Genetic and biotechnological tools to identify breeding targets for improving postharvest quality and extending shelf life of peppers.

Improved postharvest storage is a major target for pepper-crop production. The three main components of postharvest improvement of pepper fruit are reducing water-loss rate, reducing chilling susceptibility, and increasing resistance to pathogens. To date, a small number of Quantitative Trait Locus (QTL) studies have been reported for reduced water loss and enhanced tolerance to chilling and anthracnose. More effort is needed to screen germplasm collections for accessions with improved postharvest traits. Molecular studies have enabled the identification of candidate genes conferring reduced susceptibility to chilling injury and pathogen infection in pepper fruit, and in related crops such as tomato - which may be implemented in pepper. Manipulation of the activity of these genes by genome editing can improve postharvest pepper quality.

Physiological genetic variation in tomato fruit chilling tolerance during postharvest storage.

Storage at low temperatures is a common practice to prolong postharvest life of fruit and vegetables with a minimal negative impact on human/environmental health. Storage at low temperatures, however, can be restricted due to produce susceptibility to non-freezing chilling temperatures, when injuries such as physiological disorders and decays may result in unmarketable produce. We have investigated tomato fruit response to postharvest chilling stress in a recombinant inbred line (RIL) population developed from a cross between a chilling-sensitive cultivated tomato (Solanum lycopersicum L.) breeding line and a chilling-tolerant inbred accession of the tomato wild species S. pimpinellifolium L. Screening of the fruit of 148 RILs under cold storage (1.5°C) indicated presence of significant variations in chilling tolerance, manifested by varying degrees of fruit injury. Two extremely contrasting groups of RILs were identified, chilling-tolerant and chilling-sensitive RILs. The RILs in the two groups were further investigated under chilling stress conditions, and several physiological parameters, including weight loss, chlorophyll fluorescence parameters Fv/Fm, and Performance Index (PI), were determined to be efficient markers for identifying response to chilling stress in postharvest fruit. The Fv/Fm values reflected the physiological damages endured by the fruit after cold storage, and PI was a sensitive marker for early changes in photosystem II function. These two parameters were early indicators of chilling response before occurrence of visible chilling injuries. Antioxidant activities and ascorbic acid content were significantly higher in the chilling-tolerant than the chilling-sensitive lines. Further, the expression of C-repeat/DREB binding factors (CBFs) genes swiftly changed within 1-hr of fruit exposure to the chilling temperature, and the SlCBF1 transcript level was generally higher in the chilling-tolerant than chilling-sensitive lines after 2-hr exposure to the low temperature. This research demonstrates the presence of potential genetic variation in fruit chilling tolerance in the tomato RIL population. Further investigation of the RIL population is underway to better understand the genetic, physiological, and biochemical mechanisms involved in postharvest fruit chilling tolerance in tomato.

Downregulation of GeBP-like α factor by MiR827 suggests their involvement in senescence and phosphate homeostasis.

BACKGROUND: Leaf senescence is a genetically controlled degenerative process intimately linked to phosphate homeostasis during plant development and responses to environmental conditions. Senescence is accelerated by phosphate deficiency, with recycling and mobilization of phosphate from senescing leaves serving as a major phosphate source for sink tissues. Previously, miR827 was shown to play a significant role in regulating phosphate homeostasis, and induction of its expression was also observed during Arabidopsis leaf senescence. However, whether shared mechanisms underlie potentially common regulatory roles of miR827 in both processes is not understood. Here, we dissect the regulatory machinery downstream of miR827. RESULTS: Overexpression or inhibited expression of miR827 led to an acceleration or delay in the progress of senescence, respectively. The transcriptional regulator GLABRA1 enhancer-binding protein (GeBP)-like (GPLα) gene was identified as a possible target of miR827. GPLα expression was elevated in miR827-suppressed lines and reduced in miR827-overexpressing lines. Furthermore, heterologous co-expression of pre-miR827 in tobacco leaves reduced GPLα transcript levels, but this effect was eliminated when pre-miR827 recognition sites in GPLα were mutated. GPLα expression is induced during senescence and its inhibition or overexpression resulted in senescence acceleration and inhibition, accordingly. Furthermore, GPLα expression was induced by phosphate deficiency, and overexpression of GPLα led to reduced expression of phosphate transporter 1 genes, lower leaf phosphate content, and related root morphology. The encoded GPLα protein was localized to the nucleus. CONCLUSIONS: We suggest that MiR827 and the transcription factor GPLα may be functionally involved in senescence and phosphate homeostasis, revealing a potential new role for miR827 and the function of the previously unstudied GPLα. The close interactions between senescence and phosphate homeostasis are further emphasized by the functional involvement of the two regulatory components, miR827 and GPLα, in both processes and the interactions between them.

Coding of Non-coding RNA: Insights Into the Regulatory Functions of Pri-MicroRNA-Encoded Peptides in Plants.

Peptides composed of a short chain of amino acids can play significant roles in plant growth, development, and stress responses. Most of these functional peptides are derived by either processing precursor proteins or direct translation of small open reading frames present in the genome and sometimes located in the untranslated region sequence of a messenger RNA. Generally, canonical peptides serve as local signal molecules mediating short- or long-distance intercellular communication. Also, they are commonly used as ligands perceived by an associated receptor, triggering cellular signaling transduction. In recent years, increasing pieces of evidence from studies in both plants and animals have revealed that peptides are also encoded by RNAs currently defined as non-coding RNAs (ncRNAs), including long ncRNAs, circular RNAs, and primary microRNAs. Primary microRNAs (miRNAs) have been reported to encode regulatory peptides in Arabidopsis, grapevine, soybean, and Medicago, called miRNA-encoded peptides (miPEPs). Remarkably, overexpression or exogenous applications of miPEPs specifically increase the expression level of their corresponding miRNAs by enhancing the transcription of the MIRNA (MIR) genes. Here, we first outline the current knowledge regarding the coding of putative ncRNAs. Notably, we review in detail the limited studies available regarding the translation of miPEPs and their relevant regulatory mechanisms. Furthermore, we discuss the potential cellular and molecular mechanisms in which miPEPs might be involved in plants and raise problems that needed to be solved.

Tomato T2 ribonuclease LE is involved in the response to pathogens.

T2 ribonucleases (RNases) are RNA-degrading enzymes that function in various cellular processes, mostly via RNA metabolism. T2 RNase-encoding genes have been identified in various organisms, from bacteria to mammals, and are most diverse in plants. The existence of T2 RNase genes in almost every organism suggests an important biological function that has been conserved through evolution. In plants, T2 RNases are suggested to be involved in phosphate scavenging and recycling, and are implicated in defence responses to pathogens. We investigated the function of the tomato T2 RNase LE, known to be induced by phosphate deficiency and wounding. The possible involvement of LE in pathogen responses was examined. Expression analysis showed LE induction during fungal infection and by stimuli known to be associated with pathogen inoculation, including oxalic acid and hydrogen peroxide. Analysis of LE-suppressed transgenic tomato lines revealed higher susceptibility to oxalic acid, a cell death-inducing factor, compared to the wild type. This elevated sensitivity of LE-suppressed lines was evidenced by visual signs of necrosis, and increased ion leakage and reactive oxygen species levels, indicating acceleration of cell death. Challenge of the LE-suppressed lines with the necrotrophic pathogen Botrytis cinerea resulted in accelerated development of disease symptoms compared to the wild type, associated with suppressed expression of pathogenesis-related marker genes. The results suggest a role for plant endogenous T2 RNases in antifungal activity.

Cell Death in Cells Overlying Lateral Root Primordia Facilitates Organ Growth in Arabidopsis.

Plant organ growth is widely accepted to be determined by cell division and cell expansion, but, unlike that in animals, the contribution of cell elimination has rarely been recognized. We investigated this paradigm during Arabidopsis lateral root formation, when the lateral root primordia (LRP) must traverse three overlying cell layers within the parent root. A subset of LRP-overlying cells displayed the induction of marker genes for cell types undergoing developmental cell death, and their cell death was detected by electron, confocal, and light sheet microscopy techniques. LRP growth was delayed in cell-death-deficient mutants lacking the positive cell death regulator ORESARA1/ANAC092 (ORE1). LRP growth was restored in ore1-2 knockout plants by genetically inducing cell elimination in cells overlying the LRP or by physically killing LRP-overlying cells by ablation with optical tweezers. Our results support that, in addition to previously discovered mechanisms, cell elimination contributes to regulating lateral root emergence.

Differential response to heat stress in outer and inner onion bulb scales.

The formation of brown protective skin in onion bulbs can be induced by rapid post-harvest heat treatment. Onions that are peeled to different depths and are exposed to heat stress show that only the outer scales form the dry brown skin, whereas the inner scales maintain high water content and do not change color. Our study demonstrates that browning of the outer scale during heat treatment is due to an enzymatic process that is associated with high levels of oxidation components, such as peroxidase and quercetin glucoside. De novo transcriptome analysis revealed differential molecular responses of the outer and inner scales to heat stress. Genes involved in lipid metabolism, oxidation pathways, and cell-wall modification were highly expressed in the outer scale during heating. Defense response-related genes such as those encoding heat-shock proteins, antioxidative stress defense, or production of osmoprotectant metabolites were mostly induced in the inner scale in response to heat exposure. These transcriptomic data led to a conceptual model that suggests sequential processes for the development of browning and desiccation of the outer scale versus processes associated with defense response and heat tolerance in the inner scales.

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance.

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.

Stronger sink demand for metabolites supports dominance of the apical bud in etiolated growth.

The potato tuber is a swollen underground stem that can sprout under dark conditions. Sprouting initiates in the tuber apical bud (AP), while lateral buds (LTs) are repressed by apical dominance (AD). Under conditions of lost AD, removal of tuber LTs showed that they partially inhibit AP growth only at the AD stage. Detached buds were inhibited by exogenous application of naphthaleneacetic acid (NAA), whereas 6-benzyladenine (6-BA) and gibberellic acid (GA3) induced bud burst and elongation, respectively. NAA, applied after 6-BA or GA3, nullified the latters' growth-stimulating effect in both the AP and LTs. GA3 applied to the fifth-position LT was transported mainly to the tuber's AP. GA3 treatment also resulted in increased indole-3-acetic acid (IAA) concentration and cis-zeatin O-glucoside in the AP. In a tuber tissue strip that included two or three buds connected by the peripheral vascular system, treatment of a LT with GA3 affected only the AP side of the strip, suggesting that the AP is the strongest sink for GA3, which induces its etiolated elongation. Dipping etiolated sprouts in labeled GA3 showed specific accumulation of the signal in the AP. Transcriptome analysis of GA3's effect showed that genes related to the cell cycle, cell proliferation, and hormone transport are up-regulated in the AP as compared to the LT. Sink demand for metabolites is suggested to support AD in etiolated stem growth by inducing differential gene expression in the AP.

Cellular and Molecular Changes Associated with Onion Skin Formation Suggest Involvement of Programmed Cell Death.

Skin formation of onion (Allium cepa L.) bulb involves scale desiccation accompanied by scale senescence, resulting in cell death and tissue browning. Understanding the mechanism of skin formation is essential to improving onion skin and bulb qualities. Although onion skin plays a crucial role in postharvest onion storage and shelf life, its formation is poorly understood. We investigated the mode of cell death in the outermost scales that are destined to form the onion skin. Surprisingly, fluorescein diacetate staining and scanning electron microscopy indicated that the outer scale desiccates from the inside out. This striking observation suggests that cell death in the outer scales, during skin formation, is an internal and organized process that does not derive only from air desiccation. DNA fragmentation, a known hallmark of programmed cell death (PCD), was revealed in the outer scales and gradually decreased toward the inner scales of the bulb. Transmission electron microscopy further revealed PCD-related structural alterations in the outer scales which were absent from the inner scales. De novo transcriptome assembly for three different scales: 1st (outer), 5th (intermediate) and 8th (inner) fleshy scales identified 2,542 differentially expressed genes among them. GO enrichment for cluster analysis revealed increasing metabolic processes in the outer senescent scale related to defense response, PCD processes, carbohydrate metabolism and flavonoid biosynthesis, whereas increased metabolism and developmental growth processes were identified in the inner scales. High expression levels of PCD-related genes were identified in the outer scale compared to the inner ones, highlighting the involvement of PCD in outer-skin development. These findings suggest that a program to form the dry protective skin exists and functions only in the outer scales of onion.

miR408 is involved in abiotic stress responses in Arabidopsis.

MicroRNAs (miRNAs) are small RNAs that regulate the expression of target genes post-transcriptionally; they are known to play major roles in development and responses to abiotic stress. miR408 is a highly conserved miRNA in plants that responds to the availability of copper and targets genes encoding copper-containing proteins. It was recently recognized to be an important component of the HY5-SPL7 gene network that mediates a coordinated response to light and copper, illustrating its central role in the response of plants to the environment. Expression of miR408 is significantly affected by a variety of developmental and environmental conditions; however, its biological function is unknown. Involvement of miR408 in the abiotic stress response was investigated in Arabidopsis. Expression of miR408, as well as its target genes, was investigated in response to salinity, cold, oxidative stress, drought and osmotic stress. Analyses of transgenic plants with modulated miR408 expression revealed that higher miR408 expression leads to improved tolerance to salinity, cold and oxidative stress, but enhanced sensitivity to drought and osmotic stress. Cellular antioxidant capacity was enhanced in plants with elevated miR408 expression, as manifested by reduced levels of reactive oxygen species and induced expression of genes associated with antioxidative functions, including Cu/Zn superoxide dismutases (CSD1 and CSD2) and glutathione-S-transferase (GST-U25), as well as auxiliary genes: the copper chaperone CCS1 and the redox stress-associated gene SAP12. Overall, the results demonstrate significant involvement of miR408 in abiotic stress responses, emphasizing the central function of miR408 in plant survival.

Differential expression of miRNAs and their target genes in senescing leaves and siliques: insights from deep sequencing of small RNAs and cleaved target RNAs.

MicroRNAs (miRNAs) are a class of small RNAs, which typically function by guiding cleavage of target mRNAs. They are known to play roles in a variety of plant processes including development, responses to environmental stresses and senescence. To identify senescence regulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel analysis of RNA ends libraries, which enable the large-scale examination of miRNA-guided cleavage products, were constructed and sequenced, resulting in over 750 million genome-matched sequences. These large datasets led to the identification a new senescence-inducible small RNA locus, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence.

De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment.

BACKGROUND: The mango belongs to the genus Mangifera, consisting of numerous tropical fruiting trees in the flowering plant family, Anacardiaceae. Postharvest treatment by hot water brushing (HWB) for 15-20 s was introduced commercially to improve fruit quality and reduce postharvest disease. This treatment enabled successful storage for 3-4 weeks at 12°C, with improved color and reduced disease development, but it enhanced lenticel discoloration on the fruit peel. We investigated global gene expression induced in fruit peel by HWB treatment, and identified key genes involved in mechanisms potentially associated with fruit resistance to pathogens, peel color improvement, and development of lenticel discoloration; this might explain the fruit's phenotypic responses. RESULTS: The mango transcriptome assembly was created and characterized by application of RNA-seq to fruit-peel samples. RNA-seq-based gene-expression profiling identified three main groups of genes associated with HWB treatment: 1) genes involved with biotic and abiotic stress responses and pathogen-defense mechanisms, which were highly expressed; 2) genes associated with chlorophyll degradation and photosynthesis, which showed transient and low expression; and 3) genes involved with sugar and flavonoid metabolism, which were highly expressed. CONCLUSIONS: We describe a new transcriptome of mango fruit peel of cultivar Shelly. The existence of three main groups of genes that were differentially expressed following HWB treatment suggests a molecular basis for the biochemical and physiological consequences of the postharvest HWB treatment, including resistance to pathogens, improved color development, and occurrence of lenticel discoloration.

NAC transcription factor ORE1 and senescence-induced BIFUNCTIONAL NUCLEASE1 (BFN1) constitute a regulatory cascade in Arabidopsis.

Senescence is a highly regulated process that involves the action of a large number of transcription factors. The NAC transcription factor ORE1 (ANAC092) has recently been shown to play a critical role in positively controlling senescence in Arabidopsis thaliana; however, no direct target gene through which it exerts its molecular function has been identified previously. Here, we report that BIFUNCTIONAL NUCLEASE1 (BFN1), a well-known senescence-enhanced gene, is directly regulated by ORE1. We detected elevated expression of BFN1 already 2 h after induction of ORE1 in estradiol-inducible ORE1 overexpression lines and 6 h after transfection of Arabidopsis mesophyll cell protoplasts with a 35S:ORE1 construct. ORE1 and BFN1 expression patterns largely overlap, as shown by promoter-reporter gene (GUS) fusions, while BFN1 expression in senescent leaves and the abscission zones of maturing flower organs was virtually absent in ore1 mutant background. In vitro binding site assays revealed a bipartite ORE1 binding site, similar to that of ORS1, a paralog of ORE1. A bipartite ORE1 binding site was identified in the BFN1 promoter; mutating the cis-element within the context of the full-length BFN1 promoter drastically reduced ORE1-mediated transactivation capacity in transiently transfected Arabidopsis mesophyll cell protoplasts. Furthermore, chromatin immunoprecipitation (ChIP) demonstrates in vivo binding of ORE1 to the BFN1 promoter. We also demonstrate binding of ORE1 in vivo to the promoters of two other senescence-associated genes, namely SAG29/SWEET15 and SINA1, supporting the central role of ORE1 during senescence.

Endoreduplication preferentially occurs at the proximal side of the abscission zone during abscission of tomato leaf.

Endoreduplication is a cell cycle variant in which multiple rounds of DNA replication occur without subsequent mitosis, resulting in polyploid cells. Although cells with endoreduplicated nuclei were ubiquitously distributed throughout the abscission zone (AZ) of tomato leaf before abscission induction by ethylene, endoreduplication was detected mostly on the proximal side of the AZ after induction. The possible association between endoreduplication and intensive membrane trafficking in cells at the proximal side of the AZ is discussed.

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem.

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.

Programmed cell death occurs asymmetrically during abscission in tomato.

Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.

Localization of the Arabidopsis senescence- and cell death-associated BFN1 nuclease: from the ER to fragmented nuclei.

Plant senescence- or PCD-associated nucleases share significant homology with nucleases from different organisms. However, knowledge of their function is limited. Intracellular localization of the Arabidopsis senescence- and PCD-associated nuclease BFN1 was investigated. Analysis of BFN1-GFP localization in transiently transformed tobacco protoplasts revealed initial localization in filamentous structures spread throughout the cytoplasm, which then clustered around the nuclei as the protoplasts senesced. These filamentous structures were identified as being of ER origin. In BFN1-GFP-transgenic Arabidopsis plants, similar localization of BFN1-GFP was observed in young leaves, that is, in filamentous structures that reorganized around the nuclei only in senescing cells. In late senescence, BFN1-GFP was localized with fragmented nuclei in membrane-wrapped vesicles. BFN1's postulated function as a nucleic acid-degrading enzyme in senescence and PCD is supported by its localization pattern. Our results suggest the existence of a dedicated compartment mediating nucleic acid degradation in senescence and PCD processes.

Identification of defense-related genes newly-associated with tomato flower abscission.

The current abscission model suggests the formation of a post-abscission trans-differentiation of a protective layer as the last step of the process. The present report expands the repertoire of genes activated in the tomato flower abscission zone (AZ), which are likely to be involved in defense responses. We identified four different defense-related genes, including: Cysteine-type endopeptidase, α-Dioxygenase 1 (α-DOX1), HopW-1-1-Interacting protein2 (WIN2), and Stomatal-derived factor-2 (SDF2), that are newly-associated with the late stage of the abscission process. The late expression of these genes, induced at 8-14 h after flower removal when pedicel abscission was already in progress, was AZ-specific, and was inhibited by treatments that prevented pedicel abscission, including 1-methylcyclopropene pretreatment or IAA application. This information supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses.

Microarray analysis of the abscission-related transcriptome in the tomato flower abscission zone in response to auxin depletion.

The abscission process is initiated by changes in the auxin gradient across the abscission zone (AZ) and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the molecular and biochemical basis of the increased AZ sensitivity to ethylene. We examined transcriptome changes in the tomato (Solanum lycopersicum 'Shiran 1335') flower AZ during the rapid acquisition of ethylene sensitivity following flower removal, which depletes the AZ from auxin, with or without preexposure to 1-methylcyclopropene or application of indole-3-acetic acid after flower removal. Microarray analysis using the Affymetrix Tomato GeneChip revealed changes in expression, occurring prior to and during pedicel abscission, of many genes with possible regulatory functions. They included a range of auxin- and ethylene-related transcription factors, other transcription factors and regulatory genes that are transiently induced early, 2 h after flower removal, and a set of novel AZ-specific genes. All gene expressions initiated by flower removal and leading to pedicel abscission were inhibited by indole-3-acetic acid application, while 1-methylcyclopropene pretreatment inhibited only the ethylene-induced expressions, including those induced by wound-associated ethylene signals. These results confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes resulting from auxin depletion. Our results shed light on the regulatory control of abscission at the molecular level and further expand our knowledge of auxin-ethylene cross talk during the initial controlling stages of the process.