Stewart Postharvest Review 2015 22 wwwstewartpostharvestcom ISSN 1745-9656

copy 2015 SPS (UK) Ltd

Introduction Abscission is a process in which vegetative and reproductive organs are detached from the mother plant at a specific layer in response to developmental environmental hormonal and molecular cues [1-12] It is generally accepted that the basipe-tal polar flux of auxin from the distal organ towards the ab-scission zone (AZ) renders it insensitive to ethylene thereby delaying or preventing abscission The reduced auxin flow to the AZ as a result of decreased biosynthesis in the source tis-sue (the abscising organ) or inhibition of its polar auxin transport (PAT) are the main factors that render the AZ com-petent to respond to abscission signals [13-17 18 19 20] A widely accepted working model of abscission [7 12] de-fines four major stages in the abscission process 1) differenti-ation of the AZ in the future site of organ detachment 2) ac-quisition of competence of the AZ cells to react to abscission signals 3) activation of the abscission process in the AZ and execution of organ detachment 4) formation of a protective layer on the proximal surface of the separation layer

It is well established that plant hormones apart from auxin and ethylene regulate abscission Abscisic acid jasmonates and cytokinins act as abscission-accelerating signals [6 8 12 21-26] while gibberellins polyamines and brassinosteroids act as abscission inhibiting signals [6 7 12 27-31] but these effects will not be covered here This review focuses on evidence that auxin depletion in the AZ cells is the main signal leading to acquisition of their competence to respond to ethylene by execution of organ abscission The following issues will be addressed 1) regulation of auxin levels in plants biosynthesis metabolism transport and signaling processes 2) auxin deple-tion during natural and stress-induced abscission 3) elucida-tion of the sequence of events leading to organ abscission following artificial auxin depletion in tomato (Solanum lycopersi-cum) New data derived from recent customized tomato AZ-specific microarray experiments show regulated changes in expression of auxin-related genes in the AZs following remov-al of their auxin source These data reveal new auxin-related genes that were not associated until now with the abscission process

Purpose of review Abscission is a programmed developmental process initiated by auxin depletion This review summarizes the mechanisms leading to auxin depletion in the abscission zone (AZ) evaluates the methods for estimation of the spatio-temporal auxin levels demonstrates how auxin depletion occurs during natural stress-induced and artificially-induced organ abscission and presents new evidence for early and late events resulting from auxin depletion which lead to organ abscission Findings Auxin depletion occurs during natural developmental processes which end in organ abscission (leaf and flower senescence fruit ripening and self-pruning) and stress-induced abscission and following artificial organ removal in the tomato model system Stress-induced auxin depletion is mediated by increased ethylene and reac-tive oxygen species (ROS) production and carbohydrate starvation Similar changes in auxin-related genes oc-curred in both flower AZ (FAZ) and leaf AZ (LAZ) following flower or leaf removal respectively suggesting a similar regulation of the abscission process of these organs Auxin depletion resulted from decreased indole-3-acetic acid (IAA) biosynthesis and transport as well as from enhanced IAA transport autoinhibition (ATA) conju-gation and oxidative IAA catabolism Functional analyses of several target genes delaying abscission such as Knot-ted-Like Homeobox Protein1 (KD1) Tomato Proline Rich Protein (TPRP) Ethylene Responsive Factor52 (ERF52) and Ribo-nuclease LX (LX) shed light on various events operating in response to auxin depletion in tomato FAZ andor LAZ The information gained allows a better understanding of the abscission process driven by auxin depletion and might lead to development of improved methods for abscission control in horticultural crops Direction for future research A better understanding of abscission regulation as it pertains to auxin depletion will require advanced molecular tools such as microarrays new generation sequencing (NGS) transcriptomic functional and proteomic analyses of target genes and proteins found to operate in the abscission process Keywords abscission zone auxin homeostasis carbohydrates ethylene functional analysis of target genes IAA ROS tomato transcriptome

Role of auxin depletion in abscission control

Shimon Meir1 Srivignesh Sundaresan12 Joseph Riov2 Ishangi Agarwal3 and Sonia Philosoph-Hadas1 1Department of Postharvest Science of Fresh Produce Agricultural Research Organization (ARO) The Volcani Center Bet-Dagan Israel

2The Robert H Smith Institute of Plant Sciences and Genetics in Agriculture The Robert H Smith Faculty of Agriculture Food and Environment The Hebrew University of Jerusalem Rehovot Israel

3Genotypic Technology Pvt Ltd Bangalore India

Correspondence to Shimon Meir Email shimonmvolcaniagrigovil doi102212spr201522

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Meir et al Stewart Postharvest Review 2015 22

Regulation of auxin levels in plants biosynthesis metabolism transport and signaling processes

The processes of auxin biosynthesis metabolism transport and signaling have been extensively reviewed in recent years [32-35 36-40 41 42 43-46 47 48] Tryptophan (Trp) is the main precursor for indole-3-acetic acid (IAA) biosynthesis in plants There are several proposed IAA biosynthetic pathways [37 42 48] In Arabidopsis the predominant pathway is the indole-3-pyruvic acid two-step linear IAA biosynthetic pathway which is operated by Trp aminotransferase of Arabidopsis and

flavin monooxygenase enzyme families encoded by the YUC-CA gene family Another two-step biosynthetic pathway that operates in bacteria and plants is the indole-3-acetamide (IAM) pathway In this pathway Trp is converted to IAM by Trp-2-monooxygenase (iaaM) which is subsequently hydrolyzed to IAA by indole-3-acetamide hydrolase The distribution and homeostasis of IAA depend on both me-tabolism (biosynthesis conjugation and catabolism) and cellu-lar transport IAA conjugates play an important role as inactive storage forms of IAA [35 37] In its free active form IAA comprises only 5-25 of the total amount of IAA depending on the tissue and plant species The major forms of IAA conju-gates are low molecular weight esters such as IAA-glucose synthesized by IAA-glucose synthase and amide forms synthe-sized by the enzyme Gretchen Hagen3 (GH3) The IAA conju-gates can be hydrolyzed to form free IAA by IAA-Leu resistant (ILR) IAA-Ala resistant or ILR-like enzymes whereas indole-3-acetyl aspartate and indole-3-acetyl glutamate act as precursors of a non-hydrolytic degradation pathway [37 42 45 48] IAA catabolism has been shown to occur either by an oxidative de-carboxylation pathway leading to modifications of both the side chain and the indole ring or by a non-decarboxylative oxi-dation of the indole moiety Oxidative degradation of auxin appears to be developmentally important mainly during fruit ripening and plant responses to oxidative stress [37] Most aux-in biosynthetic and metabolic pathways occur in low rates ranging between 10 nMh to 1 microMh with the exception of auxin conjugation which has rates as high as 100 microMh [45] Molecular and biochemical data discussed later in this review provide evidence that components of auxin homeostasis are regulated in the AZ tissues From the sites of biosynthesis IAA is transported to other parts of the plant by diffusion or through active transport The directional PAT system distributes auxin from the sites of bio-synthesis to basipetal parts of the plant and is mediated by influx and efflux carriers The active influx of auxin in plant cells is mediated by the influx carriers Auxin resistant 1Like aux1 (AUXLAX) while efflux carriers belong to the pin-formed (PIN) family of proteins [37 38 46] The PIN family includes eight members in Arabidopsis thaliana (AtPIN1ndash8) and ten members in both tomato and potato (SlPIN1-10 StPIN1-10 respectively) [49] PINs are divided into ldquolongrdquo and ldquoshortrdquo PINs according to the length of the hydrophilic protein domain [37 46 49] In contrast to the long PIN proteins which are located in the plasma membrane (PM) the short AtPIN5 pro-tein is localized in the endoplasmic reticulum (ER) and it par-ticipates in the compartmental localization and homeostasis of auxin In recent years a novel putative auxin transport facilitat-ing family that also regulates intracellular auxin homeostasis in plants has been identified [41] Named PIN-LIKES (PILS) this family includes seven members in A thaliana [41 46] The PILS proteins contribute to auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin PILS proteins regulate intracellular auxin accumulation in the ER and thus auxin availability for nuclear auxin signaling [41 46] Data presented later in this review provide direct evi-dence for PIN and PILS signaling in tomato AZ tissues Auxin enhances its own efflux by rapid modulation of the abundance of PIN proteins The transcription of PIN is under

ARF Auxin Response Factor ATA IAA Transport Autoinhibition AuxIAA

Auxin ResistantIndole-3-Acetic Acid-Inducible

AUXLAX Auxin ResistantLike Aux AZ Abscission Zone DAP Days After Pollination DPA Days Post Anthesis ER Endoplasmic Reticulum ERF Ethylene Responsive Factor FAZ Flower AZ GH3 Gretchen Hagen3 GUS β-Glucuronidase IAA Indole-3-Acetic Acid iaaM Tryptophan-2-Monooxygenase

PM Plasma Membrane qPCR Quantitative PCR REV REVOLUTA ROS Reactive Oxygen Species TAPG4 Tomato Abscission PG4

ILR IAA-Leu Resistant KD1 Knotted-Like Homeobox Protein1 LAX Like Aux LAZ Leaf AZ LX Ribonuclease LX 1-MCP 1-Methylcyclopropene NAZ Non-AZ NGS New Generation Sequencing PAT Polar Auxin Transport PCD Programmed Cell Death PCR Polymerase Chain Reaction PG Polygalacturonase PILS PIN-LIKES PIN Pin-formed

SAUR Small Auxin Upregulated RNA TF Transcription Factor TIR1AFB Transport Inhibitor Response1Auxin

Signaling F-Box TPRP Tomato Proline Rich Protein Trp L-Tryptophan

Abbreviations

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Meir et al Stewart Postharvest Review 2015 22

the direct control of auxin and PIN proteins are also post-transcriptionally regulated by auxin The auxin feedback regu-lation of its transport direction and capacity is closely linked to the canalization hypothesis proposed by Sachs [50] The cellu-lar and molecular mechanisms for canalization involve the auxin resistantIAA-inducible - Auxin Response Factor (AuxIAA-ARF) signaling pathway [51] A similar phenomenon of canalization is the high velocity IAA transport from a domi-nant organ which inhibits the IAA export out of the dominat-ed organ thereby causing IAA transport autoinhibition (ATA) in the junction of auxin transport from the two organs [52] ATA is involved in apical dominance and abscission of domi-nated organs [52-55] Other plant hormones (ethylene cyto-kinins gibberellins) small secretory peptides and environmen-tal signals (light gravity abiotic and biotic stresses) appear to modulate auxin intracellular compartmentalization by affecting PIN expression and sub-cellular PIN trafficking [37 46] Ethylene cytokinins jasmonates and strigolactone modulate PAT via transcriptional and posttranscriptional regulatory mechanisms of auxin carrier transcription or traf-ficking By regulating PIN1 levels in the PM strigolactone can influence the capacity of bud-derived auxin to canalize towards the stem and thus modulates bud activity and shoot architec-ture [54] Two transcription factors (TFs) were found to regulate auxin biosynthesis transport and signaling KANADI which has a Myb-like domain and REVOLUTA (REV) a Class III Home-odomain-Leucine Zipper TF [56 57 58-62] These two TFs have been shown to play antagonistic roles KANADI acts as a transcriptional repressor and negatively regulates PIN1 expres-sion while REV promotes PIN expression and function Both TF genes are expressed in the auxin transport routes through the procambium cambium and phloem thereby playing an important role in vascular tissue formation and canalization [60] Microarray evidence for regulated REV expression in to-mato AZs responding to auxin depletion is described below in this review Auxin regulation of transcription events involves a core path-way consisting of the transport inhibitor response1Auxin sig-naling F-box (TIR1AFB) proteins the AuxIAA transcrip-tional repressors and the ARF TFs Perception of auxin stabi-lizes the co-receptor complex of TIR1AFB and AuxIAA proteins which trigger the proteasome-dependent degradation of the AuxIAA transcriptional regulators AuxIAAs regulate auxin-dependent gene transcription by forming dimers with ARF proteins The auxin-dependent release of the ARF TFs leads to the onset of auxin-mediated transcriptional reprogram-ming [33 37 39 43 47 48] In Arabidopsis six TIR1AFB can interact with 23 different AuxIAAs to form numerous co-receptor complexes Each of the AuxIAA may interact with up to 19 ARFs to regulate distinct sets of target genes that con-trol different physiological processes Genome-wide identifica-tion functional analysis and expression profiling of the AuxIAA gene family in tomato were previously reviewed [40] Most of the AuxIAA genes are rapidly induced by auxin and the level of several AuxIAA transcripts increases within a few minutes following auxin treatment Therefore the expression of these auxin-induced genes can be used as indicators for aux-in activity in various tissues including AZs [63 64]

Auxin depletion during natural and stress-induced abscission

Determination of IAA levels

Direct measurements of endogenous levels of IAA (free and conjugated) using analytical [65 66] or immunological [66] methods were developed Few publications used this analytical method to demonstrate IAA depletion in the AZ during the initiation of the abscission process [67 68 69 70 71 72] Most determinations of auxin activity in the AZs were based on the expression of auxin-induced genes Initially few auxin-induced genes were cloned from the AZ and their expression in the AZ was analyzed by polymerase chain reaction (PCR) [63 64] Later expression profiles of auxin-induced multi-gene families in various abscission systems were analyzed using mi-croarray [20 37 47 57 67 77] [or NGS [78 79] techniques In transgenic plants the synthetic auxin-responsive promoter element DR5 fused to β-glucuronidase (GUS) DR5GUS was used for evaluation of IAA levels in the AZs [70 71 80 81] The use of DR5GUS has the advantage of visual assessment but has a disadvantage for determining IAA depletion due to the high stability of the GUS protein as com-pared to the green fluorescent protein which dissipates rapidly Therefore the green fluorescent protein was used as a reporter system for studying the temporal expression profiles of pro-moters for estimation of IAA depletion [82] In recent years a new auxin sensor DII-VENUS has been developed This sen-sor which is composed of domain II of the AuxIAA fused to the VENUS fluorescent protein provides high-resolution spa-tio-temporal information about auxin distribution and response [83] Unlike the DR5 response the DII-VENUS sensor is closely related to auxin concentrations and does not depend on the levels of AuxIAA and ARF proteins thereby enabling its use as a quantitative tool All of these methods monitor auxin concentration indirectly via the expression of auxin-induced genes In our opinion they act as in vivo bioassays which have some weaknesses Moreover measuring auxin activity requires to consider the effects of inhibitors and repressors in the tissues andor in the extracts Hence the analytical auxin measure-ments based on gas chromatography-mass spectrometry deter-mination [65] are more accurate for the determination of low levels of IAA and are therefore preferential for estimation of auxin depletion in the AZ tissues

Auxin depletion during natural abscission Abscission of leaves flowers floral parts and overripe fruits is the last developmental stage of these organs and is therefore regarded as natural abscission The present review examines whether auxin depletion in these organs which serves as auxin sources to their AZs precedes the execution of ethylene-controlled abscission IAA depletion during senescence-induced flower abscis-

sion Flower senescence and abscission can be ethylene-dependent or independent [84 85] Since the basic premise of this review is that auxin depletion in the AZ increases the ethylene sensitivity of the AZ cells we will focus on ethylene-dependent abscission of flower buds open flowers and petals Endogenous IAA levels usually decrease during flower senescence in most flow-ers that subsequently abscise [86 87] Indeed the abscission of

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Meir et al Stewart Postharvest Review 2015 22

unfertilized or male flowers may be ascribed to the low levels of endogenous IAA which is produced in the ovary [88] In Begon-ia abscising male flower buds contain only 1 of the IAA pre-sent in non-abscising female flowers and the seasonal variation in male bud abscission coincided with reduction of the IAA content in the buds [89] In Lilium IAA levels decreased in the gynoecium and petals when the petals started to wilt and se-nesce prior to their abscission [90] IAA level and the expres-sion of 50 auxin-related genes were analyzed in the outer tepals of two Lilium cultivars that differ in their abscission timing Although both cultivars have fully formed AZs Lilium longiflo-rum flowers wilted substantially during senescence prior to their late abscission while those of the closely related Lilium longiflo-rum Asiatic hybrid (LA) abscised early without wilting [91] A clear correlation between auxin levels and abscission timing of both cultivars was found in relation to senescence markers Thus in L longiflorum both free and conjugated-IAA signifi-cantly increased as senescence progressed while free IAA levels in Lilium LA remained low at all developmental stages from closed bud to abscission and the portion of IAA-amide conju-gate gradually increased Consistent with the view that declining IAA levels precede abscission the ARF719-like gene was up-regulated in the delayed-abscising genotype while in the early-abscising genotype it was down-regulated [91] In Dendrobium cut flowers the floral buds at the top of the in-florescence stalk exhibit early yellowing and abscise Applica-tion of an auxin transport inhibitor or an auxin action inhibitor to the stigma of open flowers induced high flower abscission rates [92] Removal of the open flowers at the distal end of the pedicel reduced the time to abscission of the remaining pedicel IAA placed on the cut surface of the pedicel counteracted the effect of flower removal Application of different auxins de-layed senescence and inhibited the abscission of open Dendrobi-um flowers [92 93] These results support the view that auxin is an endogenous regulator of abscission of Dendrobium floral buds and flowers Several other reports show that exogenously ap-plied auxins prevented or delayed abscission of flowers and floral parts styles and stamens [88] Accordingly auxin is ap-plied to extend the vase life of several cut flowers such as Geraldton wax flowers Cestrum and poinsettia [63 88 94 95] Genetic experiments with Arabidopsis mutants further demon-strated the role of auxin in petal abscission Mutation in ARF2 [96] and ARF1 and ARF2 [97] delayed senescence and abscis-sion of Arabidopsis petals Flowers taken from mutants in which individual family members of the auxin influx carriers AUX1 and Like-AUX3 (LAX3) were down-regulated exhibit-ed early abscission Manipulation of IAA levels in the AZ cells by activation of the bacterial IAA biosynthetic genes IAA-Lys-synthetase and iaaM enhanced or delayed petal abscission re-spectively [81] It was shown that IAA-Lys-synthetase re-duced IAA levels in the cells by conjugating free IAA to IAA-Lysine while iaaM promoted IAA levels by converting Trp to indole acetamide IAA depletion during senescence-induced leaf abscission

Endogenous IAA decreases at the onset and during senescence of leaves and auxin treatment can delay leaf senescence in some plants [98-100] Most of the research on leaf senescence was done by using detached leaves in model systems which do not show abscission phenotypes (Arabidopsis or flag leaf in monocots) Therefore the changes in IAA levels during abscis-

sion were not studied Only one early report showed a positive correlation between the decrease in IAA content during senes-cence of Coleus leaves and their abscission [101] Moreover in monocots and some annual dicots there is no AZ at the base of the leaf and the leaves senesce wilt and remain dry on the plant a process termed marcescence Therefore the changes in IAA content during leaf senescence in these plants including the Arabidopsis model system are less relevant IAA depletion during ripening-induced fruit abscission

Fruits typically abscise at the overripe stage of development In general IAA levels decrease in fruit during ripening and this decrease coincides with maturation of the seeds in which most of the IAA is produced [80 102-105] The decrease in IAA levels starts in tomato fruit as the fruit reaches the breaker stage [80 103-104] The reduction of free IAA levels during ripening results from the decrease of auxin signal around the seeds [80 102] and expression of the auxin transport gene families LAX and PIN with advanced ripening [80] as well as from increased conjugation of free IAA to its amide conjugates as GH3 ex-pression is sharply increased with ripening [103-104] A similar decrease in IAA levels and an increase in GH3 expression and indole-3-acetyl-aspartate levels were also observed in grape berries after reaching the mature green stage [105 106] It is likely therefore that auxin depletion in fruit contributes to subsequent abscission competence Unfortunately most studies of auxin metabolism in ripening fruit were terminated at the ripe stage without evaluating fruit at the overripe stage in which most actual abscission events occur Only one exception was reported in which IAA levels were monitored in oil palm fruit until abscission Thus in developing fruit the content of IAA peaked between 60 and 100 days after pollination (DAP) and subsequently decreased to very low levels between 100 and 120 DAP just before abscission which occurred 140 DAP [107] Auxin application delayed oil palm fruit abscission [108] However IAA depletion in fruit may be species-dependent as in peach several reports demonstrated increased IAA level during fruit ripening which is required for fruit sof-tening [109-111] There has been little work in which fruit ripening was correlat-ed with events occurring in nearby AZs Recently two tran-scriptomic reports demonstrated changes in auxin-related gene expression in the AZs of mature olive and melon fruit [78 79] In olive gene expression was studied 154 days post anthe-sis (DPA) when fruit started to ripen and 217 DPA when fruit abscised Numerous auxin-related genes were down-regulated in the AZ 217 DPA including auxin biosynthesis genes ILR1 auxin transporters LAX1 LAX2 and PIN TIR1 two AUXIAA family members three ARF family members and several auxin-induced genes [78] In melon gene expression was stud-ied at three time points 36 38 and 40 DPA The ethylene cli-macteric peak and abscission occurred 37 and 40 DPA respec-tively Between 36 to 38 DPA two genes encoding for auxin effux carriers four AUXIAA genes and one ARF gene were down-regulated in the AZ [79] Between 38 to 40 DPA 13 other auxin-related genes were down-regulated These reports provide the first direct evidence for auxin depletion in the AZ during mature fruit abscission Taken together the reported data show that the processes of flower and leaf senescence and fruit ripening generally lead to

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Meir et al Stewart Postharvest Review 2015 22

reduction of endogenous auxin levels which in turn result in organ abscission Sometimes a direct evidence for auxin deple-tion prior to organ abscission is lacking in these systems as the experiments did not analyze IAA levels in the senesced organ or overripe fruit but the general pattern was validated in vari-ous systems

IAA depletion during self-pruning of spring shoots in sweet orange (Citrus sinensis)

Citrus shoot tips abscise at an anatomically distinct AZ that separates the top part of the shoots into basal and apical por-tions This natural process termed self-pruning plays an im-portant role in citrus floral bud initiation Citrus microarray was used to monitor the expression of genes at several time points 5 or 3 days before self-pruning when the shoot tips started to fall and at the beginning of self-pruning of spring shoots when the AZ was activated [20] Twenty four auxin-related genes were differentially altered during these stages of self-pruning genes encoding auxin-induced proteins were up-regulated while genes encoding ARFs were down-regulated The authors concluded that auxin depletion in the AZ of the spring shoots causes the AZ to become sensitive to ethylene and abscisic acid which accelerate the abscission process of the shoot tips [20]

Ethylene as a mediator of IAA depletion Ethylene is the main regulator of leaf and flower senescence and fruit ripening and is proposed here as a modulator of auxin depletion in these processes Unlike the synergistic interactions between ethylene and auxin controlling specific growth and developmental processes [112 113 114 115 116] the con-trol of both natural and stress-induced abscission (see below) involves antagonistic effects of ethylene and auxin Several

modes of action were suggested for this negative interaction One of the regulatory effects of ethylene on auxin levels oper-ated through inhibition of auxin transport to the AZs was demonstrated long ago [117-122] Additionally in various natu-ral abscission systems ethylene was reported to reduce auxin levels by increasing the rate of auxin conjugation [120-122]

Auxin depletion during stress-induced abscission

Abscission of different organs is a common response to various biotic abiotic and physiological stresses [2 4 6 119 123] Stress-induced abscission initiated by auxin depletion is mediat-ed by three main intermediate modulators ethylene ROS and carbohydrate starvation [119 123 124 125] Ethylene pro-duction increases in tissues subjected to many stresses subse-quently regulating their auxin levels [126-128] Stress-induced ethylene may promote auxin depletion in various abscissing systems via inhibition of auxin transport to the AZs [117-122 129 130]

ROS

ROS can induce organ abscission [131-133] and application of antioxidants and ROS scavengers can inhibit abscission [67 68 134-137] Several publications reviewed the interplay between ROS and auxin [124 138-140] ROS induced by stress con-ditions have an impact on auxin signaling by affecting auxin homeostasis at the levels of auxin biosynthesis metabolism and distribution ROS stimulated auxin catabolism by increasing decarboxylative and non-decarboxylative oxidation of IAA IAA conjugation (ester and amid forms) and GH3 expression and inhibited IAA transport by decreasing PIN expression [67 68 124 138 139 140] ROS scavenging genes were induced by ethylene during abscission of citrus leaves suggesting that ethylene induces ROS in this system [138]

Table 1 Examples of auxin depletion mechanisms operated in various stress-induced abscission systems

Stress type Plant and abscission system

Mechanisms for auxin depletion Reference

Domination Apple fruitlets in clusters Decrease in PAT decrease in ATA 13 16 17 143 157

Grapevine berries in clusters Decrease in PAT decrease in ATA 19

Cowpea flowers and fruitlets Decrease in PAT decrease in ATA 14

Carbohydrate starvation (phloem-girdling)

Litchi fruitlets Decrease in PAT 142

High temperatures Bell pepper flowers and fruitlets Decrease in IAA level decrease in PAT 130

Mango fruitlets Decrease in PAT 141

Chilling temperatures Ixora leaves Increase in IAA oxidation Decrease in IAA level Decrease in PAT

67

Chilling + high light Ixora leaves Increase in IAA oxidation Decrease in IAA level

68

Water stress Cotton leaves Decrease in PAT 158 159

Cotton buds flowers and fruits Decrease in IAA level Increase in IAA conjugates level

160 161

Citrus leaves Decrease in expression of IAA signaling genes 162

Poplar leaves Decreased expression of IAA signaling genes 163

Balsam fir needles Decrease in IAA level 164

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Meir et al Stewart Postharvest Review 2015 22

Carbohydrate starvation Numerous stressors like water stress high and low light high or chilling temperatures and high sink organ domination result in carbohydrate starvation Abscission of premature fruit leaves flowers and flower buds is related to low sugar content [13 15-17 141-146] Auxin application can inhibit some stress-induced organ abscission [15 146-148] suggesting that the shortage of carbohydrate is correlated with auxin depletion In non-abscissing systems sugars have also been shown to play pivotal roles as signaling molecules [149-155156] Thus solu-ble sugars were reported to increase IAA biosynthesis free IAA levels and IAA transport in these systems [154-155] as well as to up-regulate early auxin biosynthesis and PIN1 genes to pro-mote PIN1 abundance in the PM and to down-regulate two genes of PAT inhibitors [156] We anticipate that sugar sig-naling may ultimately be found to accompany stress-induced decrease in carbohydrates that result in auxin depletion in source organs and their AZs The mechanisms of auxin deple-tion in various stress-induced abscission systems are summa-rized in Table 1 Auxin depletion is expected to increase AZ sensitivity to stress-induced ethylene thereby resulting in ab-scission Elucidation of the sequence of events leading to organ abscission following artificial auxin depletion in tomato

Tomato has been extensively used to study flower and fruit abscission processes Tomato is a very convenient model sys-tem since tomato plants develop a distinct AZ in the midpoint of the flower pedicel referred to as a pedicel AZ or FAZ in different publications The anatomy and development of the tomato FAZ is well established [165-167] as are the proteins involved in the regulation of the FAZ differentiation and devel-opment [168-173] Since the eighties of the previous century in-depth studies have defined enzymes involved in cell separation in various species The AZ-specific polygalacturonases (PGs) and cellulases are particularly well characterized [174-184] The AZ cells are defined as specialized cell types that differ from their adjacent cells in perception and response to ethylene and auxin [2 4 185-187] Therefore it is expected that specific TFs and genes will be specifically expressed in the AZ and not in the adjacent non-AZ (NAZ) cells Indeed in the first transcrip-tome microarray analysis of the tomato FAZ performed follow-ing auxin depletion several genes were found to be specifically expressed in the FAZ and not in the basal portion (proximal) of the pedicel NAZ region [74] These genes include several TFs such as TOMATO KNOTTED4 Homeobox-Leu zipper13 TOMATO AGAMOUSE-LIKE212 TOMATO PROLINE RICH PROTEIN (TPRP-F1) KNOTTED1-LIKE HOMEO-BOX PROTEIN1 (KD1) Phantastica and Ovate [74] In two transcriptome analyses comparing gene expression in the toma-to FAZ versus the NAZ including the basal portion (proximal) and apical portion (distal) pedicel regions 89 and 1255 genes respectively were found to be specifically expressed at anthesis in the FAZ cells including genes encoding for TFs hormone-related proteins cell wall modification enzymes lipid metabo-lism and others [76 77] Most interestingly the AZ-specific gene set include TF genes that encode key regulators of meri-stem-associated functions which may be regulated by a signal-ing pathway that requires auxin supplied from the flower before the onset of abscission Suppression of one of the tomato shoot meristem-associated TF genes tomato ETHYLENE-

RESPONSIVE FACTOR52 (SIERF52) by RNAi did not af-fect FAZ development but significantly delayed pedicel abscis-sion This suggests that SlERF52 plays a pivotal role in tran-scriptional regulation in the FAZ [188] In an attempt to perform a functional analysis of 45 AZ-specific genes whose expression changed early in the tomato FAZ following auxin depletion experiments based on virus-induced gene silencing in the FAZ and LAZ were conducted Silencing nine of these 45 genes led to a significant retardation of pedicel andor petiole abscission following abscission induc-tion The role of these nine genes was further examined by silencing each of them in plants stably transformed with anti-sense or RNAi constructs driven by an AZ-specific promoter Tomato Abscission PG4 (TAPG4) The results showed that TAPG4antisense constructs of KD1 [71] and of TPRP-F1 [189] strongly inhibited both pedicel and petiole abscission Conversely up-regulation of KD1 showed accelerated pedicel and petiole abscission [71] Complementary DNA microarray and quantitative PCR (qPCR) analyses indicated that regulation of abscission by KD1 was associated with a change in the abun-dance of genes related to auxin transporters and signaling com-ponents Measurement of IAA content using the DR5GUS auxin reporter assay confirmed by analytical auxin determina-tion showed that change s in KD1 expression modulated the auxin concentration and response gradient in the FAZ [71] In a transcriptomic analysis comparing gene expression in the tomato FAZ and NAZ in the proximal and distal flanking pedi-cel regions at anthesis [76] a region-specific expression of auxin-related genes was found 1) METHYLESTERASE1 which converts IAA methyl ester to IAA DWARF IN LIGHT1 which encodes an IAA-amido synthetase and GH36 showed higher transcript levels in the FAZ than in the NAZ 2) ARF9 was expressed at higher levels in the proximal (basal) than in the distal (apical) NAZ region 3) GH31 and a Hookless1 homolog were expressed at higher levels in the distal than in the proximal NAZ region This region-specific differential expres-sion of genes involved in the determination of auxin levels sug-gests that a gradient of auxin concentration formed along the pedicel regions may be a key factor in regulating the timing of pedicel abscission [76] Indeed by using the DR5GUS re-porter such a gradient of IAA concentration was recently found in VF36 tomato plants in which the FAZ was also divided into distal and proximal regions [71] Consistent with the initial location of the auxin-producing source tissue this gradient oc-curred according to the following sequence distal side of NAZ gt distal side of FAZ gt proximal side of FAZ gt proximal side of NAZ [71] In order to elucidate the molecular changes occurring in the tomato artificial auxin depletion model system during acquisi-tion of abscission competence in the FAZ following auxin de-pletion and during execution of pedicel abscission our group performed a microarray analysis using the Affymetrix 10K oligonucleotide Tomato GeneChip [74] In this system the flower that serves as an auxin source is removed Applying IAA after flower removal or inhibiting ethylene action using 1-methylcyclopropene (1-MCP) prior to flower removal inhibited pedicel abscission suggesting that pedicel abscission results from auxin depletion and is ethylene-dependent [74] Based

7

Meir et al Stewart Postharvest Review 2015 22

on the transcriptomic results following the application of the abscission modulators an abscission-inducing model was pro-posed in which the sequence of events occurring during tomato pedicel abscission was divided into two phases early events (0 to 4 h after flower removal) and late events (8 to 14 h after flower removal) The early events probably lead to acquisition of ethylene sensitivity and abscission competence while the late events include processes leading to the execution of pedicel abscission and development of the defense layer According to this model the decrease in IAA provides the first signal for abscission Responses to auxin depletion included down-regulation of genes induced by auxin such as AuxIAA and other TF genes and up-regulation of genes repressed by auxin The late events included increased ethylene production due to up-regulation of ethylene biosynthesis genes such as the genes encoding 1-amino-cyclopropane-1-carboxylate synthase which lead in turn to AZ-specific up-regulation of abscission-related genes These genes include genes encoding cell wall-modifying proteins and pathogenesis-related proteins [74] as well as genes related to the development of a protective layer on the surface of the remaining tissue [190] The late events which are ethylene-induced were inhibited by 1-MCP pretreatment while the early events were not affected by the inhibitor On the oth-er hand IAA application immediately after flower removal in-hibited all the cascade of abscission events and the changes in the expression of auxin-induced or auxin-repressed genes [74] Two later studies showed a rapid decrease of AuxIAA [191] and ARF [70] genes after tomato flower removal Addi-tionally quantitative and DR5GUS data demonstrated a fast IAA depletion after flower removal [70] These authors adjust-ed the proposed abscission model [74] to the slower abscis-sion rate obtained in their tomato system (0-8 h for the early events and 16-32 h for the late events) and included the vari-ous ARFs in the model [70] Programmed cell death (PCD) was shown to be another late event involved in the abscission process This was based on the data showing that inhibiting the activity of LX ribonuclease (LX) an enzyme associated with PCD delayed tomato leaf abscission [192] Indeed hallmarks of PCD were identified in the tomato LAZ and FAZ during the late stage of abscission

and data showing that different abscission-related processes occurred asymmetrically between the FAZ proximal and distal regions were presented [193] This asymmetric distribution of various abscission-related processes might be related to the auxin gradient demonstrated recently in the tomato FAZ [71] Leaf deblading removes the natural source of auxin to an AZ and promotes abscission [64] The only report so far describing the effects of leaf deblading on expression of auxin-related genes in the LAZ was performed in Mirabilis jalapa [64] In this system transcripts of two auxin-induced genes Mj-AuxIAA1 and Mj-AuxIAA2 were down-regulated as a result of IAA depletion by leaf deblading or treatment with the IAA transport inhibitor 1-naphthylphthalamic acid Application of IAA to the cut end of the petioles inhibited their abscission and prevented the decline in the transcript levels in the LAZ [64] To further define auxin-relevant members of tomato LAZ and FAZ transcriptomes we recently refined an experimental sys-tem based on the abscission responses of debladed tomato leaves and removed flowers Deblading of tomato leaves led to abscission of attached petioles during a period of 8-12 days Exposure of the debladed-leaf explants to ethylene treatment for 24 h accelerated petiole abscission which was completed within three days after the ethylene treatment (Figure 1A) at a similar rate to that of flower pedicel abscission without ethylene treatment (Figure 1B) RNA collected during petiole and pedi-cel abscission allowed us to compare transcriptomic changes in the tomato LAZ and FAZ respectively through the use of a customized AZ-specific microarray chip The chip included transcripts identified using NGS of RNA isolated from tomato AZs at various time points during organ abscission as well as transcripts from the Solanaceae genomics network and Nation-al Center for Biotechnology Information databases [194] Here we present for the first time results from these microarray anal-yses describing the changes in expression of auxin-related genes (Figures 2-6) Generally there is a high similarity in the abscis-sion process of tomato leaves and flowers with a few excep-tions Most of the auxin-related genes are expressed in both AZs but some members of different gene families are ex-pressed specifically in the FAZ or the LAZ The results presented in Figure 2 show that most genes encod-ing auxin influx (LAX family) and auxin efflux (PIN family) carriers were rapidly down-regulated in both AZs following IAA depletion thereby supporting the requirement for auxin for activation of its transport carriers The few exceptions were related to genes that have very low expression levels such as LAX45 and PIN28 in the FAZ and PIN3 in the LAZ The down-regulation of PIN5 and PILS family members in the two AZs provides the first indication that the regulation of intracel-lular auxin accumulation in the ER expected to control auxin availability for auxin signaling in the nuclei of AZ cells may be important for abscission regulation at the cellular level Two PILS were up-regulated in the FAZ and one in the LAZ fol-lowing flower removal or leaf deblading respectively (Figure 2) further supporting the role of this novel auxin carrier family in regulating auxin homeostasis [41] The REV TF was quickly down-regulated in the FAZ and LAZ after abscission induc-tion suggesting that there is a regulatory mechanism for de-creasing PIN expression and function in both AZs as a result of

Figure 1 Effect of leaf deblading and ethylene treatment (A) and flower removal (B) on the kinetics of petiole and pedicel abscission respective-ly in tomato explants The debladed-leaf explants held in vials with water were prepared as previously described for the flower explants [72] and exposed to ethylene (10 ppm for 24 h) The percentage of accumu-lated pedicel or petiole abscission was monitored at various time inter-vals following organ removal The results are means of four replicates (n=30 explants) plusmn SE

8

Meir et al Stewart Postharvest Review 2015 22

Figure 2 Microarray expression profiles of tomato auxin transport-related genes in the FAZ and LAZ at various time points following abscission induc-tion The assay included family members of the auxin influx carriers (AUXLAX) auxin efflux carriers (PINs PILS) genes and an auxin-related TF gene (REV) Samples were taken from the FAZ at 0 2 4 8 and 14 h after flower removal and from the LAZ at 0 24 48 72 and 96 h after leaf deblading and ethylene treatment as detailed in the legend of Figure 1 Gene expression levels are indicated with the colored code bars ranging from 0 (light blue) to 100 (dark blue) Expression levels are based on the percentage of change of the average intensity values of the replicated samples for each time point The numbers indicated in the first box of each sample represent average intensities values for all replicates at 0 h Corresponding Solanum lyco-persicum (Solyc) ID and gene names are presented in the left and right sides respectively

Figure 3 Microarray expression profiles of tomato IAA-related genes associated with IAA conjugation in the FAZ and LAZ at various time points follow-ing abscission induction The assay included members of the GH3 and ILRs gene families All other details are as described in the legend of Figure 2

9

Meir et al Stewart Postharvest Review 2015 22

Figure 4 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the AuxIAA gene family All other details are as described in the legend of Figure 2

Figure 5 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the ARF gene family All other details are as described in the legend of Figure 2

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Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

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Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

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Meir et al Stewart Postharvest Review 2015 22

the direct control of auxin and PIN proteins are also post-transcriptionally regulated by auxin The auxin feedback regu-lation of its transport direction and capacity is closely linked to the canalization hypothesis proposed by Sachs [50] The cellu-lar and molecular mechanisms for canalization involve the auxin resistantIAA-inducible - Auxin Response Factor (AuxIAA-ARF) signaling pathway [51] A similar phenomenon of canalization is the high velocity IAA transport from a domi-nant organ which inhibits the IAA export out of the dominat-ed organ thereby causing IAA transport autoinhibition (ATA) in the junction of auxin transport from the two organs [52] ATA is involved in apical dominance and abscission of domi-nated organs [52-55] Other plant hormones (ethylene cyto-kinins gibberellins) small secretory peptides and environmen-tal signals (light gravity abiotic and biotic stresses) appear to modulate auxin intracellular compartmentalization by affecting PIN expression and sub-cellular PIN trafficking [37 46] Ethylene cytokinins jasmonates and strigolactone modulate PAT via transcriptional and posttranscriptional regulatory mechanisms of auxin carrier transcription or traf-ficking By regulating PIN1 levels in the PM strigolactone can influence the capacity of bud-derived auxin to canalize towards the stem and thus modulates bud activity and shoot architec-ture [54] Two transcription factors (TFs) were found to regulate auxin biosynthesis transport and signaling KANADI which has a Myb-like domain and REVOLUTA (REV) a Class III Home-odomain-Leucine Zipper TF [56 57 58-62] These two TFs have been shown to play antagonistic roles KANADI acts as a transcriptional repressor and negatively regulates PIN1 expres-sion while REV promotes PIN expression and function Both TF genes are expressed in the auxin transport routes through the procambium cambium and phloem thereby playing an important role in vascular tissue formation and canalization [60] Microarray evidence for regulated REV expression in to-mato AZs responding to auxin depletion is described below in this review Auxin regulation of transcription events involves a core path-way consisting of the transport inhibitor response1Auxin sig-naling F-box (TIR1AFB) proteins the AuxIAA transcrip-tional repressors and the ARF TFs Perception of auxin stabi-lizes the co-receptor complex of TIR1AFB and AuxIAA proteins which trigger the proteasome-dependent degradation of the AuxIAA transcriptional regulators AuxIAAs regulate auxin-dependent gene transcription by forming dimers with ARF proteins The auxin-dependent release of the ARF TFs leads to the onset of auxin-mediated transcriptional reprogram-ming [33 37 39 43 47 48] In Arabidopsis six TIR1AFB can interact with 23 different AuxIAAs to form numerous co-receptor complexes Each of the AuxIAA may interact with up to 19 ARFs to regulate distinct sets of target genes that con-trol different physiological processes Genome-wide identifica-tion functional analysis and expression profiling of the AuxIAA gene family in tomato were previously reviewed [40] Most of the AuxIAA genes are rapidly induced by auxin and the level of several AuxIAA transcripts increases within a few minutes following auxin treatment Therefore the expression of these auxin-induced genes can be used as indicators for aux-in activity in various tissues including AZs [63 64]

Auxin depletion during natural and stress-induced abscission

Determination of IAA levels

Direct measurements of endogenous levels of IAA (free and conjugated) using analytical [65 66] or immunological [66] methods were developed Few publications used this analytical method to demonstrate IAA depletion in the AZ during the initiation of the abscission process [67 68 69 70 71 72] Most determinations of auxin activity in the AZs were based on the expression of auxin-induced genes Initially few auxin-induced genes were cloned from the AZ and their expression in the AZ was analyzed by polymerase chain reaction (PCR) [63 64] Later expression profiles of auxin-induced multi-gene families in various abscission systems were analyzed using mi-croarray [20 37 47 57 67 77] [or NGS [78 79] techniques In transgenic plants the synthetic auxin-responsive promoter element DR5 fused to β-glucuronidase (GUS) DR5GUS was used for evaluation of IAA levels in the AZs [70 71 80 81] The use of DR5GUS has the advantage of visual assessment but has a disadvantage for determining IAA depletion due to the high stability of the GUS protein as com-pared to the green fluorescent protein which dissipates rapidly Therefore the green fluorescent protein was used as a reporter system for studying the temporal expression profiles of pro-moters for estimation of IAA depletion [82] In recent years a new auxin sensor DII-VENUS has been developed This sen-sor which is composed of domain II of the AuxIAA fused to the VENUS fluorescent protein provides high-resolution spa-tio-temporal information about auxin distribution and response [83] Unlike the DR5 response the DII-VENUS sensor is closely related to auxin concentrations and does not depend on the levels of AuxIAA and ARF proteins thereby enabling its use as a quantitative tool All of these methods monitor auxin concentration indirectly via the expression of auxin-induced genes In our opinion they act as in vivo bioassays which have some weaknesses Moreover measuring auxin activity requires to consider the effects of inhibitors and repressors in the tissues andor in the extracts Hence the analytical auxin measure-ments based on gas chromatography-mass spectrometry deter-mination [65] are more accurate for the determination of low levels of IAA and are therefore preferential for estimation of auxin depletion in the AZ tissues

Auxin depletion during natural abscission Abscission of leaves flowers floral parts and overripe fruits is the last developmental stage of these organs and is therefore regarded as natural abscission The present review examines whether auxin depletion in these organs which serves as auxin sources to their AZs precedes the execution of ethylene-controlled abscission IAA depletion during senescence-induced flower abscis-

sion Flower senescence and abscission can be ethylene-dependent or independent [84 85] Since the basic premise of this review is that auxin depletion in the AZ increases the ethylene sensitivity of the AZ cells we will focus on ethylene-dependent abscission of flower buds open flowers and petals Endogenous IAA levels usually decrease during flower senescence in most flow-ers that subsequently abscise [86 87] Indeed the abscission of

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Meir et al Stewart Postharvest Review 2015 22

unfertilized or male flowers may be ascribed to the low levels of endogenous IAA which is produced in the ovary [88] In Begon-ia abscising male flower buds contain only 1 of the IAA pre-sent in non-abscising female flowers and the seasonal variation in male bud abscission coincided with reduction of the IAA content in the buds [89] In Lilium IAA levels decreased in the gynoecium and petals when the petals started to wilt and se-nesce prior to their abscission [90] IAA level and the expres-sion of 50 auxin-related genes were analyzed in the outer tepals of two Lilium cultivars that differ in their abscission timing Although both cultivars have fully formed AZs Lilium longiflo-rum flowers wilted substantially during senescence prior to their late abscission while those of the closely related Lilium longiflo-rum Asiatic hybrid (LA) abscised early without wilting [91] A clear correlation between auxin levels and abscission timing of both cultivars was found in relation to senescence markers Thus in L longiflorum both free and conjugated-IAA signifi-cantly increased as senescence progressed while free IAA levels in Lilium LA remained low at all developmental stages from closed bud to abscission and the portion of IAA-amide conju-gate gradually increased Consistent with the view that declining IAA levels precede abscission the ARF719-like gene was up-regulated in the delayed-abscising genotype while in the early-abscising genotype it was down-regulated [91] In Dendrobium cut flowers the floral buds at the top of the in-florescence stalk exhibit early yellowing and abscise Applica-tion of an auxin transport inhibitor or an auxin action inhibitor to the stigma of open flowers induced high flower abscission rates [92] Removal of the open flowers at the distal end of the pedicel reduced the time to abscission of the remaining pedicel IAA placed on the cut surface of the pedicel counteracted the effect of flower removal Application of different auxins de-layed senescence and inhibited the abscission of open Dendrobi-um flowers [92 93] These results support the view that auxin is an endogenous regulator of abscission of Dendrobium floral buds and flowers Several other reports show that exogenously ap-plied auxins prevented or delayed abscission of flowers and floral parts styles and stamens [88] Accordingly auxin is ap-plied to extend the vase life of several cut flowers such as Geraldton wax flowers Cestrum and poinsettia [63 88 94 95] Genetic experiments with Arabidopsis mutants further demon-strated the role of auxin in petal abscission Mutation in ARF2 [96] and ARF1 and ARF2 [97] delayed senescence and abscis-sion of Arabidopsis petals Flowers taken from mutants in which individual family members of the auxin influx carriers AUX1 and Like-AUX3 (LAX3) were down-regulated exhibit-ed early abscission Manipulation of IAA levels in the AZ cells by activation of the bacterial IAA biosynthetic genes IAA-Lys-synthetase and iaaM enhanced or delayed petal abscission re-spectively [81] It was shown that IAA-Lys-synthetase re-duced IAA levels in the cells by conjugating free IAA to IAA-Lysine while iaaM promoted IAA levels by converting Trp to indole acetamide IAA depletion during senescence-induced leaf abscission

Endogenous IAA decreases at the onset and during senescence of leaves and auxin treatment can delay leaf senescence in some plants [98-100] Most of the research on leaf senescence was done by using detached leaves in model systems which do not show abscission phenotypes (Arabidopsis or flag leaf in monocots) Therefore the changes in IAA levels during abscis-

sion were not studied Only one early report showed a positive correlation between the decrease in IAA content during senes-cence of Coleus leaves and their abscission [101] Moreover in monocots and some annual dicots there is no AZ at the base of the leaf and the leaves senesce wilt and remain dry on the plant a process termed marcescence Therefore the changes in IAA content during leaf senescence in these plants including the Arabidopsis model system are less relevant IAA depletion during ripening-induced fruit abscission

Fruits typically abscise at the overripe stage of development In general IAA levels decrease in fruit during ripening and this decrease coincides with maturation of the seeds in which most of the IAA is produced [80 102-105] The decrease in IAA levels starts in tomato fruit as the fruit reaches the breaker stage [80 103-104] The reduction of free IAA levels during ripening results from the decrease of auxin signal around the seeds [80 102] and expression of the auxin transport gene families LAX and PIN with advanced ripening [80] as well as from increased conjugation of free IAA to its amide conjugates as GH3 ex-pression is sharply increased with ripening [103-104] A similar decrease in IAA levels and an increase in GH3 expression and indole-3-acetyl-aspartate levels were also observed in grape berries after reaching the mature green stage [105 106] It is likely therefore that auxin depletion in fruit contributes to subsequent abscission competence Unfortunately most studies of auxin metabolism in ripening fruit were terminated at the ripe stage without evaluating fruit at the overripe stage in which most actual abscission events occur Only one exception was reported in which IAA levels were monitored in oil palm fruit until abscission Thus in developing fruit the content of IAA peaked between 60 and 100 days after pollination (DAP) and subsequently decreased to very low levels between 100 and 120 DAP just before abscission which occurred 140 DAP [107] Auxin application delayed oil palm fruit abscission [108] However IAA depletion in fruit may be species-dependent as in peach several reports demonstrated increased IAA level during fruit ripening which is required for fruit sof-tening [109-111] There has been little work in which fruit ripening was correlat-ed with events occurring in nearby AZs Recently two tran-scriptomic reports demonstrated changes in auxin-related gene expression in the AZs of mature olive and melon fruit [78 79] In olive gene expression was studied 154 days post anthe-sis (DPA) when fruit started to ripen and 217 DPA when fruit abscised Numerous auxin-related genes were down-regulated in the AZ 217 DPA including auxin biosynthesis genes ILR1 auxin transporters LAX1 LAX2 and PIN TIR1 two AUXIAA family members three ARF family members and several auxin-induced genes [78] In melon gene expression was stud-ied at three time points 36 38 and 40 DPA The ethylene cli-macteric peak and abscission occurred 37 and 40 DPA respec-tively Between 36 to 38 DPA two genes encoding for auxin effux carriers four AUXIAA genes and one ARF gene were down-regulated in the AZ [79] Between 38 to 40 DPA 13 other auxin-related genes were down-regulated These reports provide the first direct evidence for auxin depletion in the AZ during mature fruit abscission Taken together the reported data show that the processes of flower and leaf senescence and fruit ripening generally lead to

5

Meir et al Stewart Postharvest Review 2015 22

reduction of endogenous auxin levels which in turn result in organ abscission Sometimes a direct evidence for auxin deple-tion prior to organ abscission is lacking in these systems as the experiments did not analyze IAA levels in the senesced organ or overripe fruit but the general pattern was validated in vari-ous systems

IAA depletion during self-pruning of spring shoots in sweet orange (Citrus sinensis)

Citrus shoot tips abscise at an anatomically distinct AZ that separates the top part of the shoots into basal and apical por-tions This natural process termed self-pruning plays an im-portant role in citrus floral bud initiation Citrus microarray was used to monitor the expression of genes at several time points 5 or 3 days before self-pruning when the shoot tips started to fall and at the beginning of self-pruning of spring shoots when the AZ was activated [20] Twenty four auxin-related genes were differentially altered during these stages of self-pruning genes encoding auxin-induced proteins were up-regulated while genes encoding ARFs were down-regulated The authors concluded that auxin depletion in the AZ of the spring shoots causes the AZ to become sensitive to ethylene and abscisic acid which accelerate the abscission process of the shoot tips [20]

Ethylene as a mediator of IAA depletion Ethylene is the main regulator of leaf and flower senescence and fruit ripening and is proposed here as a modulator of auxin depletion in these processes Unlike the synergistic interactions between ethylene and auxin controlling specific growth and developmental processes [112 113 114 115 116] the con-trol of both natural and stress-induced abscission (see below) involves antagonistic effects of ethylene and auxin Several

modes of action were suggested for this negative interaction One of the regulatory effects of ethylene on auxin levels oper-ated through inhibition of auxin transport to the AZs was demonstrated long ago [117-122] Additionally in various natu-ral abscission systems ethylene was reported to reduce auxin levels by increasing the rate of auxin conjugation [120-122]

Auxin depletion during stress-induced abscission

Abscission of different organs is a common response to various biotic abiotic and physiological stresses [2 4 6 119 123] Stress-induced abscission initiated by auxin depletion is mediat-ed by three main intermediate modulators ethylene ROS and carbohydrate starvation [119 123 124 125] Ethylene pro-duction increases in tissues subjected to many stresses subse-quently regulating their auxin levels [126-128] Stress-induced ethylene may promote auxin depletion in various abscissing systems via inhibition of auxin transport to the AZs [117-122 129 130]

ROS

ROS can induce organ abscission [131-133] and application of antioxidants and ROS scavengers can inhibit abscission [67 68 134-137] Several publications reviewed the interplay between ROS and auxin [124 138-140] ROS induced by stress con-ditions have an impact on auxin signaling by affecting auxin homeostasis at the levels of auxin biosynthesis metabolism and distribution ROS stimulated auxin catabolism by increasing decarboxylative and non-decarboxylative oxidation of IAA IAA conjugation (ester and amid forms) and GH3 expression and inhibited IAA transport by decreasing PIN expression [67 68 124 138 139 140] ROS scavenging genes were induced by ethylene during abscission of citrus leaves suggesting that ethylene induces ROS in this system [138]

Table 1 Examples of auxin depletion mechanisms operated in various stress-induced abscission systems

Stress type Plant and abscission system

Mechanisms for auxin depletion Reference

Domination Apple fruitlets in clusters Decrease in PAT decrease in ATA 13 16 17 143 157

Grapevine berries in clusters Decrease in PAT decrease in ATA 19

Cowpea flowers and fruitlets Decrease in PAT decrease in ATA 14

Carbohydrate starvation (phloem-girdling)

Litchi fruitlets Decrease in PAT 142

High temperatures Bell pepper flowers and fruitlets Decrease in IAA level decrease in PAT 130

Mango fruitlets Decrease in PAT 141

Chilling temperatures Ixora leaves Increase in IAA oxidation Decrease in IAA level Decrease in PAT

67

Chilling + high light Ixora leaves Increase in IAA oxidation Decrease in IAA level

68

Water stress Cotton leaves Decrease in PAT 158 159

Cotton buds flowers and fruits Decrease in IAA level Increase in IAA conjugates level

160 161

Citrus leaves Decrease in expression of IAA signaling genes 162

Poplar leaves Decreased expression of IAA signaling genes 163

Balsam fir needles Decrease in IAA level 164

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Meir et al Stewart Postharvest Review 2015 22

Carbohydrate starvation Numerous stressors like water stress high and low light high or chilling temperatures and high sink organ domination result in carbohydrate starvation Abscission of premature fruit leaves flowers and flower buds is related to low sugar content [13 15-17 141-146] Auxin application can inhibit some stress-induced organ abscission [15 146-148] suggesting that the shortage of carbohydrate is correlated with auxin depletion In non-abscissing systems sugars have also been shown to play pivotal roles as signaling molecules [149-155156] Thus solu-ble sugars were reported to increase IAA biosynthesis free IAA levels and IAA transport in these systems [154-155] as well as to up-regulate early auxin biosynthesis and PIN1 genes to pro-mote PIN1 abundance in the PM and to down-regulate two genes of PAT inhibitors [156] We anticipate that sugar sig-naling may ultimately be found to accompany stress-induced decrease in carbohydrates that result in auxin depletion in source organs and their AZs The mechanisms of auxin deple-tion in various stress-induced abscission systems are summa-rized in Table 1 Auxin depletion is expected to increase AZ sensitivity to stress-induced ethylene thereby resulting in ab-scission Elucidation of the sequence of events leading to organ abscission following artificial auxin depletion in tomato

Tomato has been extensively used to study flower and fruit abscission processes Tomato is a very convenient model sys-tem since tomato plants develop a distinct AZ in the midpoint of the flower pedicel referred to as a pedicel AZ or FAZ in different publications The anatomy and development of the tomato FAZ is well established [165-167] as are the proteins involved in the regulation of the FAZ differentiation and devel-opment [168-173] Since the eighties of the previous century in-depth studies have defined enzymes involved in cell separation in various species The AZ-specific polygalacturonases (PGs) and cellulases are particularly well characterized [174-184] The AZ cells are defined as specialized cell types that differ from their adjacent cells in perception and response to ethylene and auxin [2 4 185-187] Therefore it is expected that specific TFs and genes will be specifically expressed in the AZ and not in the adjacent non-AZ (NAZ) cells Indeed in the first transcrip-tome microarray analysis of the tomato FAZ performed follow-ing auxin depletion several genes were found to be specifically expressed in the FAZ and not in the basal portion (proximal) of the pedicel NAZ region [74] These genes include several TFs such as TOMATO KNOTTED4 Homeobox-Leu zipper13 TOMATO AGAMOUSE-LIKE212 TOMATO PROLINE RICH PROTEIN (TPRP-F1) KNOTTED1-LIKE HOMEO-BOX PROTEIN1 (KD1) Phantastica and Ovate [74] In two transcriptome analyses comparing gene expression in the toma-to FAZ versus the NAZ including the basal portion (proximal) and apical portion (distal) pedicel regions 89 and 1255 genes respectively were found to be specifically expressed at anthesis in the FAZ cells including genes encoding for TFs hormone-related proteins cell wall modification enzymes lipid metabo-lism and others [76 77] Most interestingly the AZ-specific gene set include TF genes that encode key regulators of meri-stem-associated functions which may be regulated by a signal-ing pathway that requires auxin supplied from the flower before the onset of abscission Suppression of one of the tomato shoot meristem-associated TF genes tomato ETHYLENE-

RESPONSIVE FACTOR52 (SIERF52) by RNAi did not af-fect FAZ development but significantly delayed pedicel abscis-sion This suggests that SlERF52 plays a pivotal role in tran-scriptional regulation in the FAZ [188] In an attempt to perform a functional analysis of 45 AZ-specific genes whose expression changed early in the tomato FAZ following auxin depletion experiments based on virus-induced gene silencing in the FAZ and LAZ were conducted Silencing nine of these 45 genes led to a significant retardation of pedicel andor petiole abscission following abscission induc-tion The role of these nine genes was further examined by silencing each of them in plants stably transformed with anti-sense or RNAi constructs driven by an AZ-specific promoter Tomato Abscission PG4 (TAPG4) The results showed that TAPG4antisense constructs of KD1 [71] and of TPRP-F1 [189] strongly inhibited both pedicel and petiole abscission Conversely up-regulation of KD1 showed accelerated pedicel and petiole abscission [71] Complementary DNA microarray and quantitative PCR (qPCR) analyses indicated that regulation of abscission by KD1 was associated with a change in the abun-dance of genes related to auxin transporters and signaling com-ponents Measurement of IAA content using the DR5GUS auxin reporter assay confirmed by analytical auxin determina-tion showed that change s in KD1 expression modulated the auxin concentration and response gradient in the FAZ [71] In a transcriptomic analysis comparing gene expression in the tomato FAZ and NAZ in the proximal and distal flanking pedi-cel regions at anthesis [76] a region-specific expression of auxin-related genes was found 1) METHYLESTERASE1 which converts IAA methyl ester to IAA DWARF IN LIGHT1 which encodes an IAA-amido synthetase and GH36 showed higher transcript levels in the FAZ than in the NAZ 2) ARF9 was expressed at higher levels in the proximal (basal) than in the distal (apical) NAZ region 3) GH31 and a Hookless1 homolog were expressed at higher levels in the distal than in the proximal NAZ region This region-specific differential expres-sion of genes involved in the determination of auxin levels sug-gests that a gradient of auxin concentration formed along the pedicel regions may be a key factor in regulating the timing of pedicel abscission [76] Indeed by using the DR5GUS re-porter such a gradient of IAA concentration was recently found in VF36 tomato plants in which the FAZ was also divided into distal and proximal regions [71] Consistent with the initial location of the auxin-producing source tissue this gradient oc-curred according to the following sequence distal side of NAZ gt distal side of FAZ gt proximal side of FAZ gt proximal side of NAZ [71] In order to elucidate the molecular changes occurring in the tomato artificial auxin depletion model system during acquisi-tion of abscission competence in the FAZ following auxin de-pletion and during execution of pedicel abscission our group performed a microarray analysis using the Affymetrix 10K oligonucleotide Tomato GeneChip [74] In this system the flower that serves as an auxin source is removed Applying IAA after flower removal or inhibiting ethylene action using 1-methylcyclopropene (1-MCP) prior to flower removal inhibited pedicel abscission suggesting that pedicel abscission results from auxin depletion and is ethylene-dependent [74] Based

7

Meir et al Stewart Postharvest Review 2015 22

on the transcriptomic results following the application of the abscission modulators an abscission-inducing model was pro-posed in which the sequence of events occurring during tomato pedicel abscission was divided into two phases early events (0 to 4 h after flower removal) and late events (8 to 14 h after flower removal) The early events probably lead to acquisition of ethylene sensitivity and abscission competence while the late events include processes leading to the execution of pedicel abscission and development of the defense layer According to this model the decrease in IAA provides the first signal for abscission Responses to auxin depletion included down-regulation of genes induced by auxin such as AuxIAA and other TF genes and up-regulation of genes repressed by auxin The late events included increased ethylene production due to up-regulation of ethylene biosynthesis genes such as the genes encoding 1-amino-cyclopropane-1-carboxylate synthase which lead in turn to AZ-specific up-regulation of abscission-related genes These genes include genes encoding cell wall-modifying proteins and pathogenesis-related proteins [74] as well as genes related to the development of a protective layer on the surface of the remaining tissue [190] The late events which are ethylene-induced were inhibited by 1-MCP pretreatment while the early events were not affected by the inhibitor On the oth-er hand IAA application immediately after flower removal in-hibited all the cascade of abscission events and the changes in the expression of auxin-induced or auxin-repressed genes [74] Two later studies showed a rapid decrease of AuxIAA [191] and ARF [70] genes after tomato flower removal Addi-tionally quantitative and DR5GUS data demonstrated a fast IAA depletion after flower removal [70] These authors adjust-ed the proposed abscission model [74] to the slower abscis-sion rate obtained in their tomato system (0-8 h for the early events and 16-32 h for the late events) and included the vari-ous ARFs in the model [70] Programmed cell death (PCD) was shown to be another late event involved in the abscission process This was based on the data showing that inhibiting the activity of LX ribonuclease (LX) an enzyme associated with PCD delayed tomato leaf abscission [192] Indeed hallmarks of PCD were identified in the tomato LAZ and FAZ during the late stage of abscission

and data showing that different abscission-related processes occurred asymmetrically between the FAZ proximal and distal regions were presented [193] This asymmetric distribution of various abscission-related processes might be related to the auxin gradient demonstrated recently in the tomato FAZ [71] Leaf deblading removes the natural source of auxin to an AZ and promotes abscission [64] The only report so far describing the effects of leaf deblading on expression of auxin-related genes in the LAZ was performed in Mirabilis jalapa [64] In this system transcripts of two auxin-induced genes Mj-AuxIAA1 and Mj-AuxIAA2 were down-regulated as a result of IAA depletion by leaf deblading or treatment with the IAA transport inhibitor 1-naphthylphthalamic acid Application of IAA to the cut end of the petioles inhibited their abscission and prevented the decline in the transcript levels in the LAZ [64] To further define auxin-relevant members of tomato LAZ and FAZ transcriptomes we recently refined an experimental sys-tem based on the abscission responses of debladed tomato leaves and removed flowers Deblading of tomato leaves led to abscission of attached petioles during a period of 8-12 days Exposure of the debladed-leaf explants to ethylene treatment for 24 h accelerated petiole abscission which was completed within three days after the ethylene treatment (Figure 1A) at a similar rate to that of flower pedicel abscission without ethylene treatment (Figure 1B) RNA collected during petiole and pedi-cel abscission allowed us to compare transcriptomic changes in the tomato LAZ and FAZ respectively through the use of a customized AZ-specific microarray chip The chip included transcripts identified using NGS of RNA isolated from tomato AZs at various time points during organ abscission as well as transcripts from the Solanaceae genomics network and Nation-al Center for Biotechnology Information databases [194] Here we present for the first time results from these microarray anal-yses describing the changes in expression of auxin-related genes (Figures 2-6) Generally there is a high similarity in the abscis-sion process of tomato leaves and flowers with a few excep-tions Most of the auxin-related genes are expressed in both AZs but some members of different gene families are ex-pressed specifically in the FAZ or the LAZ The results presented in Figure 2 show that most genes encod-ing auxin influx (LAX family) and auxin efflux (PIN family) carriers were rapidly down-regulated in both AZs following IAA depletion thereby supporting the requirement for auxin for activation of its transport carriers The few exceptions were related to genes that have very low expression levels such as LAX45 and PIN28 in the FAZ and PIN3 in the LAZ The down-regulation of PIN5 and PILS family members in the two AZs provides the first indication that the regulation of intracel-lular auxin accumulation in the ER expected to control auxin availability for auxin signaling in the nuclei of AZ cells may be important for abscission regulation at the cellular level Two PILS were up-regulated in the FAZ and one in the LAZ fol-lowing flower removal or leaf deblading respectively (Figure 2) further supporting the role of this novel auxin carrier family in regulating auxin homeostasis [41] The REV TF was quickly down-regulated in the FAZ and LAZ after abscission induc-tion suggesting that there is a regulatory mechanism for de-creasing PIN expression and function in both AZs as a result of

Figure 1 Effect of leaf deblading and ethylene treatment (A) and flower removal (B) on the kinetics of petiole and pedicel abscission respective-ly in tomato explants The debladed-leaf explants held in vials with water were prepared as previously described for the flower explants [72] and exposed to ethylene (10 ppm for 24 h) The percentage of accumu-lated pedicel or petiole abscission was monitored at various time inter-vals following organ removal The results are means of four replicates (n=30 explants) plusmn SE

8

Meir et al Stewart Postharvest Review 2015 22

Figure 2 Microarray expression profiles of tomato auxin transport-related genes in the FAZ and LAZ at various time points following abscission induc-tion The assay included family members of the auxin influx carriers (AUXLAX) auxin efflux carriers (PINs PILS) genes and an auxin-related TF gene (REV) Samples were taken from the FAZ at 0 2 4 8 and 14 h after flower removal and from the LAZ at 0 24 48 72 and 96 h after leaf deblading and ethylene treatment as detailed in the legend of Figure 1 Gene expression levels are indicated with the colored code bars ranging from 0 (light blue) to 100 (dark blue) Expression levels are based on the percentage of change of the average intensity values of the replicated samples for each time point The numbers indicated in the first box of each sample represent average intensities values for all replicates at 0 h Corresponding Solanum lyco-persicum (Solyc) ID and gene names are presented in the left and right sides respectively

Figure 3 Microarray expression profiles of tomato IAA-related genes associated with IAA conjugation in the FAZ and LAZ at various time points follow-ing abscission induction The assay included members of the GH3 and ILRs gene families All other details are as described in the legend of Figure 2

9

Meir et al Stewart Postharvest Review 2015 22

Figure 4 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the AuxIAA gene family All other details are as described in the legend of Figure 2

Figure 5 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the ARF gene family All other details are as described in the legend of Figure 2

10

Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

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Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

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Meir et al Stewart Postharvest Review 2015 22

unfertilized or male flowers may be ascribed to the low levels of endogenous IAA which is produced in the ovary [88] In Begon-ia abscising male flower buds contain only 1 of the IAA pre-sent in non-abscising female flowers and the seasonal variation in male bud abscission coincided with reduction of the IAA content in the buds [89] In Lilium IAA levels decreased in the gynoecium and petals when the petals started to wilt and se-nesce prior to their abscission [90] IAA level and the expres-sion of 50 auxin-related genes were analyzed in the outer tepals of two Lilium cultivars that differ in their abscission timing Although both cultivars have fully formed AZs Lilium longiflo-rum flowers wilted substantially during senescence prior to their late abscission while those of the closely related Lilium longiflo-rum Asiatic hybrid (LA) abscised early without wilting [91] A clear correlation between auxin levels and abscission timing of both cultivars was found in relation to senescence markers Thus in L longiflorum both free and conjugated-IAA signifi-cantly increased as senescence progressed while free IAA levels in Lilium LA remained low at all developmental stages from closed bud to abscission and the portion of IAA-amide conju-gate gradually increased Consistent with the view that declining IAA levels precede abscission the ARF719-like gene was up-regulated in the delayed-abscising genotype while in the early-abscising genotype it was down-regulated [91] In Dendrobium cut flowers the floral buds at the top of the in-florescence stalk exhibit early yellowing and abscise Applica-tion of an auxin transport inhibitor or an auxin action inhibitor to the stigma of open flowers induced high flower abscission rates [92] Removal of the open flowers at the distal end of the pedicel reduced the time to abscission of the remaining pedicel IAA placed on the cut surface of the pedicel counteracted the effect of flower removal Application of different auxins de-layed senescence and inhibited the abscission of open Dendrobi-um flowers [92 93] These results support the view that auxin is an endogenous regulator of abscission of Dendrobium floral buds and flowers Several other reports show that exogenously ap-plied auxins prevented or delayed abscission of flowers and floral parts styles and stamens [88] Accordingly auxin is ap-plied to extend the vase life of several cut flowers such as Geraldton wax flowers Cestrum and poinsettia [63 88 94 95] Genetic experiments with Arabidopsis mutants further demon-strated the role of auxin in petal abscission Mutation in ARF2 [96] and ARF1 and ARF2 [97] delayed senescence and abscis-sion of Arabidopsis petals Flowers taken from mutants in which individual family members of the auxin influx carriers AUX1 and Like-AUX3 (LAX3) were down-regulated exhibit-ed early abscission Manipulation of IAA levels in the AZ cells by activation of the bacterial IAA biosynthetic genes IAA-Lys-synthetase and iaaM enhanced or delayed petal abscission re-spectively [81] It was shown that IAA-Lys-synthetase re-duced IAA levels in the cells by conjugating free IAA to IAA-Lysine while iaaM promoted IAA levels by converting Trp to indole acetamide IAA depletion during senescence-induced leaf abscission

Endogenous IAA decreases at the onset and during senescence of leaves and auxin treatment can delay leaf senescence in some plants [98-100] Most of the research on leaf senescence was done by using detached leaves in model systems which do not show abscission phenotypes (Arabidopsis or flag leaf in monocots) Therefore the changes in IAA levels during abscis-

sion were not studied Only one early report showed a positive correlation between the decrease in IAA content during senes-cence of Coleus leaves and their abscission [101] Moreover in monocots and some annual dicots there is no AZ at the base of the leaf and the leaves senesce wilt and remain dry on the plant a process termed marcescence Therefore the changes in IAA content during leaf senescence in these plants including the Arabidopsis model system are less relevant IAA depletion during ripening-induced fruit abscission

Fruits typically abscise at the overripe stage of development In general IAA levels decrease in fruit during ripening and this decrease coincides with maturation of the seeds in which most of the IAA is produced [80 102-105] The decrease in IAA levels starts in tomato fruit as the fruit reaches the breaker stage [80 103-104] The reduction of free IAA levels during ripening results from the decrease of auxin signal around the seeds [80 102] and expression of the auxin transport gene families LAX and PIN with advanced ripening [80] as well as from increased conjugation of free IAA to its amide conjugates as GH3 ex-pression is sharply increased with ripening [103-104] A similar decrease in IAA levels and an increase in GH3 expression and indole-3-acetyl-aspartate levels were also observed in grape berries after reaching the mature green stage [105 106] It is likely therefore that auxin depletion in fruit contributes to subsequent abscission competence Unfortunately most studies of auxin metabolism in ripening fruit were terminated at the ripe stage without evaluating fruit at the overripe stage in which most actual abscission events occur Only one exception was reported in which IAA levels were monitored in oil palm fruit until abscission Thus in developing fruit the content of IAA peaked between 60 and 100 days after pollination (DAP) and subsequently decreased to very low levels between 100 and 120 DAP just before abscission which occurred 140 DAP [107] Auxin application delayed oil palm fruit abscission [108] However IAA depletion in fruit may be species-dependent as in peach several reports demonstrated increased IAA level during fruit ripening which is required for fruit sof-tening [109-111] There has been little work in which fruit ripening was correlat-ed with events occurring in nearby AZs Recently two tran-scriptomic reports demonstrated changes in auxin-related gene expression in the AZs of mature olive and melon fruit [78 79] In olive gene expression was studied 154 days post anthe-sis (DPA) when fruit started to ripen and 217 DPA when fruit abscised Numerous auxin-related genes were down-regulated in the AZ 217 DPA including auxin biosynthesis genes ILR1 auxin transporters LAX1 LAX2 and PIN TIR1 two AUXIAA family members three ARF family members and several auxin-induced genes [78] In melon gene expression was stud-ied at three time points 36 38 and 40 DPA The ethylene cli-macteric peak and abscission occurred 37 and 40 DPA respec-tively Between 36 to 38 DPA two genes encoding for auxin effux carriers four AUXIAA genes and one ARF gene were down-regulated in the AZ [79] Between 38 to 40 DPA 13 other auxin-related genes were down-regulated These reports provide the first direct evidence for auxin depletion in the AZ during mature fruit abscission Taken together the reported data show that the processes of flower and leaf senescence and fruit ripening generally lead to

5

Meir et al Stewart Postharvest Review 2015 22

reduction of endogenous auxin levels which in turn result in organ abscission Sometimes a direct evidence for auxin deple-tion prior to organ abscission is lacking in these systems as the experiments did not analyze IAA levels in the senesced organ or overripe fruit but the general pattern was validated in vari-ous systems

IAA depletion during self-pruning of spring shoots in sweet orange (Citrus sinensis)

Citrus shoot tips abscise at an anatomically distinct AZ that separates the top part of the shoots into basal and apical por-tions This natural process termed self-pruning plays an im-portant role in citrus floral bud initiation Citrus microarray was used to monitor the expression of genes at several time points 5 or 3 days before self-pruning when the shoot tips started to fall and at the beginning of self-pruning of spring shoots when the AZ was activated [20] Twenty four auxin-related genes were differentially altered during these stages of self-pruning genes encoding auxin-induced proteins were up-regulated while genes encoding ARFs were down-regulated The authors concluded that auxin depletion in the AZ of the spring shoots causes the AZ to become sensitive to ethylene and abscisic acid which accelerate the abscission process of the shoot tips [20]

Ethylene as a mediator of IAA depletion Ethylene is the main regulator of leaf and flower senescence and fruit ripening and is proposed here as a modulator of auxin depletion in these processes Unlike the synergistic interactions between ethylene and auxin controlling specific growth and developmental processes [112 113 114 115 116] the con-trol of both natural and stress-induced abscission (see below) involves antagonistic effects of ethylene and auxin Several

modes of action were suggested for this negative interaction One of the regulatory effects of ethylene on auxin levels oper-ated through inhibition of auxin transport to the AZs was demonstrated long ago [117-122] Additionally in various natu-ral abscission systems ethylene was reported to reduce auxin levels by increasing the rate of auxin conjugation [120-122]

Auxin depletion during stress-induced abscission

Abscission of different organs is a common response to various biotic abiotic and physiological stresses [2 4 6 119 123] Stress-induced abscission initiated by auxin depletion is mediat-ed by three main intermediate modulators ethylene ROS and carbohydrate starvation [119 123 124 125] Ethylene pro-duction increases in tissues subjected to many stresses subse-quently regulating their auxin levels [126-128] Stress-induced ethylene may promote auxin depletion in various abscissing systems via inhibition of auxin transport to the AZs [117-122 129 130]

ROS

ROS can induce organ abscission [131-133] and application of antioxidants and ROS scavengers can inhibit abscission [67 68 134-137] Several publications reviewed the interplay between ROS and auxin [124 138-140] ROS induced by stress con-ditions have an impact on auxin signaling by affecting auxin homeostasis at the levels of auxin biosynthesis metabolism and distribution ROS stimulated auxin catabolism by increasing decarboxylative and non-decarboxylative oxidation of IAA IAA conjugation (ester and amid forms) and GH3 expression and inhibited IAA transport by decreasing PIN expression [67 68 124 138 139 140] ROS scavenging genes were induced by ethylene during abscission of citrus leaves suggesting that ethylene induces ROS in this system [138]

Table 1 Examples of auxin depletion mechanisms operated in various stress-induced abscission systems

Stress type Plant and abscission system

Mechanisms for auxin depletion Reference

Domination Apple fruitlets in clusters Decrease in PAT decrease in ATA 13 16 17 143 157

Grapevine berries in clusters Decrease in PAT decrease in ATA 19

Cowpea flowers and fruitlets Decrease in PAT decrease in ATA 14

Carbohydrate starvation (phloem-girdling)

Litchi fruitlets Decrease in PAT 142

High temperatures Bell pepper flowers and fruitlets Decrease in IAA level decrease in PAT 130

Mango fruitlets Decrease in PAT 141

Chilling temperatures Ixora leaves Increase in IAA oxidation Decrease in IAA level Decrease in PAT

67

Chilling + high light Ixora leaves Increase in IAA oxidation Decrease in IAA level

68

Water stress Cotton leaves Decrease in PAT 158 159

Cotton buds flowers and fruits Decrease in IAA level Increase in IAA conjugates level

160 161

Citrus leaves Decrease in expression of IAA signaling genes 162

Poplar leaves Decreased expression of IAA signaling genes 163

Balsam fir needles Decrease in IAA level 164

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Meir et al Stewart Postharvest Review 2015 22

Carbohydrate starvation Numerous stressors like water stress high and low light high or chilling temperatures and high sink organ domination result in carbohydrate starvation Abscission of premature fruit leaves flowers and flower buds is related to low sugar content [13 15-17 141-146] Auxin application can inhibit some stress-induced organ abscission [15 146-148] suggesting that the shortage of carbohydrate is correlated with auxin depletion In non-abscissing systems sugars have also been shown to play pivotal roles as signaling molecules [149-155156] Thus solu-ble sugars were reported to increase IAA biosynthesis free IAA levels and IAA transport in these systems [154-155] as well as to up-regulate early auxin biosynthesis and PIN1 genes to pro-mote PIN1 abundance in the PM and to down-regulate two genes of PAT inhibitors [156] We anticipate that sugar sig-naling may ultimately be found to accompany stress-induced decrease in carbohydrates that result in auxin depletion in source organs and their AZs The mechanisms of auxin deple-tion in various stress-induced abscission systems are summa-rized in Table 1 Auxin depletion is expected to increase AZ sensitivity to stress-induced ethylene thereby resulting in ab-scission Elucidation of the sequence of events leading to organ abscission following artificial auxin depletion in tomato

Tomato has been extensively used to study flower and fruit abscission processes Tomato is a very convenient model sys-tem since tomato plants develop a distinct AZ in the midpoint of the flower pedicel referred to as a pedicel AZ or FAZ in different publications The anatomy and development of the tomato FAZ is well established [165-167] as are the proteins involved in the regulation of the FAZ differentiation and devel-opment [168-173] Since the eighties of the previous century in-depth studies have defined enzymes involved in cell separation in various species The AZ-specific polygalacturonases (PGs) and cellulases are particularly well characterized [174-184] The AZ cells are defined as specialized cell types that differ from their adjacent cells in perception and response to ethylene and auxin [2 4 185-187] Therefore it is expected that specific TFs and genes will be specifically expressed in the AZ and not in the adjacent non-AZ (NAZ) cells Indeed in the first transcrip-tome microarray analysis of the tomato FAZ performed follow-ing auxin depletion several genes were found to be specifically expressed in the FAZ and not in the basal portion (proximal) of the pedicel NAZ region [74] These genes include several TFs such as TOMATO KNOTTED4 Homeobox-Leu zipper13 TOMATO AGAMOUSE-LIKE212 TOMATO PROLINE RICH PROTEIN (TPRP-F1) KNOTTED1-LIKE HOMEO-BOX PROTEIN1 (KD1) Phantastica and Ovate [74] In two transcriptome analyses comparing gene expression in the toma-to FAZ versus the NAZ including the basal portion (proximal) and apical portion (distal) pedicel regions 89 and 1255 genes respectively were found to be specifically expressed at anthesis in the FAZ cells including genes encoding for TFs hormone-related proteins cell wall modification enzymes lipid metabo-lism and others [76 77] Most interestingly the AZ-specific gene set include TF genes that encode key regulators of meri-stem-associated functions which may be regulated by a signal-ing pathway that requires auxin supplied from the flower before the onset of abscission Suppression of one of the tomato shoot meristem-associated TF genes tomato ETHYLENE-

RESPONSIVE FACTOR52 (SIERF52) by RNAi did not af-fect FAZ development but significantly delayed pedicel abscis-sion This suggests that SlERF52 plays a pivotal role in tran-scriptional regulation in the FAZ [188] In an attempt to perform a functional analysis of 45 AZ-specific genes whose expression changed early in the tomato FAZ following auxin depletion experiments based on virus-induced gene silencing in the FAZ and LAZ were conducted Silencing nine of these 45 genes led to a significant retardation of pedicel andor petiole abscission following abscission induc-tion The role of these nine genes was further examined by silencing each of them in plants stably transformed with anti-sense or RNAi constructs driven by an AZ-specific promoter Tomato Abscission PG4 (TAPG4) The results showed that TAPG4antisense constructs of KD1 [71] and of TPRP-F1 [189] strongly inhibited both pedicel and petiole abscission Conversely up-regulation of KD1 showed accelerated pedicel and petiole abscission [71] Complementary DNA microarray and quantitative PCR (qPCR) analyses indicated that regulation of abscission by KD1 was associated with a change in the abun-dance of genes related to auxin transporters and signaling com-ponents Measurement of IAA content using the DR5GUS auxin reporter assay confirmed by analytical auxin determina-tion showed that change s in KD1 expression modulated the auxin concentration and response gradient in the FAZ [71] In a transcriptomic analysis comparing gene expression in the tomato FAZ and NAZ in the proximal and distal flanking pedi-cel regions at anthesis [76] a region-specific expression of auxin-related genes was found 1) METHYLESTERASE1 which converts IAA methyl ester to IAA DWARF IN LIGHT1 which encodes an IAA-amido synthetase and GH36 showed higher transcript levels in the FAZ than in the NAZ 2) ARF9 was expressed at higher levels in the proximal (basal) than in the distal (apical) NAZ region 3) GH31 and a Hookless1 homolog were expressed at higher levels in the distal than in the proximal NAZ region This region-specific differential expres-sion of genes involved in the determination of auxin levels sug-gests that a gradient of auxin concentration formed along the pedicel regions may be a key factor in regulating the timing of pedicel abscission [76] Indeed by using the DR5GUS re-porter such a gradient of IAA concentration was recently found in VF36 tomato plants in which the FAZ was also divided into distal and proximal regions [71] Consistent with the initial location of the auxin-producing source tissue this gradient oc-curred according to the following sequence distal side of NAZ gt distal side of FAZ gt proximal side of FAZ gt proximal side of NAZ [71] In order to elucidate the molecular changes occurring in the tomato artificial auxin depletion model system during acquisi-tion of abscission competence in the FAZ following auxin de-pletion and during execution of pedicel abscission our group performed a microarray analysis using the Affymetrix 10K oligonucleotide Tomato GeneChip [74] In this system the flower that serves as an auxin source is removed Applying IAA after flower removal or inhibiting ethylene action using 1-methylcyclopropene (1-MCP) prior to flower removal inhibited pedicel abscission suggesting that pedicel abscission results from auxin depletion and is ethylene-dependent [74] Based

7

Meir et al Stewart Postharvest Review 2015 22

on the transcriptomic results following the application of the abscission modulators an abscission-inducing model was pro-posed in which the sequence of events occurring during tomato pedicel abscission was divided into two phases early events (0 to 4 h after flower removal) and late events (8 to 14 h after flower removal) The early events probably lead to acquisition of ethylene sensitivity and abscission competence while the late events include processes leading to the execution of pedicel abscission and development of the defense layer According to this model the decrease in IAA provides the first signal for abscission Responses to auxin depletion included down-regulation of genes induced by auxin such as AuxIAA and other TF genes and up-regulation of genes repressed by auxin The late events included increased ethylene production due to up-regulation of ethylene biosynthesis genes such as the genes encoding 1-amino-cyclopropane-1-carboxylate synthase which lead in turn to AZ-specific up-regulation of abscission-related genes These genes include genes encoding cell wall-modifying proteins and pathogenesis-related proteins [74] as well as genes related to the development of a protective layer on the surface of the remaining tissue [190] The late events which are ethylene-induced were inhibited by 1-MCP pretreatment while the early events were not affected by the inhibitor On the oth-er hand IAA application immediately after flower removal in-hibited all the cascade of abscission events and the changes in the expression of auxin-induced or auxin-repressed genes [74] Two later studies showed a rapid decrease of AuxIAA [191] and ARF [70] genes after tomato flower removal Addi-tionally quantitative and DR5GUS data demonstrated a fast IAA depletion after flower removal [70] These authors adjust-ed the proposed abscission model [74] to the slower abscis-sion rate obtained in their tomato system (0-8 h for the early events and 16-32 h for the late events) and included the vari-ous ARFs in the model [70] Programmed cell death (PCD) was shown to be another late event involved in the abscission process This was based on the data showing that inhibiting the activity of LX ribonuclease (LX) an enzyme associated with PCD delayed tomato leaf abscission [192] Indeed hallmarks of PCD were identified in the tomato LAZ and FAZ during the late stage of abscission

and data showing that different abscission-related processes occurred asymmetrically between the FAZ proximal and distal regions were presented [193] This asymmetric distribution of various abscission-related processes might be related to the auxin gradient demonstrated recently in the tomato FAZ [71] Leaf deblading removes the natural source of auxin to an AZ and promotes abscission [64] The only report so far describing the effects of leaf deblading on expression of auxin-related genes in the LAZ was performed in Mirabilis jalapa [64] In this system transcripts of two auxin-induced genes Mj-AuxIAA1 and Mj-AuxIAA2 were down-regulated as a result of IAA depletion by leaf deblading or treatment with the IAA transport inhibitor 1-naphthylphthalamic acid Application of IAA to the cut end of the petioles inhibited their abscission and prevented the decline in the transcript levels in the LAZ [64] To further define auxin-relevant members of tomato LAZ and FAZ transcriptomes we recently refined an experimental sys-tem based on the abscission responses of debladed tomato leaves and removed flowers Deblading of tomato leaves led to abscission of attached petioles during a period of 8-12 days Exposure of the debladed-leaf explants to ethylene treatment for 24 h accelerated petiole abscission which was completed within three days after the ethylene treatment (Figure 1A) at a similar rate to that of flower pedicel abscission without ethylene treatment (Figure 1B) RNA collected during petiole and pedi-cel abscission allowed us to compare transcriptomic changes in the tomato LAZ and FAZ respectively through the use of a customized AZ-specific microarray chip The chip included transcripts identified using NGS of RNA isolated from tomato AZs at various time points during organ abscission as well as transcripts from the Solanaceae genomics network and Nation-al Center for Biotechnology Information databases [194] Here we present for the first time results from these microarray anal-yses describing the changes in expression of auxin-related genes (Figures 2-6) Generally there is a high similarity in the abscis-sion process of tomato leaves and flowers with a few excep-tions Most of the auxin-related genes are expressed in both AZs but some members of different gene families are ex-pressed specifically in the FAZ or the LAZ The results presented in Figure 2 show that most genes encod-ing auxin influx (LAX family) and auxin efflux (PIN family) carriers were rapidly down-regulated in both AZs following IAA depletion thereby supporting the requirement for auxin for activation of its transport carriers The few exceptions were related to genes that have very low expression levels such as LAX45 and PIN28 in the FAZ and PIN3 in the LAZ The down-regulation of PIN5 and PILS family members in the two AZs provides the first indication that the regulation of intracel-lular auxin accumulation in the ER expected to control auxin availability for auxin signaling in the nuclei of AZ cells may be important for abscission regulation at the cellular level Two PILS were up-regulated in the FAZ and one in the LAZ fol-lowing flower removal or leaf deblading respectively (Figure 2) further supporting the role of this novel auxin carrier family in regulating auxin homeostasis [41] The REV TF was quickly down-regulated in the FAZ and LAZ after abscission induc-tion suggesting that there is a regulatory mechanism for de-creasing PIN expression and function in both AZs as a result of

Figure 1 Effect of leaf deblading and ethylene treatment (A) and flower removal (B) on the kinetics of petiole and pedicel abscission respective-ly in tomato explants The debladed-leaf explants held in vials with water were prepared as previously described for the flower explants [72] and exposed to ethylene (10 ppm for 24 h) The percentage of accumu-lated pedicel or petiole abscission was monitored at various time inter-vals following organ removal The results are means of four replicates (n=30 explants) plusmn SE

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Meir et al Stewart Postharvest Review 2015 22

Figure 2 Microarray expression profiles of tomato auxin transport-related genes in the FAZ and LAZ at various time points following abscission induc-tion The assay included family members of the auxin influx carriers (AUXLAX) auxin efflux carriers (PINs PILS) genes and an auxin-related TF gene (REV) Samples were taken from the FAZ at 0 2 4 8 and 14 h after flower removal and from the LAZ at 0 24 48 72 and 96 h after leaf deblading and ethylene treatment as detailed in the legend of Figure 1 Gene expression levels are indicated with the colored code bars ranging from 0 (light blue) to 100 (dark blue) Expression levels are based on the percentage of change of the average intensity values of the replicated samples for each time point The numbers indicated in the first box of each sample represent average intensities values for all replicates at 0 h Corresponding Solanum lyco-persicum (Solyc) ID and gene names are presented in the left and right sides respectively

Figure 3 Microarray expression profiles of tomato IAA-related genes associated with IAA conjugation in the FAZ and LAZ at various time points follow-ing abscission induction The assay included members of the GH3 and ILRs gene families All other details are as described in the legend of Figure 2

9

Meir et al Stewart Postharvest Review 2015 22

Figure 4 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the AuxIAA gene family All other details are as described in the legend of Figure 2

Figure 5 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the ARF gene family All other details are as described in the legend of Figure 2

10

Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

11

Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

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Meir et al Stewart Postharvest Review 2015 22

reduction of endogenous auxin levels which in turn result in organ abscission Sometimes a direct evidence for auxin deple-tion prior to organ abscission is lacking in these systems as the experiments did not analyze IAA levels in the senesced organ or overripe fruit but the general pattern was validated in vari-ous systems

IAA depletion during self-pruning of spring shoots in sweet orange (Citrus sinensis)

Citrus shoot tips abscise at an anatomically distinct AZ that separates the top part of the shoots into basal and apical por-tions This natural process termed self-pruning plays an im-portant role in citrus floral bud initiation Citrus microarray was used to monitor the expression of genes at several time points 5 or 3 days before self-pruning when the shoot tips started to fall and at the beginning of self-pruning of spring shoots when the AZ was activated [20] Twenty four auxin-related genes were differentially altered during these stages of self-pruning genes encoding auxin-induced proteins were up-regulated while genes encoding ARFs were down-regulated The authors concluded that auxin depletion in the AZ of the spring shoots causes the AZ to become sensitive to ethylene and abscisic acid which accelerate the abscission process of the shoot tips [20]

Ethylene as a mediator of IAA depletion Ethylene is the main regulator of leaf and flower senescence and fruit ripening and is proposed here as a modulator of auxin depletion in these processes Unlike the synergistic interactions between ethylene and auxin controlling specific growth and developmental processes [112 113 114 115 116] the con-trol of both natural and stress-induced abscission (see below) involves antagonistic effects of ethylene and auxin Several

modes of action were suggested for this negative interaction One of the regulatory effects of ethylene on auxin levels oper-ated through inhibition of auxin transport to the AZs was demonstrated long ago [117-122] Additionally in various natu-ral abscission systems ethylene was reported to reduce auxin levels by increasing the rate of auxin conjugation [120-122]

Auxin depletion during stress-induced abscission

Abscission of different organs is a common response to various biotic abiotic and physiological stresses [2 4 6 119 123] Stress-induced abscission initiated by auxin depletion is mediat-ed by three main intermediate modulators ethylene ROS and carbohydrate starvation [119 123 124 125] Ethylene pro-duction increases in tissues subjected to many stresses subse-quently regulating their auxin levels [126-128] Stress-induced ethylene may promote auxin depletion in various abscissing systems via inhibition of auxin transport to the AZs [117-122 129 130]

ROS

ROS can induce organ abscission [131-133] and application of antioxidants and ROS scavengers can inhibit abscission [67 68 134-137] Several publications reviewed the interplay between ROS and auxin [124 138-140] ROS induced by stress con-ditions have an impact on auxin signaling by affecting auxin homeostasis at the levels of auxin biosynthesis metabolism and distribution ROS stimulated auxin catabolism by increasing decarboxylative and non-decarboxylative oxidation of IAA IAA conjugation (ester and amid forms) and GH3 expression and inhibited IAA transport by decreasing PIN expression [67 68 124 138 139 140] ROS scavenging genes were induced by ethylene during abscission of citrus leaves suggesting that ethylene induces ROS in this system [138]

Table 1 Examples of auxin depletion mechanisms operated in various stress-induced abscission systems

Stress type Plant and abscission system

Mechanisms for auxin depletion Reference

Domination Apple fruitlets in clusters Decrease in PAT decrease in ATA 13 16 17 143 157

Grapevine berries in clusters Decrease in PAT decrease in ATA 19

Cowpea flowers and fruitlets Decrease in PAT decrease in ATA 14

Carbohydrate starvation (phloem-girdling)

Litchi fruitlets Decrease in PAT 142

High temperatures Bell pepper flowers and fruitlets Decrease in IAA level decrease in PAT 130

Mango fruitlets Decrease in PAT 141

Chilling temperatures Ixora leaves Increase in IAA oxidation Decrease in IAA level Decrease in PAT

67

Chilling + high light Ixora leaves Increase in IAA oxidation Decrease in IAA level

68

Water stress Cotton leaves Decrease in PAT 158 159

Cotton buds flowers and fruits Decrease in IAA level Increase in IAA conjugates level

160 161

Citrus leaves Decrease in expression of IAA signaling genes 162

Poplar leaves Decreased expression of IAA signaling genes 163

Balsam fir needles Decrease in IAA level 164

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Meir et al Stewart Postharvest Review 2015 22

Carbohydrate starvation Numerous stressors like water stress high and low light high or chilling temperatures and high sink organ domination result in carbohydrate starvation Abscission of premature fruit leaves flowers and flower buds is related to low sugar content [13 15-17 141-146] Auxin application can inhibit some stress-induced organ abscission [15 146-148] suggesting that the shortage of carbohydrate is correlated with auxin depletion In non-abscissing systems sugars have also been shown to play pivotal roles as signaling molecules [149-155156] Thus solu-ble sugars were reported to increase IAA biosynthesis free IAA levels and IAA transport in these systems [154-155] as well as to up-regulate early auxin biosynthesis and PIN1 genes to pro-mote PIN1 abundance in the PM and to down-regulate two genes of PAT inhibitors [156] We anticipate that sugar sig-naling may ultimately be found to accompany stress-induced decrease in carbohydrates that result in auxin depletion in source organs and their AZs The mechanisms of auxin deple-tion in various stress-induced abscission systems are summa-rized in Table 1 Auxin depletion is expected to increase AZ sensitivity to stress-induced ethylene thereby resulting in ab-scission Elucidation of the sequence of events leading to organ abscission following artificial auxin depletion in tomato

Tomato has been extensively used to study flower and fruit abscission processes Tomato is a very convenient model sys-tem since tomato plants develop a distinct AZ in the midpoint of the flower pedicel referred to as a pedicel AZ or FAZ in different publications The anatomy and development of the tomato FAZ is well established [165-167] as are the proteins involved in the regulation of the FAZ differentiation and devel-opment [168-173] Since the eighties of the previous century in-depth studies have defined enzymes involved in cell separation in various species The AZ-specific polygalacturonases (PGs) and cellulases are particularly well characterized [174-184] The AZ cells are defined as specialized cell types that differ from their adjacent cells in perception and response to ethylene and auxin [2 4 185-187] Therefore it is expected that specific TFs and genes will be specifically expressed in the AZ and not in the adjacent non-AZ (NAZ) cells Indeed in the first transcrip-tome microarray analysis of the tomato FAZ performed follow-ing auxin depletion several genes were found to be specifically expressed in the FAZ and not in the basal portion (proximal) of the pedicel NAZ region [74] These genes include several TFs such as TOMATO KNOTTED4 Homeobox-Leu zipper13 TOMATO AGAMOUSE-LIKE212 TOMATO PROLINE RICH PROTEIN (TPRP-F1) KNOTTED1-LIKE HOMEO-BOX PROTEIN1 (KD1) Phantastica and Ovate [74] In two transcriptome analyses comparing gene expression in the toma-to FAZ versus the NAZ including the basal portion (proximal) and apical portion (distal) pedicel regions 89 and 1255 genes respectively were found to be specifically expressed at anthesis in the FAZ cells including genes encoding for TFs hormone-related proteins cell wall modification enzymes lipid metabo-lism and others [76 77] Most interestingly the AZ-specific gene set include TF genes that encode key regulators of meri-stem-associated functions which may be regulated by a signal-ing pathway that requires auxin supplied from the flower before the onset of abscission Suppression of one of the tomato shoot meristem-associated TF genes tomato ETHYLENE-

RESPONSIVE FACTOR52 (SIERF52) by RNAi did not af-fect FAZ development but significantly delayed pedicel abscis-sion This suggests that SlERF52 plays a pivotal role in tran-scriptional regulation in the FAZ [188] In an attempt to perform a functional analysis of 45 AZ-specific genes whose expression changed early in the tomato FAZ following auxin depletion experiments based on virus-induced gene silencing in the FAZ and LAZ were conducted Silencing nine of these 45 genes led to a significant retardation of pedicel andor petiole abscission following abscission induc-tion The role of these nine genes was further examined by silencing each of them in plants stably transformed with anti-sense or RNAi constructs driven by an AZ-specific promoter Tomato Abscission PG4 (TAPG4) The results showed that TAPG4antisense constructs of KD1 [71] and of TPRP-F1 [189] strongly inhibited both pedicel and petiole abscission Conversely up-regulation of KD1 showed accelerated pedicel and petiole abscission [71] Complementary DNA microarray and quantitative PCR (qPCR) analyses indicated that regulation of abscission by KD1 was associated with a change in the abun-dance of genes related to auxin transporters and signaling com-ponents Measurement of IAA content using the DR5GUS auxin reporter assay confirmed by analytical auxin determina-tion showed that change s in KD1 expression modulated the auxin concentration and response gradient in the FAZ [71] In a transcriptomic analysis comparing gene expression in the tomato FAZ and NAZ in the proximal and distal flanking pedi-cel regions at anthesis [76] a region-specific expression of auxin-related genes was found 1) METHYLESTERASE1 which converts IAA methyl ester to IAA DWARF IN LIGHT1 which encodes an IAA-amido synthetase and GH36 showed higher transcript levels in the FAZ than in the NAZ 2) ARF9 was expressed at higher levels in the proximal (basal) than in the distal (apical) NAZ region 3) GH31 and a Hookless1 homolog were expressed at higher levels in the distal than in the proximal NAZ region This region-specific differential expres-sion of genes involved in the determination of auxin levels sug-gests that a gradient of auxin concentration formed along the pedicel regions may be a key factor in regulating the timing of pedicel abscission [76] Indeed by using the DR5GUS re-porter such a gradient of IAA concentration was recently found in VF36 tomato plants in which the FAZ was also divided into distal and proximal regions [71] Consistent with the initial location of the auxin-producing source tissue this gradient oc-curred according to the following sequence distal side of NAZ gt distal side of FAZ gt proximal side of FAZ gt proximal side of NAZ [71] In order to elucidate the molecular changes occurring in the tomato artificial auxin depletion model system during acquisi-tion of abscission competence in the FAZ following auxin de-pletion and during execution of pedicel abscission our group performed a microarray analysis using the Affymetrix 10K oligonucleotide Tomato GeneChip [74] In this system the flower that serves as an auxin source is removed Applying IAA after flower removal or inhibiting ethylene action using 1-methylcyclopropene (1-MCP) prior to flower removal inhibited pedicel abscission suggesting that pedicel abscission results from auxin depletion and is ethylene-dependent [74] Based

7

Meir et al Stewart Postharvest Review 2015 22

on the transcriptomic results following the application of the abscission modulators an abscission-inducing model was pro-posed in which the sequence of events occurring during tomato pedicel abscission was divided into two phases early events (0 to 4 h after flower removal) and late events (8 to 14 h after flower removal) The early events probably lead to acquisition of ethylene sensitivity and abscission competence while the late events include processes leading to the execution of pedicel abscission and development of the defense layer According to this model the decrease in IAA provides the first signal for abscission Responses to auxin depletion included down-regulation of genes induced by auxin such as AuxIAA and other TF genes and up-regulation of genes repressed by auxin The late events included increased ethylene production due to up-regulation of ethylene biosynthesis genes such as the genes encoding 1-amino-cyclopropane-1-carboxylate synthase which lead in turn to AZ-specific up-regulation of abscission-related genes These genes include genes encoding cell wall-modifying proteins and pathogenesis-related proteins [74] as well as genes related to the development of a protective layer on the surface of the remaining tissue [190] The late events which are ethylene-induced were inhibited by 1-MCP pretreatment while the early events were not affected by the inhibitor On the oth-er hand IAA application immediately after flower removal in-hibited all the cascade of abscission events and the changes in the expression of auxin-induced or auxin-repressed genes [74] Two later studies showed a rapid decrease of AuxIAA [191] and ARF [70] genes after tomato flower removal Addi-tionally quantitative and DR5GUS data demonstrated a fast IAA depletion after flower removal [70] These authors adjust-ed the proposed abscission model [74] to the slower abscis-sion rate obtained in their tomato system (0-8 h for the early events and 16-32 h for the late events) and included the vari-ous ARFs in the model [70] Programmed cell death (PCD) was shown to be another late event involved in the abscission process This was based on the data showing that inhibiting the activity of LX ribonuclease (LX) an enzyme associated with PCD delayed tomato leaf abscission [192] Indeed hallmarks of PCD were identified in the tomato LAZ and FAZ during the late stage of abscission

and data showing that different abscission-related processes occurred asymmetrically between the FAZ proximal and distal regions were presented [193] This asymmetric distribution of various abscission-related processes might be related to the auxin gradient demonstrated recently in the tomato FAZ [71] Leaf deblading removes the natural source of auxin to an AZ and promotes abscission [64] The only report so far describing the effects of leaf deblading on expression of auxin-related genes in the LAZ was performed in Mirabilis jalapa [64] In this system transcripts of two auxin-induced genes Mj-AuxIAA1 and Mj-AuxIAA2 were down-regulated as a result of IAA depletion by leaf deblading or treatment with the IAA transport inhibitor 1-naphthylphthalamic acid Application of IAA to the cut end of the petioles inhibited their abscission and prevented the decline in the transcript levels in the LAZ [64] To further define auxin-relevant members of tomato LAZ and FAZ transcriptomes we recently refined an experimental sys-tem based on the abscission responses of debladed tomato leaves and removed flowers Deblading of tomato leaves led to abscission of attached petioles during a period of 8-12 days Exposure of the debladed-leaf explants to ethylene treatment for 24 h accelerated petiole abscission which was completed within three days after the ethylene treatment (Figure 1A) at a similar rate to that of flower pedicel abscission without ethylene treatment (Figure 1B) RNA collected during petiole and pedi-cel abscission allowed us to compare transcriptomic changes in the tomato LAZ and FAZ respectively through the use of a customized AZ-specific microarray chip The chip included transcripts identified using NGS of RNA isolated from tomato AZs at various time points during organ abscission as well as transcripts from the Solanaceae genomics network and Nation-al Center for Biotechnology Information databases [194] Here we present for the first time results from these microarray anal-yses describing the changes in expression of auxin-related genes (Figures 2-6) Generally there is a high similarity in the abscis-sion process of tomato leaves and flowers with a few excep-tions Most of the auxin-related genes are expressed in both AZs but some members of different gene families are ex-pressed specifically in the FAZ or the LAZ The results presented in Figure 2 show that most genes encod-ing auxin influx (LAX family) and auxin efflux (PIN family) carriers were rapidly down-regulated in both AZs following IAA depletion thereby supporting the requirement for auxin for activation of its transport carriers The few exceptions were related to genes that have very low expression levels such as LAX45 and PIN28 in the FAZ and PIN3 in the LAZ The down-regulation of PIN5 and PILS family members in the two AZs provides the first indication that the regulation of intracel-lular auxin accumulation in the ER expected to control auxin availability for auxin signaling in the nuclei of AZ cells may be important for abscission regulation at the cellular level Two PILS were up-regulated in the FAZ and one in the LAZ fol-lowing flower removal or leaf deblading respectively (Figure 2) further supporting the role of this novel auxin carrier family in regulating auxin homeostasis [41] The REV TF was quickly down-regulated in the FAZ and LAZ after abscission induc-tion suggesting that there is a regulatory mechanism for de-creasing PIN expression and function in both AZs as a result of

Figure 1 Effect of leaf deblading and ethylene treatment (A) and flower removal (B) on the kinetics of petiole and pedicel abscission respective-ly in tomato explants The debladed-leaf explants held in vials with water were prepared as previously described for the flower explants [72] and exposed to ethylene (10 ppm for 24 h) The percentage of accumu-lated pedicel or petiole abscission was monitored at various time inter-vals following organ removal The results are means of four replicates (n=30 explants) plusmn SE

8

Meir et al Stewart Postharvest Review 2015 22

Figure 2 Microarray expression profiles of tomato auxin transport-related genes in the FAZ and LAZ at various time points following abscission induc-tion The assay included family members of the auxin influx carriers (AUXLAX) auxin efflux carriers (PINs PILS) genes and an auxin-related TF gene (REV) Samples were taken from the FAZ at 0 2 4 8 and 14 h after flower removal and from the LAZ at 0 24 48 72 and 96 h after leaf deblading and ethylene treatment as detailed in the legend of Figure 1 Gene expression levels are indicated with the colored code bars ranging from 0 (light blue) to 100 (dark blue) Expression levels are based on the percentage of change of the average intensity values of the replicated samples for each time point The numbers indicated in the first box of each sample represent average intensities values for all replicates at 0 h Corresponding Solanum lyco-persicum (Solyc) ID and gene names are presented in the left and right sides respectively

Figure 3 Microarray expression profiles of tomato IAA-related genes associated with IAA conjugation in the FAZ and LAZ at various time points follow-ing abscission induction The assay included members of the GH3 and ILRs gene families All other details are as described in the legend of Figure 2

9

Meir et al Stewart Postharvest Review 2015 22

Figure 4 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the AuxIAA gene family All other details are as described in the legend of Figure 2

Figure 5 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the ARF gene family All other details are as described in the legend of Figure 2

10

Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

11

Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

6

Meir et al Stewart Postharvest Review 2015 22

Carbohydrate starvation Numerous stressors like water stress high and low light high or chilling temperatures and high sink organ domination result in carbohydrate starvation Abscission of premature fruit leaves flowers and flower buds is related to low sugar content [13 15-17 141-146] Auxin application can inhibit some stress-induced organ abscission [15 146-148] suggesting that the shortage of carbohydrate is correlated with auxin depletion In non-abscissing systems sugars have also been shown to play pivotal roles as signaling molecules [149-155156] Thus solu-ble sugars were reported to increase IAA biosynthesis free IAA levels and IAA transport in these systems [154-155] as well as to up-regulate early auxin biosynthesis and PIN1 genes to pro-mote PIN1 abundance in the PM and to down-regulate two genes of PAT inhibitors [156] We anticipate that sugar sig-naling may ultimately be found to accompany stress-induced decrease in carbohydrates that result in auxin depletion in source organs and their AZs The mechanisms of auxin deple-tion in various stress-induced abscission systems are summa-rized in Table 1 Auxin depletion is expected to increase AZ sensitivity to stress-induced ethylene thereby resulting in ab-scission Elucidation of the sequence of events leading to organ abscission following artificial auxin depletion in tomato

Tomato has been extensively used to study flower and fruit abscission processes Tomato is a very convenient model sys-tem since tomato plants develop a distinct AZ in the midpoint of the flower pedicel referred to as a pedicel AZ or FAZ in different publications The anatomy and development of the tomato FAZ is well established [165-167] as are the proteins involved in the regulation of the FAZ differentiation and devel-opment [168-173] Since the eighties of the previous century in-depth studies have defined enzymes involved in cell separation in various species The AZ-specific polygalacturonases (PGs) and cellulases are particularly well characterized [174-184] The AZ cells are defined as specialized cell types that differ from their adjacent cells in perception and response to ethylene and auxin [2 4 185-187] Therefore it is expected that specific TFs and genes will be specifically expressed in the AZ and not in the adjacent non-AZ (NAZ) cells Indeed in the first transcrip-tome microarray analysis of the tomato FAZ performed follow-ing auxin depletion several genes were found to be specifically expressed in the FAZ and not in the basal portion (proximal) of the pedicel NAZ region [74] These genes include several TFs such as TOMATO KNOTTED4 Homeobox-Leu zipper13 TOMATO AGAMOUSE-LIKE212 TOMATO PROLINE RICH PROTEIN (TPRP-F1) KNOTTED1-LIKE HOMEO-BOX PROTEIN1 (KD1) Phantastica and Ovate [74] In two transcriptome analyses comparing gene expression in the toma-to FAZ versus the NAZ including the basal portion (proximal) and apical portion (distal) pedicel regions 89 and 1255 genes respectively were found to be specifically expressed at anthesis in the FAZ cells including genes encoding for TFs hormone-related proteins cell wall modification enzymes lipid metabo-lism and others [76 77] Most interestingly the AZ-specific gene set include TF genes that encode key regulators of meri-stem-associated functions which may be regulated by a signal-ing pathway that requires auxin supplied from the flower before the onset of abscission Suppression of one of the tomato shoot meristem-associated TF genes tomato ETHYLENE-

RESPONSIVE FACTOR52 (SIERF52) by RNAi did not af-fect FAZ development but significantly delayed pedicel abscis-sion This suggests that SlERF52 plays a pivotal role in tran-scriptional regulation in the FAZ [188] In an attempt to perform a functional analysis of 45 AZ-specific genes whose expression changed early in the tomato FAZ following auxin depletion experiments based on virus-induced gene silencing in the FAZ and LAZ were conducted Silencing nine of these 45 genes led to a significant retardation of pedicel andor petiole abscission following abscission induc-tion The role of these nine genes was further examined by silencing each of them in plants stably transformed with anti-sense or RNAi constructs driven by an AZ-specific promoter Tomato Abscission PG4 (TAPG4) The results showed that TAPG4antisense constructs of KD1 [71] and of TPRP-F1 [189] strongly inhibited both pedicel and petiole abscission Conversely up-regulation of KD1 showed accelerated pedicel and petiole abscission [71] Complementary DNA microarray and quantitative PCR (qPCR) analyses indicated that regulation of abscission by KD1 was associated with a change in the abun-dance of genes related to auxin transporters and signaling com-ponents Measurement of IAA content using the DR5GUS auxin reporter assay confirmed by analytical auxin determina-tion showed that change s in KD1 expression modulated the auxin concentration and response gradient in the FAZ [71] In a transcriptomic analysis comparing gene expression in the tomato FAZ and NAZ in the proximal and distal flanking pedi-cel regions at anthesis [76] a region-specific expression of auxin-related genes was found 1) METHYLESTERASE1 which converts IAA methyl ester to IAA DWARF IN LIGHT1 which encodes an IAA-amido synthetase and GH36 showed higher transcript levels in the FAZ than in the NAZ 2) ARF9 was expressed at higher levels in the proximal (basal) than in the distal (apical) NAZ region 3) GH31 and a Hookless1 homolog were expressed at higher levels in the distal than in the proximal NAZ region This region-specific differential expres-sion of genes involved in the determination of auxin levels sug-gests that a gradient of auxin concentration formed along the pedicel regions may be a key factor in regulating the timing of pedicel abscission [76] Indeed by using the DR5GUS re-porter such a gradient of IAA concentration was recently found in VF36 tomato plants in which the FAZ was also divided into distal and proximal regions [71] Consistent with the initial location of the auxin-producing source tissue this gradient oc-curred according to the following sequence distal side of NAZ gt distal side of FAZ gt proximal side of FAZ gt proximal side of NAZ [71] In order to elucidate the molecular changes occurring in the tomato artificial auxin depletion model system during acquisi-tion of abscission competence in the FAZ following auxin de-pletion and during execution of pedicel abscission our group performed a microarray analysis using the Affymetrix 10K oligonucleotide Tomato GeneChip [74] In this system the flower that serves as an auxin source is removed Applying IAA after flower removal or inhibiting ethylene action using 1-methylcyclopropene (1-MCP) prior to flower removal inhibited pedicel abscission suggesting that pedicel abscission results from auxin depletion and is ethylene-dependent [74] Based

7

Meir et al Stewart Postharvest Review 2015 22

on the transcriptomic results following the application of the abscission modulators an abscission-inducing model was pro-posed in which the sequence of events occurring during tomato pedicel abscission was divided into two phases early events (0 to 4 h after flower removal) and late events (8 to 14 h after flower removal) The early events probably lead to acquisition of ethylene sensitivity and abscission competence while the late events include processes leading to the execution of pedicel abscission and development of the defense layer According to this model the decrease in IAA provides the first signal for abscission Responses to auxin depletion included down-regulation of genes induced by auxin such as AuxIAA and other TF genes and up-regulation of genes repressed by auxin The late events included increased ethylene production due to up-regulation of ethylene biosynthesis genes such as the genes encoding 1-amino-cyclopropane-1-carboxylate synthase which lead in turn to AZ-specific up-regulation of abscission-related genes These genes include genes encoding cell wall-modifying proteins and pathogenesis-related proteins [74] as well as genes related to the development of a protective layer on the surface of the remaining tissue [190] The late events which are ethylene-induced were inhibited by 1-MCP pretreatment while the early events were not affected by the inhibitor On the oth-er hand IAA application immediately after flower removal in-hibited all the cascade of abscission events and the changes in the expression of auxin-induced or auxin-repressed genes [74] Two later studies showed a rapid decrease of AuxIAA [191] and ARF [70] genes after tomato flower removal Addi-tionally quantitative and DR5GUS data demonstrated a fast IAA depletion after flower removal [70] These authors adjust-ed the proposed abscission model [74] to the slower abscis-sion rate obtained in their tomato system (0-8 h for the early events and 16-32 h for the late events) and included the vari-ous ARFs in the model [70] Programmed cell death (PCD) was shown to be another late event involved in the abscission process This was based on the data showing that inhibiting the activity of LX ribonuclease (LX) an enzyme associated with PCD delayed tomato leaf abscission [192] Indeed hallmarks of PCD were identified in the tomato LAZ and FAZ during the late stage of abscission

and data showing that different abscission-related processes occurred asymmetrically between the FAZ proximal and distal regions were presented [193] This asymmetric distribution of various abscission-related processes might be related to the auxin gradient demonstrated recently in the tomato FAZ [71] Leaf deblading removes the natural source of auxin to an AZ and promotes abscission [64] The only report so far describing the effects of leaf deblading on expression of auxin-related genes in the LAZ was performed in Mirabilis jalapa [64] In this system transcripts of two auxin-induced genes Mj-AuxIAA1 and Mj-AuxIAA2 were down-regulated as a result of IAA depletion by leaf deblading or treatment with the IAA transport inhibitor 1-naphthylphthalamic acid Application of IAA to the cut end of the petioles inhibited their abscission and prevented the decline in the transcript levels in the LAZ [64] To further define auxin-relevant members of tomato LAZ and FAZ transcriptomes we recently refined an experimental sys-tem based on the abscission responses of debladed tomato leaves and removed flowers Deblading of tomato leaves led to abscission of attached petioles during a period of 8-12 days Exposure of the debladed-leaf explants to ethylene treatment for 24 h accelerated petiole abscission which was completed within three days after the ethylene treatment (Figure 1A) at a similar rate to that of flower pedicel abscission without ethylene treatment (Figure 1B) RNA collected during petiole and pedi-cel abscission allowed us to compare transcriptomic changes in the tomato LAZ and FAZ respectively through the use of a customized AZ-specific microarray chip The chip included transcripts identified using NGS of RNA isolated from tomato AZs at various time points during organ abscission as well as transcripts from the Solanaceae genomics network and Nation-al Center for Biotechnology Information databases [194] Here we present for the first time results from these microarray anal-yses describing the changes in expression of auxin-related genes (Figures 2-6) Generally there is a high similarity in the abscis-sion process of tomato leaves and flowers with a few excep-tions Most of the auxin-related genes are expressed in both AZs but some members of different gene families are ex-pressed specifically in the FAZ or the LAZ The results presented in Figure 2 show that most genes encod-ing auxin influx (LAX family) and auxin efflux (PIN family) carriers were rapidly down-regulated in both AZs following IAA depletion thereby supporting the requirement for auxin for activation of its transport carriers The few exceptions were related to genes that have very low expression levels such as LAX45 and PIN28 in the FAZ and PIN3 in the LAZ The down-regulation of PIN5 and PILS family members in the two AZs provides the first indication that the regulation of intracel-lular auxin accumulation in the ER expected to control auxin availability for auxin signaling in the nuclei of AZ cells may be important for abscission regulation at the cellular level Two PILS were up-regulated in the FAZ and one in the LAZ fol-lowing flower removal or leaf deblading respectively (Figure 2) further supporting the role of this novel auxin carrier family in regulating auxin homeostasis [41] The REV TF was quickly down-regulated in the FAZ and LAZ after abscission induc-tion suggesting that there is a regulatory mechanism for de-creasing PIN expression and function in both AZs as a result of

Figure 1 Effect of leaf deblading and ethylene treatment (A) and flower removal (B) on the kinetics of petiole and pedicel abscission respective-ly in tomato explants The debladed-leaf explants held in vials with water were prepared as previously described for the flower explants [72] and exposed to ethylene (10 ppm for 24 h) The percentage of accumu-lated pedicel or petiole abscission was monitored at various time inter-vals following organ removal The results are means of four replicates (n=30 explants) plusmn SE

8

Meir et al Stewart Postharvest Review 2015 22

Figure 2 Microarray expression profiles of tomato auxin transport-related genes in the FAZ and LAZ at various time points following abscission induc-tion The assay included family members of the auxin influx carriers (AUXLAX) auxin efflux carriers (PINs PILS) genes and an auxin-related TF gene (REV) Samples were taken from the FAZ at 0 2 4 8 and 14 h after flower removal and from the LAZ at 0 24 48 72 and 96 h after leaf deblading and ethylene treatment as detailed in the legend of Figure 1 Gene expression levels are indicated with the colored code bars ranging from 0 (light blue) to 100 (dark blue) Expression levels are based on the percentage of change of the average intensity values of the replicated samples for each time point The numbers indicated in the first box of each sample represent average intensities values for all replicates at 0 h Corresponding Solanum lyco-persicum (Solyc) ID and gene names are presented in the left and right sides respectively

Figure 3 Microarray expression profiles of tomato IAA-related genes associated with IAA conjugation in the FAZ and LAZ at various time points follow-ing abscission induction The assay included members of the GH3 and ILRs gene families All other details are as described in the legend of Figure 2

9

Meir et al Stewart Postharvest Review 2015 22

Figure 4 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the AuxIAA gene family All other details are as described in the legend of Figure 2

Figure 5 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the ARF gene family All other details are as described in the legend of Figure 2

10

Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

11

Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

7

Meir et al Stewart Postharvest Review 2015 22

on the transcriptomic results following the application of the abscission modulators an abscission-inducing model was pro-posed in which the sequence of events occurring during tomato pedicel abscission was divided into two phases early events (0 to 4 h after flower removal) and late events (8 to 14 h after flower removal) The early events probably lead to acquisition of ethylene sensitivity and abscission competence while the late events include processes leading to the execution of pedicel abscission and development of the defense layer According to this model the decrease in IAA provides the first signal for abscission Responses to auxin depletion included down-regulation of genes induced by auxin such as AuxIAA and other TF genes and up-regulation of genes repressed by auxin The late events included increased ethylene production due to up-regulation of ethylene biosynthesis genes such as the genes encoding 1-amino-cyclopropane-1-carboxylate synthase which lead in turn to AZ-specific up-regulation of abscission-related genes These genes include genes encoding cell wall-modifying proteins and pathogenesis-related proteins [74] as well as genes related to the development of a protective layer on the surface of the remaining tissue [190] The late events which are ethylene-induced were inhibited by 1-MCP pretreatment while the early events were not affected by the inhibitor On the oth-er hand IAA application immediately after flower removal in-hibited all the cascade of abscission events and the changes in the expression of auxin-induced or auxin-repressed genes [74] Two later studies showed a rapid decrease of AuxIAA [191] and ARF [70] genes after tomato flower removal Addi-tionally quantitative and DR5GUS data demonstrated a fast IAA depletion after flower removal [70] These authors adjust-ed the proposed abscission model [74] to the slower abscis-sion rate obtained in their tomato system (0-8 h for the early events and 16-32 h for the late events) and included the vari-ous ARFs in the model [70] Programmed cell death (PCD) was shown to be another late event involved in the abscission process This was based on the data showing that inhibiting the activity of LX ribonuclease (LX) an enzyme associated with PCD delayed tomato leaf abscission [192] Indeed hallmarks of PCD were identified in the tomato LAZ and FAZ during the late stage of abscission

and data showing that different abscission-related processes occurred asymmetrically between the FAZ proximal and distal regions were presented [193] This asymmetric distribution of various abscission-related processes might be related to the auxin gradient demonstrated recently in the tomato FAZ [71] Leaf deblading removes the natural source of auxin to an AZ and promotes abscission [64] The only report so far describing the effects of leaf deblading on expression of auxin-related genes in the LAZ was performed in Mirabilis jalapa [64] In this system transcripts of two auxin-induced genes Mj-AuxIAA1 and Mj-AuxIAA2 were down-regulated as a result of IAA depletion by leaf deblading or treatment with the IAA transport inhibitor 1-naphthylphthalamic acid Application of IAA to the cut end of the petioles inhibited their abscission and prevented the decline in the transcript levels in the LAZ [64] To further define auxin-relevant members of tomato LAZ and FAZ transcriptomes we recently refined an experimental sys-tem based on the abscission responses of debladed tomato leaves and removed flowers Deblading of tomato leaves led to abscission of attached petioles during a period of 8-12 days Exposure of the debladed-leaf explants to ethylene treatment for 24 h accelerated petiole abscission which was completed within three days after the ethylene treatment (Figure 1A) at a similar rate to that of flower pedicel abscission without ethylene treatment (Figure 1B) RNA collected during petiole and pedi-cel abscission allowed us to compare transcriptomic changes in the tomato LAZ and FAZ respectively through the use of a customized AZ-specific microarray chip The chip included transcripts identified using NGS of RNA isolated from tomato AZs at various time points during organ abscission as well as transcripts from the Solanaceae genomics network and Nation-al Center for Biotechnology Information databases [194] Here we present for the first time results from these microarray anal-yses describing the changes in expression of auxin-related genes (Figures 2-6) Generally there is a high similarity in the abscis-sion process of tomato leaves and flowers with a few excep-tions Most of the auxin-related genes are expressed in both AZs but some members of different gene families are ex-pressed specifically in the FAZ or the LAZ The results presented in Figure 2 show that most genes encod-ing auxin influx (LAX family) and auxin efflux (PIN family) carriers were rapidly down-regulated in both AZs following IAA depletion thereby supporting the requirement for auxin for activation of its transport carriers The few exceptions were related to genes that have very low expression levels such as LAX45 and PIN28 in the FAZ and PIN3 in the LAZ The down-regulation of PIN5 and PILS family members in the two AZs provides the first indication that the regulation of intracel-lular auxin accumulation in the ER expected to control auxin availability for auxin signaling in the nuclei of AZ cells may be important for abscission regulation at the cellular level Two PILS were up-regulated in the FAZ and one in the LAZ fol-lowing flower removal or leaf deblading respectively (Figure 2) further supporting the role of this novel auxin carrier family in regulating auxin homeostasis [41] The REV TF was quickly down-regulated in the FAZ and LAZ after abscission induc-tion suggesting that there is a regulatory mechanism for de-creasing PIN expression and function in both AZs as a result of

Figure 1 Effect of leaf deblading and ethylene treatment (A) and flower removal (B) on the kinetics of petiole and pedicel abscission respective-ly in tomato explants The debladed-leaf explants held in vials with water were prepared as previously described for the flower explants [72] and exposed to ethylene (10 ppm for 24 h) The percentage of accumu-lated pedicel or petiole abscission was monitored at various time inter-vals following organ removal The results are means of four replicates (n=30 explants) plusmn SE

8

Meir et al Stewart Postharvest Review 2015 22

Figure 2 Microarray expression profiles of tomato auxin transport-related genes in the FAZ and LAZ at various time points following abscission induc-tion The assay included family members of the auxin influx carriers (AUXLAX) auxin efflux carriers (PINs PILS) genes and an auxin-related TF gene (REV) Samples were taken from the FAZ at 0 2 4 8 and 14 h after flower removal and from the LAZ at 0 24 48 72 and 96 h after leaf deblading and ethylene treatment as detailed in the legend of Figure 1 Gene expression levels are indicated with the colored code bars ranging from 0 (light blue) to 100 (dark blue) Expression levels are based on the percentage of change of the average intensity values of the replicated samples for each time point The numbers indicated in the first box of each sample represent average intensities values for all replicates at 0 h Corresponding Solanum lyco-persicum (Solyc) ID and gene names are presented in the left and right sides respectively

Figure 3 Microarray expression profiles of tomato IAA-related genes associated with IAA conjugation in the FAZ and LAZ at various time points follow-ing abscission induction The assay included members of the GH3 and ILRs gene families All other details are as described in the legend of Figure 2

9

Meir et al Stewart Postharvest Review 2015 22

Figure 4 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the AuxIAA gene family All other details are as described in the legend of Figure 2

Figure 5 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the ARF gene family All other details are as described in the legend of Figure 2

10

Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

11

Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

8

Meir et al Stewart Postharvest Review 2015 22

Figure 2 Microarray expression profiles of tomato auxin transport-related genes in the FAZ and LAZ at various time points following abscission induc-tion The assay included family members of the auxin influx carriers (AUXLAX) auxin efflux carriers (PINs PILS) genes and an auxin-related TF gene (REV) Samples were taken from the FAZ at 0 2 4 8 and 14 h after flower removal and from the LAZ at 0 24 48 72 and 96 h after leaf deblading and ethylene treatment as detailed in the legend of Figure 1 Gene expression levels are indicated with the colored code bars ranging from 0 (light blue) to 100 (dark blue) Expression levels are based on the percentage of change of the average intensity values of the replicated samples for each time point The numbers indicated in the first box of each sample represent average intensities values for all replicates at 0 h Corresponding Solanum lyco-persicum (Solyc) ID and gene names are presented in the left and right sides respectively

Figure 3 Microarray expression profiles of tomato IAA-related genes associated with IAA conjugation in the FAZ and LAZ at various time points follow-ing abscission induction The assay included members of the GH3 and ILRs gene families All other details are as described in the legend of Figure 2

9

Meir et al Stewart Postharvest Review 2015 22

Figure 4 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the AuxIAA gene family All other details are as described in the legend of Figure 2

Figure 5 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the ARF gene family All other details are as described in the legend of Figure 2

10

Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

11

Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

9

Meir et al Stewart Postharvest Review 2015 22

Figure 4 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the AuxIAA gene family All other details are as described in the legend of Figure 2

Figure 5 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the ARF gene family All other details are as described in the legend of Figure 2

10

Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

11

Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

10

Meir et al Stewart Postharvest Review 2015 22

auxin depletion Interestingly it was recently reported that over-expression of tomato SlREV TF caused the transgenic plants to produce ectopic flowers from the pedicel AZ [195] The highly expressed GH3 genes significantly decreased after leaf deblading (Figure 3) further confirming that GH3 is an auxin-induced gene [33 35 37] On the other hand most GH3 genes in the FAZ were up-regulated confirming that in-creased IAA conjugation is involved in the process of auxin depletion [104 105] To the best of our knowledge there are no reports regarding changes in GH3 gene expression in the AZs after auxin depletion In contrast to our earlier report de-scribing up-regulation of ILR genes in the FAZ and the proxi-mal NAZ [74] in the current experiment the ILR genes were down-regulated in both the FAZ and LAZ after removing the auxin sources (Figure 3) Further investigation is required to understand the significance of these changes All the AuxIAA genes that were expressed in the FAZ were also expressed in the LAZ (Figure 4) IAA14 IAA9 IAA6 and IAA 26 were the most highly expressed genes in both AZs All IAA-related genes were down-regulated in both AZs within 2 h following abscission induction except for IAA6 IAA14 and IAA26 whose expression in the FAZ was reduced after 8

h These results are in agreement with previous reports regard-ing the timing of changes in the expression of IAA-responsive genes in the FAZ following flower removal [74 191] Six ARF genes were exclusively expressed in the FAZ and three in the LAZ and all ARF genes were down-regulated after IAA depletion in both the FAZ and LAZ (Figure 5) These results cannot be compared to previously reported results for ARF genes in the tomato FAZ [70] because in the previous system ethylene was applied after explant preparation Out of 38 Small Auxin Upregulated RNA (SAUR) genes 19 genes were exclusively expressed in the FAZ and four in the LAZ and a high variability in the changes of the expression pattern of SAUR genes was found (Figure 6) There are no previous re-ports on the expression of SAUR genes in the AZ The present research compared for the first time the FAZ with the LAZ showing similar changes in auxin-related genes in both AZs Additionally the results show that AuxIAA genes are the best markers for determining auxin depletion in the AZs and that the polar and intracellular auxin transport mecha-nisms are impaired after auxin depletion Several IAA-related genes were shown for the first time to be involved in the ab-scission process including PILS PIN5 REV and SAUR

Figure 6 Microarray expression profiles of tomato auxin-signaling genes in the FAZ and LAZ at various time points following abscission induction The assay included members of the SAUR gene family All other details are as described in the legend of Figure 2

11

Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

11

Meir et al Stewart Postharvest Review 2015 22

genes Taken together the new data expand the range of vari-ous IAA-related genes that are rapidly down-regulated in the tomato AZs following auxin depletion as a result of flower re-moval or leaf deblading Several recent articles further expand-ed the previously suggested model [74] for the sequence of events leading to pedicel abscission following artificial auxin depletion in tomato flowers The new data were based on func-tional analyses of few target genes including KD1 TPRP SlERF52 and LX whose silencing by virus-induced gene si-lencing antisense or RNAi delayed abscission Thus the early events following auxin depletion include down-regulation of IAA-related genes encoding PAT components auxin carriers KD1 and TPRP while the late events include up-regulation of ethylene-related genes such as SlERF52 These lead in turn to activation of cell wall hydrolyzing enzymes and PCD processes which coincided with the increased expression of genes encod-ing cell wall degrading enzymes and up-regulation of LX re-spectively All these events finally result in the execution of pedicel or petiole abscission and formation of a protective de-fense layer on the remaining tissue

Conclusions This review provides evidence that auxin depletion in the AZs is required for AZ cells to react to ethylene as an abscission signal This is a general phenomenon that occurs in natural abscission in abscission induced by various stresses and in the artificial system of tomato organ abscission after removal of the auxin source The stress-induced auxin depletion is mediated by three main intermediate modulators ethylene ROS and carbo-hydrate starvation Auxin depletion can result from decreased IAA biosynthesis and transport accelerated ATA and in-creased oxidative IAA catabolism and conjugation More target genes for functional analysis were elucidated by transcriptomic studies using NGS or microarray in different abscission sys-tems The artificial auxin depletion system in tomato was found to be a very useful system for elucidating the sequence of events leading to organ abscission

Direction for future research In this stage of abscission research with multiple target genes that might affect the abscission process five future research directions are necessary 1) performing transcriptomic analyses with more time points following abscission induction by auxin depletion similar to those performed for the tomato FAZ 2) analyzing the full transcriptome of the tomato LAZ for eluci-dating the sequence of events as in the tomato FAZ 3) contin-uing the functional analyses of more target genes found in vari-ous abscission systems 4) evaluating the importance of PIN5 and PILS genes family members for auxin signaling in the nu-cleus of the AZ cells for abscission regulation at the cellular level 5) establishing a proteomic system to elucidate the chang-es in proteins occurring in the AZs following auxin depletion and examine if these changes are related to the changes of their gene expression

Acknowledgements Contribution No 72315 from the ARO The Volcani Center Bet Dagan Israel The authors would like to acknowledge the support from the United StatesndashIsrael Binational Agricultural Research and Development Fund (BARD) [grant no US-4571-12C] and the Chief Scientist of the Israeli Ministry of Agricul-

ture Fund [grant no 203-0898-10] SS would like to thank the Indian Council of Agricultural Research for providing him with an International Fellowship (ICAR-IF) as partial support of his PhD studies

References Papers of interest have been highlighted as Marginal importance Essential reading 1 Jackson MB and Osborne DJ Ethylene the natural regulator of leaf abscis-

sion Nature 1970 2251019ndash1022 2 Addicott FT Abscission London University of California Press Ltd 1982 3 Sexton R and Roberts JA Cell biology of abscission Annual Review of Plant

Physiology 1982 33133-162 4 Osborne DJ Abscission Critical Reviews in Plant Science 1989 8103ndash129 5 Roberts JA Whitelaw CA Gonzalez-Carranza ZH and McManus MT Cell

separation processes in plants Models mechanisms and manipulation Annals of Botany 2000 86 223ndash235

6 Taylor JE and Whitelaw CA 2001 Signals in abscission New Phytologist 2001 151 323-339

7 Patterson SE Cutting loose Abscission and dehiscence in Arabidopsis Plant Physiology 2001 126 494ndash500

8 Roberts JA Elliott KA and Gonzalez-Carranza ZH Abscission dehiscence and other cell separation processes Annual Review of Plant Biology 2002 53131ndash158 ME

9 Brown K Ethylene and abscission Physiologia Plantarum 2006 100 567ndash576 10 Leslie ME Lewis MW and Liljegren SJ Organ abscission In Plant Cell Sepa-

ration and Adhesion Edited by Roberts JA Gonzalez-Carranza ZH Oxford Blackwell Publishing Ltd 2007 pp 106ndash136

11 Lashbrook CC Functional genomic approaches to abscission Stewart Post-harvest Review 2009 14

12 Estornell LH Agustί J Merelo P Talόn M and Tadeo FR Elucidating mechanisms underlying organ abscission Plant Science 2013 199ndash20048ndash60

A Very good review article on abscission 13 Bangerth F Dominance among fruitssinks and the search for a correlative

signal Physiologia Plantarum 1989 76 608-614 14 Dhanalakshmi R Prasad TG and Udayakumar M Is auxin a diffusible signal

mediating abscission of recessive sinks Plant Science 2003 164689-696 15 Blanusa T Else MA Atkinson CJ and Davies WJ The regulation of sweet

cherry fruit abscission by polar auxin transport Plant Growth Regulation 2005 45189ndash198

16 Dal Cin V Velasco R and Ramina A Dominance induction of fruitlet shed-ding in Malus times domestica (L Borkh) Molecular changes associated with polar auxin transport BioMed Central Plant Biology 2009 9139

17 Celton J-M Dheilly E Guillou M-C Simonneau F Juchaux M Costes E Laurens F and Renou J-P Additional amphivasal bundles in pedicel pith exac-erbate central fruit dominance and induce self-thinning of lateral fruitlets in apple Plant Physiology 2014 1641930ndash1951

An excellent updated report regarding abscission of lateral apple fruitlets The report includes histological and transcriptomic data which permitted a detailed characterization of AZ development and identification of key genes involved in this process 18 Else MA Stankiewicz-Davies AP Crisp CM and Atkinson CJ The role of

polar auxin transport through pedicels of Prunus avium L in relation to fruit development and retention Journal of Experimental Botany 2004 552099ndash2109

19 Kuumlhn N Abello C Godoy F Delrot S and Arce-Johnson P Differential behavior within a grapevine cluster Decreased ethylene-related gene expres-sion dependent on auxin transport is correlated with low abscission of first developed berries PLoS ONE 2014 9 e111258 doi101371journalpone 0111258

This article provides an excellent example for decreased PAT in the late developed barriers within a grapevine cluster 20 Zhang J-Z Zhao K Ai X-Y and Hu C-G Involvements of PCD and changes

in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis) BioMed Central Genomics 2014 15892

21 Dal Cin V Boschetti A Dorigoni A and Ramina A Benzylaminopurine appli-cation on two different apple cultivars (Malus domestica) displays new and unex-pected fruitlet abscission features Annals of Botany 2007 99 1195ndash1202

22 Hartmond H Yuan R Burns JK Grant A and Kender WJ Citrus fruit abscis-sion induced by methyl jasomonate Journal of the American Society for Horti-cultural Science 2000 125 547ndash552

23 Sipes D and Einset J Cytokinin stimulation of abscission in lemon pistil ex-

12

Meir et al Stewart Postharvest Review 2015 22

plants Journal of Plant Growth Regulators 1983 273ndash80 24 Aneja M Gianfagna T and Ng E The roles of abscisic acid and ethylene in the

abscission and senescence of cocoa flowers Plant Growth Regulation 1999 27149ndash155

25 Kim J Dotson B Rey C Lindsey J Bleecker AB Brad M Binder BM and Patterson SE New clothes for the jasmonic acid receptor COI1 Delayed abscission meristem arrest and apical dominance PLoS ONE 2013 8e60505

26 Kim J Four shades of detachment Regulation of floral organ abscission Plant S i g n a l i n g a n d B e h a v i o r 2 0 1 4 9 e 9 7 6 1 5 4 D O I 104161155923242014976154

27 Stutte GW and Gage J Gibberellin inhibits fruit abscission following seed abortion in peach Journal of The American Society for Horticultural Science 1990 115107ndash110

28 Ben-Cheikh W Perez-Botella J Tadeo FR Talon M and Primo-Millo E Pollination increases gibberellin levels in developing ovaries of seeded varieties of citrus Plant Physiology 1997 114557ndash564

29 Khripach VA Zhabinskii VN and de Groot AE Brassinosteroids a New Class of Plant Hormones London Academic Press 1999

30 Aziz A Spermidine and related-metabolic inhibitors modulate sugar and ami-no acid levels in Vitis vinifera L Possible relationships with initial fruitlet abscission Journal of Experimental Botany 2003 54355ndash363

31 Parra-Lobato MC and Gomez-Jimenez MC Polyamine-induced modulation of genes involved in ethylene biosynthesis and signaling pathways and nitric oxide production during olive mature fruit abscission Journal of Experimental Botany 2011 624447ndash4465

32 Vanneste S and Friml J Auxin A trigger for change in plant development Cell 2009 1361005ndash1016

33 Tromas A and Perrot-Rechenmann C Recent progress in auxin biology Comptes Rendus Biologies 2010 333 297ndash306

34 Zhao Y Auxin biosynthesis and its role in plant development Annual Reviews of Plant Biology 2010 6149ndash64

35 Ludwig-Muumlller J Auxin conjugates Their role for plant development and in the evolution of land plants Journal of Experimental Botany 2011 621757ndash1773

This article provides an excellent review on cellular auxin homeostasis 36 Mano Y and Nemoto K The pathway of auxin biosynthesis in plants Journal

of Experimental Botany 2012 632853ndash2872 37 Rosquete MR Barbez E and Kleine-Vehn J Cellular auxin homeostasis Gate-

keeping is housekeeping Molecular Plant 2012 5772ndash786 38 Swarup R and Peacuteret B AUXLAX family of auxin influx carriers An over-

view Frontiers in Plant Science 2012 3225 doi 103389fpls201200225 39 Guilfoyle TJ and Hagen G Getting a grasp on domain IIIIV responsible for

Auxin Response FactorndashIAA protein interactions Plant Science 2012 19082-88

40 Audran-Delalande C Bassa C Mila I Farid Regad F Zouine M and Bouzayen M Genome-wide identification functional analysis and expression pofiling of the AuxIAA gene family in tomato Plant and Cell Physiology 2012 53659ndash672

41 Barbez E Kubes M Rolcik J Beziat C Pencık A Wang B Rosquete MR Zhu J Dobrev PI Lee Y Zazımalova E Jan Petrasek J Markus Geisler M J Friml and Kleine-Vehn J A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants Nature 2012 485119-124

The first report describing the discovery of the intracellular auxin carrier family ndash PILS 42 Tivendale ND Ross JJ and Cohen JD The shifting paradigms of auxin bio-

synthesis Trends in Plant Science 2014 1944-51 An updated review showing the recent findings in auxin biosynthesis 43 Salehin M Bagchi R and Estelle M SCFTIR1AFB-based auxin perception

Mechanism and role in plant growth and development The Plant Cell 2015 279-19

44 Korasick DA Westfall CS Lee SG Nanao MH Dumas R Hagen G Guil-foyle TJ Jez JM and Strader LC Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression Proceedings of The National Academy of Science USA 2014 111 5427ndash5432

45 Kramer EM and Ackelsberg EM Auxin metabolism rates and implications for plants Frontiers in Plant Science 2015 6150 doi 103389fpls201500150

46 Adamowski M and Friml J PIN-dependent auxin transport Action regula-tion and evolution The Plant Cell 2015 2720-32

An excellent updated review on auxin transport 47 Guilfoyle TJ The PB1 domain in Auxin Response Factor and AuxIAA

proteins A versatile protein interaction module in the auxin response The Plant Cell 2015 2733-43

48 Ljung K Auxin metabolism and homeostasis during plant development Development 2013 140943-950

49 Roumeliotis E Bjorn Kloosterman B Oortwijn M Visser RGF and Bachem CWB The PIN family of proteins in potato and their putative role in tuberiza-tion Frontiers in Plant Science 2013 4524 doi 103389fpls201300524

50 Sachs T Cell polarity and tissue patterning in plants Development 1991 1133ndash93

51 Sauer M Balla J Luschnig C Wisniewska J Reinoumlhl V Friml J and Benkovaacute E Canalization of auxin flow by AuxIAA-ARF-dependent feedback regula-tion of PIN polarity Genes and Development 2006 202902-2911

52 Lia C-J and Bangerth KF Autoinhibition of indoleacetic acid transport in the shoots of two-branched pea (Pisum sativum) plants and its relationship to correl-ative dominance Physiologia Plantarum 1990 106415-420

53 Schroder M Linka H and Bangerth KF Correlative polar auxin transport to explain the thinning mode of action of benzyladenine on apple Scientia Horti-culturae 2013 15384-92

54 Ongaro V and Leyser O Hormonal control of shoot branching Journal of Experimental Botany 2008 5967ndash74

55 Shinohara N Taylor C and Leyser O Strigolactone can promote or inhibit shoot branching by triggering rapid depletion of the auxin efflux protein PIN1 from the plasma membrane PLoS Biology 2013 11 e1001474

56 Huang T Harrar Y Lin C Reinhart B Newell NR Talavera-Rauh F Samuel A Hokin SA Barton MK and Kerstettera RA Arabidopsis KANADI1 acts as a transcriptional repressor by interacting with a specific cis-element and regu-lates auxin biosynthesis transport and signaling in opposition to HD-ZIPIII factors The Plant Cell 2014 26246-262

57 Porth I Klaacutepšte J McKown AD La Mantia J Hamelin RC Skyba O Unda F Friedmann MC Cronk QBC Ehlting J Guy RD Mansfield SD El-Kassaby YA and Douglas CJ Extensive functional pleiotropy of REVOLUTA sub-stantiated through forward genetics Plant Physiology 2014 164 548-554

Important article describing the role of REVOLUTA protein in auxin transport and signaling 58 Robischon M Du J Eriko Miura E and Groover A The populus class III HD

-ZIP pop REVOLUTA influences cambium initiation and patterning of woody stems Plant Physiology 2011 1551214ndash1225

59 Zhong R and Ye Z-H Alteration of auxin polar transport in the Arabidopsis ifl1 mutants Plant Physiology 2001 126549ndash563

60 Ilegems M Douet V Meylan-Bettex M Uyttewaal M Brand L Bowman JL and Stieger PA Interplay of auxin KANADI and Class III HD-ZIP transcrip-tion factors in vascular tissue formation Development 2010 137975-984

61 Izhakia A and Bowman JL KANADI and class III HD-Zip gene families regulate embryo patterning and modulate auxin flow during embryogenesis in Arabidopsis The Plant Cell 2007 19495ndash508

62 Merelo P Yakun Xie Y Brand L Ott F Weigel D Bowman JL Heisler MG and Wenkel S Genome-wide identification of KANADI1 target genes PLoS ONE 2013 8e77341

63 Abebie B Lers A Philosoph-Hadas S Goren R Riov J and Meir S Differen-tial effects of NAA and 24-D in reducing floret abscission in Cestrum (Cestrum elegans) cut flowers are associated with their differential activation of AuxIAA homologous genes Annals of Botany 2008 101 249ndash259

64 Meir S Hunter DA Chen J-C Halaly V and Reid MS Molecular changes occurring during acquisition of abscission competence following auxin deple-tion in Mirabilis jalapa Plant Physiology 2006 141 1604ndash1616

65 Liu X Hegeman AD Gardner G and Cohen JD Protocol High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples Plant Methods 2012 831ndash48

66 Cohen JD Bausher MG Bialek K Buta JG Gocal GFW Janzen LM Pharis RP Reed AN and Slovin J Comparison of a commercial ELISA assay for indole-3-acetic acid at several stages of purification and analysis by gas chroma-tography-selected ion monitoring-mass spectrometry using a 13C6-labeled internal standard Plant Physiology 1987 84 982-986

67 Michaeli R Riov J Philosoph-Hadas S and Meir S Chilling-induced leaf ab-scission of Ixora coccinea plants II Alteration of auxin economy by oxidative stress Physiologia Plantarum 1999 107174ndash180

68 Michaeli R Philosoph-Hadas S Riov J Shahak Y Ratner K and Meir S Chilling-induced leaf abscission of Ixora coccinea plants III Enhancement by high light via increased oxidative processes Physiologia Plantarum 2001 113338ndash345

69 Wang Y Li T Hanyong Meng H and Sun X Optimal and spatial analysis of hormones degrading enzymes and isozyme profiles in tomato pedicel explants during ethylene-induced abscission Plant Growth Regulation 2005 4697ndash107

An important article describing the role of ARF during tomato flower abscission The article includes determination of IAA levels by analytical measurements and by using the auxin reporter P5GUS 70 Guan X Tao Xu T Gao S Qi M Wang Y Liu X and Li T Temporal and

spatial distribution of Auxin Response Factor genes during tomato flower abscis-sion Journal of Plant Growth Regulation 2014 33317-327

71 Ma C Meir S Xiao L Tong J Liu Q Reid MS and Jiang C-Z A KNOT-TED1-LIKE HOMEOBOX protein regulates abscission in tomato by modu-lating the auxin pathway Plant Physiology 2015 167844ndash853

An important article elucidating the function of KD1 in auxin transport during flower and leaf abscission The article also includes a visible demonstration of the

13

Meir et al Stewart Postharvest Review 2015 22

auxin gradient in the flower pedicel and the flower AZ 72 Guinn G Brummett DL Changes in free and conjugated indole 3-acetic acid

and abscisic acid in young cotton fruits and their abscission zones in relation to fruit retention during and after moisture stress Plant Physiology 1988 8628-31

73 Cai S and Lashbrook CC Stamen abscission zone transcriptome profiling reveals new candidates for abscission control Enhanced retention of floral organs in transgenic plants overexpressing Arabidopsis ZINC FINGER PRO-TEIN2 Plant Physiology 2008 146 1305ndash1321

74 Meir S Philosoph-Hadas S Sundaresan S Selvaraj KSV Burd S Ophir R Kochanek B Reid MS Jiang C-Z and Lers A Microarray analysis of the ab-scission-related transcriptome in tomato flower abscission zone in response to auxin depletion Plant Physiology 2010 1541929-1956

An intensive study showing changes in gene expression in the AZ and NAZ during various time-points following auxin depletion by flower removal and in response to 1-MCP and IAA application This study served as the basis for the suggested detailed sequence of events operating from abscission initiation until the abscission execution 75 Agusti J Merelo P Cercoacutes M Tadeo FR and Taloacuten M Comparative transcrip-

tional survey between laser-microdissected cells from laminar abscission zone and petiolar tissue during ethylene-promoted abscission in citrus leaves Bio-Med Central Plant Biology 2009 9127 doi1011861471-2229-9-127

76 Nakano T Fujisawa M Shima Y and Ito Y Expression profiling of tomato pre-abscission pedicels provides insights into abscission zone properties in-cluding competence to respond to abscission signals BioMed Central Plant Biology 2013 1340 doi1011861471-2229-13-40

This study reveals at anthesis a region-specific gene expression in the tomato flower AZ and NAZ (proximal and distal regions to AZ) 77 Wang X Liu D Li A Sun X Zhang R Wu L Liang Y and Mao L Transcrip-

tome analysis of tomato flower pedicel tissues reveals abscission zone-specific modulation of key meristem activity genes PLoS ONE 2013 8e55238

78 Gil-Amado JA and Gomez-Jimenez MC Transcriptome analysis of mature fruit abscission control in olive Plant and Cell Physiology 2013 54244-269

This study describes a transcriptomic analysis in the AZ during mature olive fruit abscission 79 Corbacho J Romojaro F Pech J-C Latcheacute and Gomez-Jimenez MC Tran-

scriptomic events involved in melon mature-fruit abscission comprise the sequential induction of cell-wall degrading genes coupled to a stimulation of endo and exocytosis PLoS ONE 2013 8(3)e58363

A description of a transcriptomic analysis in the AZ during abscission of mature melon fruit 80 Pattison RJ and Catala C Evaluating auxin distribution in tomato (Solanum

lycopersicum) through an analysis of the PIN and AUXLAX gene families The Plant Journal 2012 70585ndash598

81 Basu MM Gonzaacutelez-Carranza ZH Azam-Ali S Tang S Shahid AA and Roberts JA The manipulation of auxin in the abscission zone cells of Ara-bidopsis flowers reveals that indoleacetic acid signaling is a prerequisite for organ shedding Plant Physiology 2013 16296-106

A very important article demonstrating the effects of genetic manipulation of auxin levels on petal abscission 82 Kavita P and Burma PK A comparative analysis of green fluorescent protein

and β-glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters Journal of Biosciences 2008 33337ndash343

83 Brunoud G Wells DM Oliva M Larrieu A Mirabet V Burrow AH Beeck-man T Kepinski S Traas J Bennett MJ and Vernoux T A novel sensor to map auxin response and distribution at high spatio-temporal resolution Na-ture 2012 482103-108

84 Woltering EJ and Van Doorn WG Role of ethylene in senescence of petals -morphological and taxonomical relationships Journal of Experimental Botany 1988 391605-1616

85 Van Doorn WG and Woltering EJ Categories of petal senescence and abscis-sion A re-evaluation Annals of Botany 2001 87447-456

86 Borochov A and Woodson WR Physiology and biochemistry of flower petal senescence Horticultural Reviews 1989 1115-43

87 Rogers HJ From models to ornamentals How is flower senescence regulated Plant Molecular Biology 2013 82563ndash574

88 Van Doorn WG and Stead AD Abscission of flowers and floral parts Journal of Experimental Botany 1997 309821-837

89 Haumlnisch ten Cate CHH Berghoef J Van der Hoorn AMH and Bruinsma J Hormonal regulation of pedicel abscission in Begonia flower buds Physiologia Plantarum 1975 33280-284

90 Arrom L and Munneacute-Bosch S Hormonal changes during flower development in floral tissues of Lilium Planta 2012 236343ndash354

91 Lombardi L Arrom L Mariotti L Battelli R Picciarelli P Kille P Stead T Munneacute-Bosch S and Rogers HJ Auxin involvement in tepal senescence and abscission in Lilium a tale of two lilies Journal of Experimental Botany 2015

66945ndash956 The authors demonstrate auxin depletion as an initial event of natural abscission of tepals in lily flowers 92 Rungruchkanont K Ketsa S Chatchawankanphanich O and Van Doorn WG

Endogenous auxin regulates the sensitivity of Dendrobium (cv Miss Teen) flower pedicel abscission to ethylene Functional Plant Biology 2007 34885ndash894

93 Rungruchkanont K Auxins and cytokinins regulate abscission and physiologi-cal changes of flowers in cut Dendrobium cv Eiskul inflorescences Science Journal Ubonratchathani University 2011 21-11

94 Gilbert DA and Sink KC The effect of exogenous growth regulators on keeping quality of poinsettia Journal of the American Society for Horticultural Science 1970 95784-787

95 Gilbert DA and Sink KC Regulation of endogenous indoleacetic acid and keeping quality of poinsettia Journal of The American Society for Horticultur-al Science 1971 963-7

96 Okushima Y Mitina I Quach HL and Theologis A AUXIN RESPONSE FACTOR 2 (ARF2) A pleiotropic developmental regulator The Plant Journal 2005 4329ndash46

97 Ellis CM Nagpal P Young JC Hagen G Guilfoyle TJ and Reed JW AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission in Arabidopsis thaliana Development 2005 1324563-4574

98 Noodeacuten LD Abscisic acid auxin and other regulators of senescence In Senescence and Aging in Plants Edited by Noodeacuten LD and Leopold AC San Diego Academic Press 1988 pp 329-367

99 Gan S The hormonal regulation of senescence In Plant Hormones Edited by Davies PJ Dordrecht The Netherlands Kluwer Academic Publishers 2004 pp 561-580

100 Schippers JHM Jing H-C Hill J and Dijkwel PP Developmental and hormo-nal control of leaf senescence In Senescence Processes in Plants Edited by Gan S Oxford UK Blackwell Publishing Ltd 2007 pp 149-170

101 Dela Fuente RK and Leopold AC Senescence processes in leaf abscission Plant Physiology 1968 431496-1502

102 Gillaspy G Ben-David H and Gruissem W Fruits A developmental perspec-tive The Plant Cell 1993 51439-1451

103 Kumar R Khurana A and Sharma AK Role of plant hormones and their interplay in development and ripening of fleshy fruits Journal of Experimental Botany 2014 654561ndash4575

104 Kumar R Agarwal P Tyagi AK and Sharma AK Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive GH3 genes during development and response to different stimuli in tomato (Solanum lycopersicum) Molecular Genetics and Genomics 2012 287221ndash235

105 Boumlttcher C Keyzers RA Boss PK and Davies C Sequestration of auxin by the indole-3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L) and the proposed role of auxin conjugation during ripening Journal of Experi-mental Botany 2010 61 3615ndash3625

106 Gouthu S and Delu LG Timing of ripening initiation in grape berries and its relationship to seed content and pericarp auxin levels BioMedical Central Plant Biology 2015 1546 DOI 101186s12870-015-0440-6

107 Tranbarger TJ Dussert S Joeumlt T Argout X Summo M Champion A Cros D Omore A Nouy B and Morcillo F Regulatory mechanisms underlying oil palm fruit mesocarp maturation ripening and functional specialization in lipid and carotenoid metabolism Plant Physiology 2011 156564ndash584

The authors showed dramatic decrease in levels of IAA and IAA-conjugates just prior to the natural abscission of oil palm fruit 108 Henderson J and Osborne DJ Inter-tissue signalling during the two-phase

abscission in oil palm fruit Journal of Experimental Botany 1994 45943-951 109 Trainotti L Tadiello A and Casadoro G The involvement of auxin in the

ripening of climacteric fruits comes of age The hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches Journal of Experimental Botany 2007 583299ndash3308

110 Torrigiani P Bressanin D Ruiz KB Tadiello A Trainotti L Claudio Bonghi C Ziosi V and Costa G Spermidine application to young developing peach fruits leads to a slowing down of ripening by impairing ripening-related ethylene and auxin metabolism and signaling Physiologia Plantarum 2012 146 86ndash98

111 Tatsuki M Nakajima N Fujii H Shimada T Nakano M Hayashi K-I Hayama H Yoshioka H and Nakamura Y Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L Batsch) Journal of Experimental Botany 2013 641049ndash1059

112 Vandenbussche F Smalle J Le J Joseacute N Saibo M De Paepe A Chaerle L Tietz O Smets R Laarhoven LJJ Harren FMJ Van Onckelen H Palme K Verbelen J-P and Van Der Straeten D The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin Plant Physiology 2003 1311228ndash1238

113 Stepanova AN Yun J Likhacheva AV and Alonso JM Multilevel interactions between ethylene and auxin in Arabidopsis roots The Plant Cell 2007 192169

14

Meir et al Stewart Postharvest Review 2015 22

ndash2185 114 Muday GK Rahman A and Binder BM Auxin and ethylene Collaborators or

competitors Trends in Plant Science 2012 17181-195 An excellent review describing auxin-ethylene crosstalk with examples of ethylene effects on auxin biosynthesis transport and signaling 115 Rahman A Auxin A regulator of cold stress response Physiologia Plantarum

2013 147 28ndash35 116 Vidoz ML Loreti E Mensuali A Alpi A and Perata P Hormonal interplay

during adventitious root formation in flooded tomato plants The Plant Journal 2010 63551ndash562

117 Beyer EM Jr and Morgan PW Abscission The role of ethylene modification of auxin transport Plant Physiology 1971 48 208-212

118 Morgan PW and Gausman HW Effect of ethylene on auxin transport Plant Physiology 1966 4145-52

119 Sanyal D and Bangerth F Stress-induced ethylene evolution and its possible relationship to auxin-transport cytokinin levels and flower bud induction in shoots of apple seedlings and bearing apple trees Plant Growth Regulation 1998 24127-134

120 Beyer EM and Morgan PW Effect of ethylene on the uptake distribution and metabolism of indoleacetic acid-1-14C and -2-14C and naphthaleneacetic acid-1-l4C Plant Physiology 1970 46157-162

121 Ernest LC and Valdovinos JG Regulation of auxin levels in Coleus blumei by ethylene Plant Physiology 1971 48402-406

122 Riov J and Goren R Effect of ethylene on auxin transport and metabolism in midrib sections in relation to leaf abscission of woody plants Plant Cell and Environment 1979 283-89

123 Sawicki M Barka EA Cleacutement C Vaillant-Gaveau N and Jacquard C Crosstalk between environmental stresses and plant metabolism during repro-ductive organ abscission Journal of Experimental Botany 2015 661707ndash1719

An excellent updated review which describes the effects of various environmental stresses on ethylene production carbohydrate levels and auxin depletion 124 Tognetti VB per Muumlhlenbock P and Van Breusegem F Stress homeostasis ndash

the redox and auxin perspective Plant Cell and Environment 2012 35321ndash333

An excellent review showing effects of environmental stresses on ROS-induced auxin depletion 125 Kazan K Auxin and the integration of environmental signals into plant root

development Annals of Botany 2013 1121655ndash1665 126 Yang SF and Hoffman NE Ethylene biosynthesis and its regulation in higher

plants Annual Review of Plant Physiology 1984 35155-89 127 Groen SC and Whiteman NK The evolution of ethylene signaling in plant

chemical ecology Journal of Chemical Ecology 2014 40700ndash716 128 Kazan K Diverse roles of Jasmonates and ethylene in abiotic stress tolerance

Trends in Plant Science 2015 20219-229 129 Agusti J Merelo P Cercόs M Tadeo FR and Talόn M Ethylene-induced

differential gene expression during abscission of citrus leaves Journal of Ex-perimental Botany 2008 592717ndash2733

130 Huberman M Riov J Aloni B and Goren R Role of ethylene biosynthesis and auxin content and transport in high temperature-induced abscission of pepper reproductive organs Journal of Plant Growth Regulation 1997 16129ndash135

131 Swarup R Parry G Graham N Allen T and Bennett M Auxin crosstalk Integration of signaling pathways to control plant development Plant Molecu-lar Biology 2002 49411ndash426

132 Sakamoto M Munemura I Tomita R and Kobayashi K Reactive oxygen species in leaf abscission signaling Plant Signaling and Behavior 2008 31014-1015

133 Sakamoto M Munemura I Tomita R and Kobayashi K Involvement of hydrogen peroxide in leaf abscission signaling revealed by analysis with an in vitro abscission system in Capsicum plants The Plant Journal 2008 5613ndash27

134 Michaeli R Philosoph-Hadas S Riov J and Meir S Chilling-induced leaf ab-scission of Ixora coccinea plants I Induction by oxidative stress via increased sensitivity to ethylene Physiologia Plantarum 1999 107166ndash173

135 Ueda J Morita Y and Kato J Promotive effect of C18-unsaturated fatty acid on the abscission of bean petiole explants Plant and Cell Physiology 1991 32983ndash987

136 Djanaguiraman M Devi DD Shanker AK Sheeba JA and Bangarusamy U The role of nitrophenol on delaying abscission of tomato flowers and fruits Food Agriculture and Environment 2004 2183-186

137 Djanaguiraman M Sheeba JA Devi DD Bangarusamy U and Prasad PVV Nitrophenolates spray can alter boll abscission rate in cotton through en-hanced peroxidase activity and increased ascorbate and phenolics levels Jour-nal of Plant Physiology 2010 1671ndash9

138 Krishnamurthy A and Rathinasabapathi B Oxidative stress tolerance in plants Novel interplay between auxin and reactive oxygen species signaling Plant Signaling and Behavior 2013 810 e25761

139 Peer WA Cheng Y and Murphy AS Evidence of oxidative attenuation of auxin signaling Journal of Experimental Botany 2013 642629ndash2639

An excellent review summarizing ROS effects on auxin depletion 140 Xia X-J Zhou Y-H Shi K Zhou J Christine H Foyer C-H and Yu J-Q

Interplay between reactive oxygen species and hormones in the control of plant development and stress tolerance Journal of Experimental Botany 2015 662839ndash2856

An excellent updated review describing ROS effects on auxin depletion 141 Roemer MG Hegele M Huong PT and Wuumlnsche JN Possible physiological

mechanisms of premature fruit drop in mango (Mangifera indica L) in northern Vietnam Acta Horticulturae 2011 903999-1006

142 Kuang J-F Wu J-Y Zhong H-Y Li C-Q Chen J-Y Lu W-J and Li J-G Car-bohydrate stress affecting fruitlet abscission and expression of genes related to auxin signal transduction pathway in litchi International Journal of Molecular Science 2012 1316084-16103

A very nice demonstration showing how carbohydrate starvation causes auxin depletion in the system of litchi fruit abscission 143 Botton A Eccher G Forcato C Ferrarini A Begheldo M Zermiani M Mos-

catello S Battistelli A Velasco R Ruperti B and Ramina A Signaling pathways mediating the induction of apple fruitlet abscission Plant Physiology 2011 155185ndash208

144 Zhirong Li Z Wakao S Fischer BB and Niyogi KK Sensing and responding to excess light Annual Review of Plant Biology 2009 60239ndash60

145 Aloni B Karni L Zaidman Z and Schaffer AA Changes of carbohydrates in pepper (Capsicum annuum L) flowers in relation to their abscission under differ-ent shading regimes Annals of Botany 1996 78163-168

146 Gago C and Monteiro JA NAA and STS effects on bract survival time carbo-hydrate content respiration rate and carbohydrate balance of potted Bougainvil-lea spectabilis Willd Postharvest Biology and Technology 2011 60235ndash243

147 Meir S Salim S Chernov Z and Philosoph-Hadas S Quality improvement of cut flowers and potted plants with postharvest treatments based on various cytokinins and auxins Acta Horticulturae 2007 755143-154

148 Al-Khalifah NS and Akderson PG The effect of auxin and ethylene on leaf abscission of Ficus benjamina In Biology and Biotechnology of the Plant Hor-mone Ethylene II Edited by Kanellis AK Chang C Klee H Bleecker AB Pech JC and Grieson D Dordrecht Kluwer Academic Publishers 1999 pp 255-265

149 Leόn P and Sheen J Sugar and hormone connections Trends in Plant Science 2003 8110-116

150 Meir S Philosoph-Hadas S Epstein E and Aharoni N Carbohydrates stimu-late ethylene production in tobacco leaf discs I Interaction with auxin and the relation to auxin metabolism Plant Physiology 1985 78131-138

151 Philosoph-Hadas S Meir S and Aharoni N Carbohydrates stimulate ethylene production in tobacco leaf discs II Sites of stimulation in the ethylene biosyn-thesis pathway Plant Physiology 1985 78139-143

152 Arru L Rognoni S Poggi A and Loreti E Effect of sugars on auxin-mediated LeEXPA2 gene expression Plant Growth Regulation 2008 5511ndash20

153 Meir S Riov J Philosoph-Hadas S and Aharoni N Carbohydrates stimulate ethylene production in tobacco leaf discs Ill Stimulation of enzymic hydrolysis of lndole-3-Acetyl-L-Alanine Plant Physiology 1989 901246-1248

154 Sairanen I Novaacutek O Pecircnčiacutek A Ikeda Y Jones B Sandberg G and Ljunga K Soluble carbohydrates regulate auxin biosynthesis via PIF proteins in Ara-bidopsis The Plant Cell 2012 244907ndash4916

155 Stewart JL Gee CW Sairanen I Ljung K and Nemhauser JL An endogenous carbon-sensing pathway triggers increased auxin flux and hypocotyl elongation Plant Physiology 2012 1602261ndash2270

156 Barbier F Peacuteron T Lecerf M Perez-Garcia M-D Barrieacutere Q Rolčίk J Boutet-Mercey S Citerne S Lemoine R Porcheron B Roman H Leduc N Gourri-erec JL Bertheloot J and Sakr S Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida Journal of Experimental Botany 2015 662569ndash2582

An important recent article describing the molecular mechanism of modulation of auxin biosynthesis and transport by endogenous sucrose 157 Bangerth F Abscission and thinning of young fruit and their regulation by

plant hormones and bioregulators Plant Growth Regulation 2000 3143ndash59 158 Davenport TL Morgan PW and Jordan WR Auxin transport as related to leaf

abscission during water stress in cotton Plant Physiology 1977 59554-557 159 Morgan PW Lordan WR Davenport TL and Durham JI Abscission respons-

es to moisture stress auxin transport inhibitors and ethephon Plant Physiolo-gy 1977 59710-712

160 Guinn G and Brummett DL Changes in free and conjugated indole 3-acetic acid and abscisic acid in young cotton fruits and their abscission zones in relation to fruit retention during and after moisture stress Plant Physiology 1988 8628-31

161 Guinn G Dunlap JR and Brummett DL Influence of water deficits on the abscisic acid and indole-3-acetic acid contents of cotton flower buds and flowers Plant Physiology 1990 931117-1120

162 Agusti J Gimeno J Merelo P Serrano R Cercόs M Conesa A Talόn M and Tadeo FR Early gene expression events in the laminar abscission zone of

15

Meir et al Stewart Postharvest Review 2015 22

abscission-promoted citrus leaves after a cycle of water stressrehydration Involvement of CitbHLH1 Journal of Experimental Botany 2012 636079ndash6091

163 Street NR Skogstroumlm O Andreas Sjoumldin A Tucker J Rodrίguez-Acosta M Nilsson P Jansson S and Taylor G The genetics and genomics of the drought response in Populus The Plant Journal 2006 48321ndash341

164 MacDonald MT and Lada RR Biophysical and hormonal changes linked to postharvest needle abscission in balsam fir Journal of Plant Growth Regula-tion 2014 33602ndash611

165 Roberts JA Schindler CB and Tucker GA Ethylene-promoted tomato flower abscission and the possible involvement of an inhibitor Planta 1984 160159-163

166 Tabuchi T Comparison on the development of abscission zones in the pedi-cels between two tomato cultivars Journal of the Japanese Society for Horti-cultural Science 1999 68939-999

167 Tabuchi T and Arai N Formation of the secondary cell division zone in toma-to pedicels at different fruit growing stages Journal of the Japanese Society for Horticultural Science 2000 69156-160

168 Szymkowiak EJ and Irish EE Interactions between jointless and wild type tomato tissues during development of the pedicel abscission zone and the inflorescence meristem Plant Cell 1999 11159ndash175

169 Mao L Begum D Chuang HW Budiman MA Szymkowiak EJ Irish EE and Wing RA JOINTLESS is a MADS-box gene controlling tomato flower ab-scission zone development Nature 2000 406910-913

170 Szymkowiak EJ and Irish EE JOINTLESS suppresses sympodial identity in inflorescence meristems of tomato Planta 2006 223646ndash658

171 Shalit A Rozman A Goldshmidt A Alvarez JP Bowman JL Eshed Y and Lifschitz E The flowering hormone florigen functions as a general systemic regulator of growth and termination Proceedings of the National Academy of Science USA 2009 1068392-8397

172 Nakano T Kimbara J Fujisawa M Kitagawa M Ihashi N Maeda H Kasumi T and Ito Y MACROCALYX and JOINTLESS interact in the transcriptional regulation of tomato fruit abscission zone development Plant Physiology 2012 158439-450

173 Liu D Wang D Qin Z Zhang D Yin L Wu L Colasanti J Li A and Mao L The SEPALLATA MADS-box protein SLMBP21 forms protein complexes with JOINTLESS and MACROCALYX as a transcription activator for devel-opment of the tomato flower abscission zone The Plant Journal 2014 77284-296

174 Kalaitzis P Koehler SM and Tucker ML Cloning of a tomato polygalac-turonase expressed in abscission Plant Molecular Biology 1995 28647ndash656

175 Del Campillo E and Bennett AB Pedicel break strength and cellulase gene expression during tomato flower abscission Plant Physiology 1996 111813-820

176 Kalaitzis P Solomos T and Tucker ML Three different polygalacturonases are expressed in tomato leaf and flower abscission each with a different temporal expression pattern Plant Physiology 1997 1131303ndash1308

177 Lashbrook CC Giovannoni JJ Hall BD Fisher RL and Bennett AB Trans-genic analysis of tomato endo-β-14-glucanase gene function Role of Cel1 in floral abscission The Plant Journal 1988 13303-310

178 Tucker GA Sexton R Del Campillo E and Lewis LN Bean abscission cellu-lase Characterisation of a cDNA clone and regulation of gene expression by ethylene and auxin Plant Physiology 1988 881257ndash1262

179 Gonzalez-Bosch C Del Campillo E and Bennett AB Immunodetection and characterization of tomato endo-beta-14-glucanase Cel1 protein in flower abscission zones Plant Physiology 1997 1141541ndash1546

180 Hong SB Sexton R and Tucker ML Analysis of gene promoters for two tomato polygalacturonases expressed in abscission zones and the stigma Plant Physiology 2000 123869ndash881

181 Tucker ML Whitelaw CA Lyssenko NN and Nath P Functional analysis of

regulatory elements in the gene promoter for an abscission-specific cellulase from bean and isolation expression and binding affinity of three TGA-type basic leucine zipper transcription factors Plant Physiology 2002 1301487-1496

182 Beno-Moualem D Gusev L Dvir O Pesis E Meir S and Lichter A The effects of ethylene methyl jasmonate and 1-MCP on abscission of cherry tomatoes from the bunch and expression of endo-14-[beta]-glucanases Plant Science 2004 167499ndash507

183 Jiang C-Z Lu F Imsabai W Meir S and Reid MS Silencing polygalacturonase expression inhibits tomato petiole abscission Journal of Experimental Botany 2008 59973-979

184 Qi M-F Xu T Chen W-Z and Li T-L Ultrastructural localization of polygalac-turonase in ethylene-stimulated abscission of tomato pedicel explants Hindawi Publishing Corporation Scientific World Journal 2014 Article ID 389896 httpdxdoiorg1011552014389896

185 Wright M and Osborne DJ Abscission in Phaseolus vulgaris the positional differentiation and ethylene-induced expansion growth of specialized cells Planta 1974 120163ndash170

186 Osborne DJ The ethylene regulation of cell growth in specific target tissues of plants In Plant Growth Substances Edited by Wareing PF New York Aca-demic Press 1982 pp 279-290

187 McManus MT Further examination of abscission zone cells as ethylene target cells in higher plants Annals of Botany 2008 101 285ndash292

188 Nakano T Fujisawa M Shima Y and and Ito Y The AP2ERF transcription factor SlERF52 functions in flower pedicel abscission in tomato Journal of Experimental Botany 2014 653111-3119

An important study describing the functional analysis of ERF52 in tomato flower AZ showing that it plays a pivotal role in transcriptional regulation in pedicel AZs at both pre-abscission and abscission stages 189 Meir S Sundaresan S Ma C Philosoph-Hadas S Riov J Lers A Kochanek B

Tucker M Reid MS and Jiang C-Z New Insights and approaches for elucidat-ing regulation of organ abscission Abstract and lecture in the 29th Internation-al Horticultural Congress (IHC2014) Brisbane Australia 2014 Abstract No 00625

190 Meir S Philosoph-Hadas S Sundaresan S Selvaraj KS Burd S Ophir R Kochanek B Reid MS Jiang C-Z and Lers A Identification of defense-related genes newly-associated with tomato flower abscission Plant Signaling amp Be-havior 2011 6590ndash593

191 Zuo X Xu T Qi M Lv S Jinhong Li Gao S and Li T Expression patterns of auxin-responsive genes during tomato flower pedicel abscission and potential effects of calcium Australian Journal of Botany 2012 6068ndash78

192 Lers A Sonego L Green PJ and Burd S Suppression of LX ribonuclease in tomato results in a delay of leaf senescence and abscission Plant Physiology 2006 142 710ndash721

193 Bar-Dror T Dermastia M Kladnik A Žnidarič MT Novak MP Meir S Burd S Philosoph-Hadas S Ori N Lilian Sonego L Dickman MB and Lers A Programmed cell death occurs asymmetrically during abscission in tomato The Plant Cell 2011 234146-4163

An important work showing the involvement of PCD in the final phase of tomato flower abscission the functional analysis of LX and an asymmetric distribution of PCD markers and abscission-related genes in the AZ 194 Sundaresan S Philosoph-Hadas S Riov J Kochanek B Mugasimangalam R

and Meir S Transcriptome analysis of the tomato flower and leaf abscission zones using a customized abscission zone microarray The 29th International Horticultural Congress (IHC2014) Brisbane Australia 2014 Abstract No 00751

195 Hu G Fan J Xian Z Huang W Lin D and Li Z Overexpression of SlREV alters the development of the flower pedicel abscission zone and fruit for-mation in tomato Plant Science 2014 22986ndash95