Auxin Metabolites Research Articles

Targeted Metabolites and Transcriptome Analysis Uncover the Putative Role of Auxin in Floral Sex Determination in Litchi chinensis Sonn.

Litchi exhibits a large number of flowers, many flowering batches, and an inconsistent ratio of male and female flowers, frequently leading to a low fruit-setting rate. Floral sexual differentiation is a crucial phase in perennial trees to ensure optimal fruit production. However, the mechanism behind floral differentiation remains unclear. The objective of the study was to identify the role of auxin in floral differentiation at the transcriptional level. The results showed that the ratio of female flowers treated with naphthalene acetic acid (NAA) was significantly lower than that of the control stage (M0/F0). The levels of endogenous auxin and auxin metabolites were measured in male and female flowers at different stages of development. It was found that the levels of IAA, IAA-Glu, IAA-Asp, and IAA-Ala were significantly higher in male flowers compared to female flowers. Next-generation sequencing and modeling were employed to perform an in-depth transcriptome analysis on all flower buds in litchi 'Feizixiao' cultivars (Litchi chinensis Sonn.). Plant hormones were found to exert a significant impact on the litchi flowering process and flower proliferation. Specifically, a majority of differentially expressed genes (DEGs) related to the auxin pathway were noticeably increased during male flower bud differentiation. The current findings will enhance our comprehension of the process and control mechanism of litchi floral sexual differentiation. It also offers a theoretical foundation for implementing strategies to regulate flowering and enhance fruit production in litchi cultivation.

Sphingosine Promotes Fiber Early Elongation in Upland Cotton.

Sphingolipids play an important role in cotton fiber development, but the regulatory mechanism is largely unclear. We found that serine palmitoyltransferase (SPT) enzyme inhibitors, myriocin and sphingosine (dihydrosphingosine (DHS) and phytosphingosine (PHS)), affected early fiber elongation in cotton, and we performed a sphingolipidomic and transcriptomic analysis of control and PHS-treated fibers. Myriocin inhibited fiber elongation, while DHS and PHS promoted it in a dose-effect manner. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that contents of 22 sphingolipids in the PHS-treated fibers for 10 days were changed, of which the contents of 4 sphingolipids increased and 18 sphingolipids decreased. The transcriptome analysis identified 432 differentially expressed genes (238 up-regulated and 194 down-regulated) in the PHS-treated fibers. Among them, the phenylpropanoid biosynthesis pathway is the most significant enrichment. The expression levels of transcription factors such as MYB, ERF, LBD, and bHLH in the fibers also changed, and most of MYB and ERF were up-regulated. Auxin-related genes IAA, GH3 and BIG GRAIN 1 were up-regulated, while ABPs were down-regulated, and the contents of 3 auxin metabolites were decreased. Our results provide important sphingolipid metabolites and regulatory pathways that influence fiber elongation.

Hormonal profiles in dormant turions of 22 aquatic plant species: do they reflect functional or taxonomic traits?

Turions are vegetative, dormant overwintering organs formed in aquatic plants in response to unfavourable ecological conditions. Contents of cytokinin (CK), auxin metabolites and abscisic acid (ABA) as main growth and development regulators were compared in innately dormant autumnal turions of 22 aquatic plant species of different functional ecological or taxonomic groups with those in non-dormant winter apices in three aquatic species and with those in spring turions of four species after their overwintering. The hormones were analysed in miniature turion samples using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. In innately dormant turions, the total contents of each of the four main CK types, biologically active forms and total CKs differed by two to three orders of magnitude across 22 species; the proportion of active CK forms was 0.18-67 %. Similarly, the content of four auxin forms was extremely variable and the IAA proportion as the active form was 0.014-99 %. The ABA content varied from almost zero to 54 µmol kg-1 dry weight and after overwintering it usually significantly decreased. Of all functional traits studied, hormone profiles depended most on the place of turion sprouting (surface vs bottom) and we suggest that this trait is crucial for turion ecophysiology. The key role of ABA in regulating turion dormancy was confirmed. However, the highly variable pattern of the ABA content in innately dormant and in overwintered turions indicates that the hormonal mechanism regulating the innate dormancy and its breaking in turions is not uniform within aquatic plants.

Metabolic profiles of 2-oxindole-3-acetyl-amino acid conjugates differ in various plant species.

Auxins are a group of phytohormones that play a key role in plant growth and development, mainly presented by the major member of the family - indole-3-acetic acid (IAA). The levels of free IAA are regulated, in addition to de novo biosynthesis, by irreversible oxidative catabolism and reversible conjugation with sugars and amino acids. These conjugates, which serve as inactive storage forms of auxin and/or degradation intermediates, can also be oxidized to form 2-oxindole-3-acetyl-1-O-ß-d-glucose (oxIAA-glc) and oxIAA-amino acids (oxIAA-AAs). Until now, only oxIAA conjugates with aspartate and glutamate have been identified in plants. However, detailed information on the endogenous levels of these and other putative oxIAA-amino acid conjugates in various plant species and their spatial distribution is still not well understood but is finally getting more attention. Herein, we identified and characterized two novel naturally occurring auxin metabolites in plants, namely oxIAA-leucine (oxIAA-Leu) and oxIAA-phenylalanine (oxIAA-Phe). Subsequently, a new liquid chromatography-tandem mass spectrometry method was developed for the determination of a wide range of IAA metabolites. Using this methodology, the quantitative determination of IAA metabolites including newly characterized oxIAA conjugates in roots, shoots and cotyledons of four selected plant models - Arabidopsis thaliana, pea (Pisum sativum L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) was performed to compare auxin metabolite profiles. The distribution of various groups of auxin metabolites differed notably among the studied species as well as their sections. For example, oxIAA-AA conjugates were the major metabolites found in pea, while oxIAA-glc dominated in Arabidopsis. We further compared IAA metabolite levels in plants harvested at different growth stages to monitor the dynamics of IAA metabolite profiles during early seedling development. In general, our results show a great diversity of auxin inactivation pathways among angiosperm plants. We believe that our findings will greatly contribute to a better understanding of IAA homeostasis.

Comparison of two contrasting Leymus chinensis accessions reveals the roles of the cell wall and auxin in rhizome development

Leymus chinensis, a perennial native forage grass, is widely distributed in the steppes of Inner Mongolia as the dominant species. The main reproductive strategy of this grass is clonal propagation, which occurs via the proliferation of subterranean horizontal stems known as rhizomes. To elucidate the mechanism underlying rhizome development in this grass, we collected 60 accessions of L. chinensis and evaluated their rhizome development. One accession, which we named SR-74 (Strong Rhizomes), had significantly better rhizome development capacity than the accession WR-16 (Weak Rhizomes) in terms of rhizome number, total and primary rhizome length, and number of rhizome seedlings. Rhizome elongation was positively correlated with the number of internodes in the rhizome, which affected plant biomass. Compared to WR-16, SR-74 had higher rhizome tip hardness, higher abundance of transcripts participating in the biosynthesis of cell wall components, and higher levels of the metabolites L-phenylalanine, trans-cinnamic acid, 3-coumaric acid, ferulic acid, and coniferin. These metabolites in the phenylpropanoid biosynthesis pathway are precursors of lignin. In addition, SR-74 rhizomes contained higher amounts of auxin and auxin metabolites, including L-Trp, IPA, IBA, IAA and IAA-Asp, as well as upregulated expression of the auxin biosynthesis and signaling genes YUCCA6, YUCCA8, YUCCA10, YUCCA11, PIN1, PIN2, UGT1, UGT2, UGT4, UGT10, GH3, IAA7, IAA23, and IAA30. We propose a network between auxin signaling and the cell wall underlying rhizome development in L. chinensis.

The high concentrations of abscisic, jasmonic, and salicylic acids produced under long days do not accelerate flowering in Chenopodium ficifolium 459

The survival and adaptation of angiosperms depends on the proper timing of flowering. The weedy species Chenopodium ficifolium serves as a useful diploid model for comparing the transition to flowering with the important tetraploid crop Chenopodium quinoa due to the close phylogenetic relationship. The detailed transcriptomic and hormonomic study of the floral induction was performed in the short-day accession C. ficifolium 459. The plants grew more rapidly under long days but flowered later than under short days. The high levels of abscisic, jasmonic, and salicylic acids at long days were accompanied by the elevated expression of the genes responding to oxidative stress. The increased concentrations of stress-related phytohormones neither inhibited the plant growth nor accelerated flowering in C. ficifolium 459 at long photoperiods. Enhanced content of cytokinins and the stimulation of cytokinin and gibberellic acid signaling pathways under short days may indicate the possible participation of these phytohormones in floral initiation. The accumulation of auxin metabolites suggests the presence of a dynamic regulatory network in C. ficifolium 459.

Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei.

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol.

The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various “omics” technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

The role of auxin during early berry development in grapevine as revealed by transcript profiling from pollination to fruit set

Auxin is a key phytohormone that modulates fruit formation in many fleshy fruits through the regulation of cell division and expansion. Auxin content rapidly increases after pollination and the manipulation in its levels may lead to the parthenocarpic development. ln Vitis vinifera L., little is known about the early fruit development that encompasses from pollination to fruit set. Pollination/fertilization events trigger fruit formation, and auxin treatment mimics their effect in grape berry set. However, the role of auxin in this process at the molecular level is not well understood. To elucidate the participation of auxin in grapevine fruit formation, morphological, reproductive, and molecular events from anthesis to fruit set were described in sequential days after pollination. Exploratory RNA-seq analysis at four time points from anthesis to fruit set revealed that the highest percentage of genes induced/repressed within the hormone-related gene category were auxin-related genes. Transcript profiling showed significant transcript variations in auxin signaling and homeostasis-related genes during the early fruit development. Indole acetic acid and several auxin metabolites were present during this period. Finally, application of an inhibitor of auxin action reduced cell number and the mesocarp diameter, similarly to unpollinated berries, further confirming the key role of auxin during early berry development. This work sheds light into the molecular features of the initial fruit development and highlights the auxin participation during this stage in grapevine.

DIOXYGENASE FOR AUXIN OXIDATION 1 catalyzes the oxidation of IAA amino acid conjugates.

Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.

Quantitative analysis of auxin metabolites in lychee flowers.

To investigate the modulation of endogenous indole-3-acetic acid (IAA) level by biosynthesis and inactivation during floral development, IAA and its metabolites were analyzed by LC-ESI/MS/MS in Lychee (Litchi chinensis Sonn.) flowers. In the bloomed flowers, the level of free IAA was higher in males than in females. In contrast, the total sum level of IAA metabolites was higher in females than in males, suggesting a higher biosynthetic activity of IAA in the females before the bloom. A detailed time-course analysis from the bud stage to the developing flower stage showed higher levels of IAA in females than males. The major metabolites were oxidized IAA in both sexes. The results suggest that IAA is involved in the maturation of female floral tissues in lychee, and oxidative metabolism plays an essential role in controlling the free IAA levels therein.

Studies of moss reproductive development indicate that auxin biosynthesis in apical stem cells may constitute an ancestral function for focal growth control.

Summary The plant hormone auxin is a key factor for regulation of plant development, and this function was probably reinforced during the evolution of early land plants. We have extended the available toolbox to allow detailed studies of how auxin biosynthesis and responses are regulated in moss reproductive organs, their stem cells and gametes to better elucidate the function of auxin in the morphogenesis of early land plants.We measured auxin metabolites and identified IPyA (indole‐3‐pyruvic acid) as the main biosynthesis pathway in Physcomitrium (Physcomitrella) patens and established knock‐out, overexpressor and reporter lines for biosynthesis genes which were analyzed alongside previously reported auxin‐sensing and transport reporters.Vegetative and reproductive apical stem cells synthesize auxin. Sustained stem cell activity depends on an inability to sense the auxin produced while progeny of the stem cells respond to the auxin, aiding in the control of cell division, expansion and differentiation. Gamete precursors are dependent on a certain degree of auxin sensing, while the final differentiation is a low auxin‐sensing process.Tha data presented indicate that low auxin activity may represent a conserved hallmark of land plant gametes, and that local auxin biosynthesis in apical stem cells may be part of an ancestral mechanism to control focal growth.

Auxin biosynthesis in the phytopathogenic fungus Leptosphaeria maculans is associated with enhanced transcription of indole-3-pyruvate decarboxylase LmIPDC2 and tryptophan aminotransferase LmTAM1

Auxins are hormones that regulate growth and development in plants. Besides plants, various microorganisms also produce auxins. Here we investigate whether and how the phytopathogenic fungus Leptosphaeria maculans biosynthesizes auxins. We characterized the auxin profile of in vitro grown L. maculans. The culture was further supplied with the auxin biosynthetic-precursors tryptophan and tryptamine and gene expression and phytohormone content was analyzed. L. maculans in vitro produced IAA (indole-3-acetic acid) as the predominant auxin metabolite. IAA production could be further stimulated by supplying precursors. Expression of indole-3-pyruvate decarboxylase LmIPDC2, tryptophan aminotransferase LmTAM1 and nitrilase LmNIT1 genes was mainly upregulated after adding tryptophan and correlated with IAA production, suggesting that these genes are the key components of auxin biosynthesis in L. maculans. Tryptamine acted as a potent inducer of IAA production, though a pathway independent of LmIPDC2/LmTAM1 may be involved. Despite L. maculans being a rich source of bioactive IAA, the auxin metabolic profile of host plant Brassica napus was not altered upon infection. Exogenous IAA inhibited the growth of L. maculans in vitro when supplied in high concentration. Altogether, we showed that L. maculans is capable of IAA production and we have identified biosynthetic genes that were responsive to tryptophan treatment.

GH3 Auxin-Amido Synthetases Alter the Ratio of Indole-3-Acetic Acid and Phenylacetic Acid in Arabidopsis.

Auxin is the first discovered plant hormone and is essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the main auxin and plays pivotal roles in intercellular communication through polar auxin transport. Phenylacetic acid (PAA) is another natural auxin that does not show polar movement. Although a wide range of species have been shown to produce PAA, its biosynthesis, inactivation and physiological significance in plants are largely unknown. In this study, we demonstrate that overexpression of the CYP79A2 gene, which is involved in benzylglucosinolate synthesis, remarkably increased the levels of PAA and enhanced lateral root formation in Arabidopsis. This coincided with a significant reduction in the levels of IAA. The results from auxin metabolite quantification suggest that the PAA-dependent induction of GRETCHEN HAGEN 3 (GH3) genes, which encode auxin-amido synthetases, promote the inactivation of IAA. Similarly, an increase in IAA synthesis, via the indole-3-acetaldoxime pathway, significantly reduced the levels of PAA. The same adjustment of IAA and PAA levels was also observed by applying each auxin to wild-type plants. These results show that GH3 auxin-amido synthetases can alter the ratio of IAA and PAA in plant growth and development.

Identification of Auxin Metabolites in Brassicaceae by Ultra-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry.

Auxins are signaling molecules involved in multiple stages of plant growth and development. The levels of the most important auxin, indole-3-acetic acid (IAA), are regulated by the formation of amide and ester conjugates with amino acids and sugars. In this work, IAA and IAA amide conjugates with amino acids bearing a free carboxylic group or a methyl ester group, along with some selected IAA metabolites, were studied in positive and negative electrospray ionization (ESI) modes, utilizing high-resolution mass spectrometry (HRMS) as a tool for their structural analysis. HRMS/MS spectra revealed the fragmentation patterns that enable us to identify IAA metabolites in plant extracts from eight vegetables of the Brassicaceae family using a fast and reliable ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) method. The accurate m/z (mass to charge) ratio and abundance of the molecular and fragment ions of the studied compounds in plant extracts matched those obtained from commercially available or synthesized compounds and confirmed the presence of IAA metabolites.

Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis.

Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d-1) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.

An UHPLC-MS/MS Method for Target Profiling of Stress-Related Phytohormones.

The methodology described here represents an improved strategy for analysis of a broad range of stress-related plant hormones including jasmonates, salicylic acid, abscisic acid, and auxin metabolites. The method conditions are optimized in order to reduce the background effect of complicated plant matrix, allow effective preconcentration and thus perform highly sensitive profiling of multiple plant hormones by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

The auxins, IAA and PAA, are synthesized by similar steps catalyzed by different enzymes

ABSTRACTOne of the fundamental plant growth substances, indole-3-acetic acid (IAA), belongs to a class of phytohormones known as auxins. The main IAA biosynthesis pathway involves the conversion of tryptophan to indole-3-pyruvic acid, which is in turn converted to IAA. The two enzymes responsible for these conversions, members of the TAA1 and YUCCA gene families, respectively, have recently been implicated in the synthesis of another auxin, phenylacetic acid (PAA). While there is some evidence to support this theory, there are also some concerns. Here we address the question: to what extent does the TAA1/YUCCA system contribute to the biosynthesis of PAA? In addition, we highlight the importance of measuring auxin metabolites and conjugates in addressing such questions.

Control of cytokinin and auxin homeostasis in cyanobacteria and algae.

Background and Aims The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [3H]trans-zeatin (transZ) and auxin ([3H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division.Methods Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography–electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [3H]transZ and [3H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection.Key Results The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [3H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both phytohormones in algal growth and cell division.Conclusions Our data suggest the existence and functioning of a complex network of metabolic pathways and activity control of CKs and auxins in cyanobacteria and algae that apparently differ from those in vascular plants.

AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening.

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process.