytohormone synthesis and signal-ing, such as that of auxin, resulting in the resumption of ovary growth and the initiation of fruit development. Similarly, pollination, fertiliza-tion, and/or seed set may result in the generation of signals that trigger the resumption of growth within the fleshy regions of the apple fruit. Malladi and Johnson (2011) studied growth of the floral tube region and associated cell production and expansion in pollinated and unpollinated flowers. Growth of the floral tube region resumed between 3 to 10 DAFB in pollinated flowers but not in unpollinated flowers. This resump-tion in growth was associated with a rapid increase in cell produc-tion. Re‐initiation of growth and cell production following pollination

8 ANISH MALLADI

was further associated with coordinated changes in accumulation of multiple transcripts associated with the regulation of cell production (Malladi and Johnson 2011). Many of the positive regulators of the cell cycle such as the A and B‐type CYCLINS and several CDKBs displayed a clear increase in transcript abundance during the fruit set period (8–11 DAFB). Further, their abundance was dramatically reduced in unpollinated flowers consistent with a reduction in cell production and growth in these flowers. Negative regulators such as the CDK inhibitor, KRP4, displayed severely reduced transcript accumulation during the initial phases of fruit set and enhanced accumulation in unpollinated flowers. Together, these data indicate a coordinated transcriptional reg-ulation of fruit set. Factors that coordinate such a response are not well understood in apple. In other fruits such as tomato, auxins and gibber-ellins (GAs) are known to regulate fruit set. Exogenous applications of auxin can induce parthenocarpic fruit growth in tomato ( Serrani et al. 2007) and other fruits. Transcriptome analysis of pollinated tomato flowers during fruit set indicated alteration of multiple auxin signaling‐related components (Vriezen et al. 2008). One set of these are the AUXIN RESPONSE FACTOR genes, ARF7 and ARF9, which are transcription factors that coordinate auxin‐dependent transcriptional responses (de Jong et al. 2009, 2015). ARF7 transcript abundance is reduced during the fruit set period in pollinated tomato flowers. Downregulation of ARF7 in transgenic tomato plants results in parthenocarpic fruit development (de Jong et al. 2009), indicating that it is involved in downregulating growth until pollination and fertilization occur in tomato flowers. ARF9 overexpression in transgenic tomato lines decreased fruit size through negative regulation of cell production during early fruit development (de Jong et al. 2015). Its downregulation enhanced final fruit size in a com-plementary way, further implicating it in regulating early fruit growth. These data illustrate the important role of auxin in fruit development.

Exogenous applications of GA also induced parthenocarpic fruit development in tomato and multiple other fruits (Bukovac 1963; Serrani et al. 2007; Watanabe et al. 2008; Liu et al. 2018). The mode of GA action in inducing fruit set may differ from that of auxins with GAs promoting cell expansion while auxins promote cell division dur-ing early fruit development, at least in tomato (Serrani et  al. 2007). In apple, unlike in tomato, auxin applications do not always induce parthenocarpic fruit development (Hayashi et al. 1968; Watanabe et al. 2008), while GA applications have been demonstrated to be effective in inducing parthenocarpy (Bukovac 1963; Bukovac and Nakagawa 1967). Among the various GAs, GA4 and GA7 were likely more effec-tive in initiating parthenocarpic fruit set than GA3 (Bukovac 1963; Bu-kovac and Nakagawa 1967). An interesting feature of GA applications

1. MOLECULAR PHYSIOLOGY OF FRUIT GROWTH IN APPLE 9

in inducing parthenocarpic fruit set in apple is an associated change in fruit shape. GA treated fruit often tend to display greater growth along the polar diameter and similar or lesser growth along the transverse diameter (Nakagawa et al. 1967). Increase in growth along the polar diameter is associated with a larger cortex width at the distal end of the fruit through an increase in cell number and size (Nakagawa et al. 1967). Such an inducible system for fruit set and parthenocarpic fruit growth offers an excellent system to investigate molecular components associated with quiescence in fruit growth at bloom, in fruit set, and in early fruit growth. However, this has not yet been explored in apple.

In the closely related fruit, pear, similar induction of parthenocarpic fruit growth in response to GAs has been reported and has been used to understand transcriptional processes involved in the regulation of fruit set (Liu et al. 2018). These data indicated an increase in transcript abundance of auxin transport‐related genes in pollinated and partheno-carpic fruit and a corresponding decrease in abundance of transcripts associated with ABA biosynthesis. Further, the abundance of positive regulators of cell production and expansion related transcripts was up‐regulated in pollinated and GA‐treated fruit. Together, coordinated tran-scriptional re‐programming of the developing flower/fruit appears to regulate the progression of fruit set. The data in pear are consistent with similar changes in the abundance of cell produ ction‐related gene prod-ucts in apple during the fruit set period (Malladi and Johnson 2011).

A recent study has investigated changes in the transcriptome, in relation to early flower growth and development, associated with differential chilling accumulation in apple (Kumar et al. 2017). Genes associated with post‐embryonic development were substantially enriched in transcript abundance during bud break and subsequent flower development leading to fruit set, suggesting coordinated regu-lation of this stage of fruit development. Further application of such transcriptomics and proteomics approaches to study fruit set using the inducible/parthenocarpic system is likely to provide important insights into the molecular factors that regulate this key transition in fruit growth and development.

V. FRUIT GROWTH

A. Components of Fruit Growth: Cell Production, Expansion, and Void Spaces

Organ growth is mediated by three main processes: cell production; cell expansion; and void space development. The relative contribu-tion of these three processes to growth is highly variable depending

10 ANISH MALLADI

on the organ and the plant species under consideration. However, in many organs including apple fruit, final size is often correlated with cell number rather than cell size (Harada et al. 2005; Johnson et al. 2011). However, it is important to emphasize that both factors contribute greatly to the final size of the fruit and that cell production and expan-sion are closely inter‐related processes (Harada et  al. 2005; Malladi and Hirst 2010). While regulation of cell production and expansion have been studied in detail in many plant systems, including in apple, development of void space remains a poorly understood process in spite of its relatively significant contribution to the final size of the organ. Cell production in this review refers to the generation of new cells through the process of cell division. It is distinguishable from cell division itself owing to the non‐synchronous nature of the population of cells in the organ. Cell division refers to the process of new cell generation at the level of an individual cell. In this context, an existing cell undergoes growth, duplication of the genome, and subsequently mitosis to gener-ate a daughter cell. However, in a population of cells, as in organs such as the fruit or even within a specific tissue of the fruit, not all cells are involved in division. Further, even among those cells that are involved in the division process, not all are at the same stage of the cell division cycle. These factors together influence the rate at which new cells are produced from an existing population of cells. As measurements of cell number within a tissue over time typically do not account for the above factors, it is more appropriate to use the term cell production rather than cell division (Beemster and Baskin 1998; Baskin 2000). The use of these terms is more than just semantics. A change in cell production rate can be achieved due to an increase in the proportion of cells dividing in the population. This would involve acquisition of competency to divide by more cells. Alternatively, cell production rate could increase due to an increase in the cell division rate: the rate at which individual cells within the population complete their cell cycle. These two processes are likely regulated through different mechanisms and their relative contribution to changes in cell production rates may vary depending on the tissue type. One approach to determining the average cell divi-sion rate is to divide the relative cell production rate (RCPR) by the proportion of dividing cells, when the latter information is available. During the early period of fruit growth in apple, RCPR peaks around 0.26 cells per cell per day (Dash and Malladi 2012). During this period, flow cytometry analysis indicates that around 15% of the cells display nuclear DNA content of 4C, indicating that they are involved in cell division (Malladi and Hirst 2010). Further, a small proportion of cells displays nuclear DNA content between 2C and 4C, likely representing