Indole-3-acetic Acid Conjugates Research Articles

Cultivar differences in the hormonal crosstalk regulating apple fruit development and ripening: Relationship with flavour components and postharvest susceptibility to Penicillium expansum

The hormonal interplay during the on-tree development and ripening of three apple cultivars with known differences in their postharvest ripening patterns was studied, at the biochemical and targeted gene expression level, along with characterizing the changes in main sugars, acids, phenylpropanoids and volatile organic compounds (VOCs). Our findings suggest that in ‘Royal Gala’ and ‘Opal®’ apples, a peak in indole 3-acetic acid (IAA) seems necessary to activate ethylene metabolism, being its intensity proportional to the ethylene production. The interplay between IAA and ethylene appears to be mediated by MdARF5, responsible for activating the expression of MdACS3 and triggering ethylene metabolism. On the other hand, the lack of ethylene production observed in ‘Granny Smith’ apples was likely related to the absence of an IAA peak and possibly caused by the over activation of IAA conjugation mechanisms leading to a greater accumulation of IAA inactive conjugates such as indole-3-acetyl-aspartate (IAAsp). Abscisic acid (ABA) accumulation was only observed in cultivars with the ability to accumulate sucrose and produce ethylene, suggesting a possible crosstalk among those hormones and sucrose in orchestrating apple on-tree ripening. While differences in hormone levels among cultivars led to noticeable differences in some specific VOCs, no evident associations were found between hormone changes and the accumulation or degradation of monosaccharides, organic acids or phenolic compounds during fruit development and ripening. Likewise, no clear relationship was found between the fruit susceptibility to blue mould and hormonal levels yet certain specific biochemical compounds (i.e., procyanidins and sucrose) could be acting as a source of resistance or susceptibility, respectively, to blue mould development. Overall, understanding the cultivar specific hormonal regulation of apple on-tree ripening provides valuable insights to optimize fruit quality at the time of harvest as well as to develop strategies for improved postharvest management.

Non-specific effects of the CINNAMATE-4-HYDROXYLASE inhibitor piperonylic acid.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.

IAA-decorated CuO nanocarriers significantly improve Chickpea growth by increasing antioxidative activities.

Plant growth regulators tagged on metallic oxide nanoparticles (NPs) may function as nanofertilizers with reduced toxicity of NPs. CuO NPs were synthesized to function as nanocarriers of Indole-3-acetic acid (IAA). Powder X-ray diffraction (XRD) and scanning electron microscopy (SEM) revealed 30.4nm size of NPs and sheet-like structure, respectively, of CuO-IAA NPs. Fourier-transform infrared spectroscopy (FTIR) confirmed CuO-IAA formation. IAA-decorated CuO NPs enhanced the physiological parameters of Chickpea plants, i.e., root length, shoot length, and biomass compared to naked CuO NPs. The variation in physiological response was due to change of phytochemical contents in plants. Phenolic content increased up to 17.98 and 18.13 µgGAE/mg DW at 20 and 40mg/L of CuO-IAA NPs, respectively. However, significant decrease in antioxidant enzymes' activity was recorded compared to control. Presence of CuO-IAA NPs increased the reducing potential of plants at higher concentration of NPs, while decrease in total antioxidant response was observed. This study concludes that IAA conjugation to CuO NPs reduces toxicity of NPs. Furthermore, NPs can be explored as nanocarriers for plant modulators and slow release in future studies.

Inactivation of the entire Arabidopsis group II GH3s confers tolerance to salinity and water deficit.

Summary Indole‐3‐acetic acid (IAA) controls a plethora of developmental processes. Thus, regulation of its concentration is of great relevance for plant performance. Cellular IAA concentration depends on its transport, biosynthesis and the various pathways for IAA inactivation, including oxidation and conjugation.Group II members of the GRETCHEN HAGEN 3 (GH3) gene family code for acyl acid amido synthetases catalysing the conjugation of IAA to amino acids. However, the high degree of functional redundancy among them has hampered thorough analysis of their roles in plant development.In this work, we generated an Arabidopsis gh3.1,2,3,4,5,6,9,17 (gh3oct) mutant to knock out the group II GH3 pathway. The gh3oct plants had an elaborated root architecture, showed an increased tolerance to different osmotic stresses, including an IAA‐dependent tolerance to salinity, and were more tolerant to water deficit. Indole‐3‐acetic acid metabolite quantification in gh3oct plants suggested the existence of additional GH3‐like enzymes in IAA metabolism. Moreover, our data suggested that 2‐oxindole‐3‐acetic acid production depends, at least in part, on the GH3 pathway. Targeted stress‐hormone analysis further suggested involvement of abscisic acid in the differential response to salinity of gh3oct plants.Taken together, our data provide new insights into the roles of group II GH3s in IAA metabolism and hormone‐regulated plant development.

Genome-Wide Analysis, Modeling, and Identification of Amino Acid Binding Motifs Suggest the Involvement of GH3 Genes during Somatic Embryogenesis of Coffea canephora.

Auxin plays a central role in growth and plant development. To maintain auxin homeostasis, biological processes such as biosynthesis, transport, degradation, and reversible conjugation are essential. The Gretchen Hagen 3 (GH3) family genes codify for the enzymes that esterify indole-3-acetic acid (IAA) to various amino acids, which is a key process in the induction of somatic embryogenesis (SE). The GH3 family is one of the principal families of early response to auxin genes, exhibiting IAA-amido synthetase activity to maintain optimal levels of free auxin in the cell. In this study, we carried out a systematic identification of the GH3 gene family in the genome of Coffea canephora, determining a total of 18 CcGH3 genes. Analysis of the genetic structures and phylogenetic relationships of CcGH3 genes with GH3 genes from other plant species revealed that they could be clustered in two major categories with groups 1 and 2 of the GH3 family of Arabidopsis. We analyzed the transcriptome expression profiles of the 18 CcGH3 genes using RNA-Seq analysis-based data and qRT-PCR during the different points of somatic embryogenesis induction. Furthermore, the endogenous quantification of free and conjugated indole-3-acetic acid (IAA) suggests that the various members of the CcGH3 genes play a crucial role during the embryogenic process of C. canephora. Three-dimensional modeling of the selected CcGH3 proteins showed that they consist of two domains: an extensive N-terminal domain and a smaller C-terminal domain. All proteins analyzed in the present study shared a unique conserved structural topology. Additionally, we identified conserved regions that could function to bind nucleotides and specific amino acids for the conjugation of IAA during SE in C. canephora. These results provide a better understanding of the C. canephora GH3 gene family for further exploration and possible genetic manipulation.

Metabolomic profiling of wheat genotypes resistant and susceptible to root-lesion nematode Pratylenchus thornei.

Metabolic profiling of Pratylenchus thornei resistant and susceptible wheat genotypes indicates that fatty acid, glycerolipid and flavonoid classes of metabolites constitutively expressed in resistant wheat roots could reduce nematode reproduction. The root-lesion nematode Pratylenchus thornei reduces wheat production in many parts of the world. In this study the metabolic profiles of two wheat genotypes ‘QT16258’ (moderately resistant) and ‘Janz’ (susceptible) were compared at 8 weeks post inoculation with or without P. thornei. We performed untargeted liquid chromatography mass spectrometry analysis (LC–MS) of the wheat root samples. A total of 11,704 MS features were identified, out of which 765 MS features were annotated using in-house chemical standards. Principal components analysis (PCA) and partial least square discriminant analysis (PLS-DA) indicated dissimilarity of the metabolome between P. thornei resistant and susceptible genotypes. Two-way analysis of variance indicated that metabolic differences were mainly constitutive rather than induced by inoculation with P. thornei. Eighty-four annotated metabolites were significantly (p ≤ 0.01) higher in relative concentration in ‘QT16258’ than ‘Janz’ and belonged to the following classes of metabolites: flavonoids, fatty acids, glycerolipids, alkaloids, tannins, nucleotides, steroid glycosides and terpenoids. Eighty-five annotated metabolites were significantly (p ≤ 0.01) higher in relative concentration in ‘Janz’ than ‘QT16258’ and belonged to the following classes of metabolites: amino acids, sugars, flavonoids and alkaloids. Several metabolites at higher concentration in ‘QT16258’, including quercetin-3,4'-O-di-beta-glucoside (flavonoid), linoleic acid (fatty acid), lysophosphatidylethanolamine (glycerolipid), hirsutine (alkaloid), 1-methylsulfinylbutenyl-isothiocyanate (glucosinolate), could potentially strengthen the root cell walls to inhibit nematode penetration and/or reduce nematode motility. Some metabolites at higher concentrations in susceptible ‘Janz’, including phenolics, coniferyl alcohol and indole acetic acid conjugates, could be nematode attractants as well as part of a hypersensitive browning reaction to nematode invasion.

High ammonium inhibits root growth in Arabidopsis thaliana by promoting auxin conjugation rather than inhibiting auxin biosynthesis

Ammonium (NH4+) inhibits primary root (PR) growth in most plant species when present even at moderate concentrations. Previous studies have shown that transport of indole-3-acetic acid (IAA) is critical to maintaining root elongation under high-NH4+ stress. However, the precise regulation of IAA homeostasis under high-NH4+ stress (HAS) remains unclear. In this study, qRT-PCR, RNA-seq, free IAA and IAA conjugate and PR elongation measurements were conducted in genetic mutants to investigate the role of IAA biosynthesis and conjugation under HAS. Our data clearly show that HAS decreases free IAA in roots by increasing IAA inactivation but does not decrease IAA biosynthesis, and that the IAA-conjugating genes GH3.1, GH3.2, GH3.3, GH3.4, and GH3.6 function as the key genes in regulating high-NH4+ sensitivity in the roots. Furthermore, the analysis of promoter::GUS staining in situ and genetic mutants reveals that HAS promotes IAA conjugation in the elongation zone (EZ), which may be responsible for the PR inhibition observed under HAS. This study provides potential new insight into the role of auxin in the improvement of tolerance to NH4+.

OsGRETCHENHAGEN3-2 modulates rice seed storability via accumulation of abscisic acid and protective substances.

Seed storability largely determines the vigor of seeds during storage and is significant in agriculture and ecology. However, the underlying genetic basis remains unclear. In the present study, we report the cloning and characterization of the rice (Oryza sativa) indole-3-acetic acid (IAA)-amido synthetase gene GRETCHEN HAGEN3-2 (OsGH3-2) associated with seed storability. OsGH3-2 was identified by performing a genome-wide association study in rice germplasms with linkage mapping in chromosome substitution segment lines, contributing to the wide variation of seed viability in the populations after long periods of storage and artificial ageing. OsGH3-2 was dominantly expressed in the developing seeds and catalyzed IAA conjugation to amino acids, forming inactive auxin. Transgenic overexpression, knockout, and knockdown experiments demonstrated that OsGH3-2 affected seed storability by regulating the accumulation level of abscisic acid (ABA). Overexpression of OsGH3-2 significantly decreased seed storability, while knockout or knockdown of the gene enhanced seed storability compared with the wild-type. OsGH3-2 acted as a negative regulator of seed storability by modulating many genes related to the ABA pathway and probably subsequently late embryogenesis-abundant proteins at the transcription level. These findings shed light on the molecular mechanisms underlying seed storability and will facilitate the improvement of seed vigor by genomic breeding and gene-editing approaches in rice.

Long chain acyl CoA synthetase 4 catalyzes the first step in peroxisomal indole-3-butyric acid to IAA conversion.

Indole-3-butyric acid (IBA) is an endogenous storage auxin important for maintaining appropriate indole-3-acetic acid (IAA) levels, thereby influencingprimary root elongation and lateral root development. IBA is metabolized into free IAA in peroxisomes in a multistep process similar to fatty acid β-oxidation. We identified LONG CHAIN ACYL-COA SYNTHETASE 4 (LACS4) in a screen for enhanced IBA resistance in primary root elongation in Arabidopsis thaliana. LACSs activate substrates by catalyzing the addition of CoA, the necessary first step for fatty acids to participate in β-oxidation or other metabolic pathways. Here, we describe the novel role of LACS4 in hormone metabolism and postulate that LACS4 catalyzes the addition of CoA onto IBA, the first step in its β-oxidation. lacs4 is resistant to the effects of IBA in primary root elongation and dark-grown hypocotyl elongation, and has reduced lateral root density. lacs6 also is resistant to IBA, although both lacs4 and lacs6 remain sensitive to IAA in primary root elongation, demonstrating that auxin responses are intact. LACS4 has in vitro enzymatic activity on IBA, but not IAA or IAA conjugates, and disruption of LACS4 activity reduces the amount of IBA-derived IAA in planta. We conclude that, in addition to activity on fatty acids, LACS4 and LACS6 also catalyze the addition of CoA onto IBA, the first step in IBA metabolism and a necessary step in generating IBA-derived IAA.

Salicylic acid regulates adventitious root formation via competitive inhibition of the auxin conjugation enzyme CsGH3.5 in cucumber hypocotyls.

Exogenous SA treatment at appropriate concentrations promotes adventitious root formation in cucumber hypocotyls, via competitive inhibiting the IAA-Asp synthetase activity of CsGH3.5, and increasing the local free IAA level. Adventitious root formation is critical for the cutting propagation of horticultural plants. Indole-3-acetic acid (IAA) has been shown to play a central role in regulating this process, while for salicylic acid (SA), its exact effects and regulatory mechanism have not been elucidated. In this study, we showed that exogenous SA treatment at the concentrations of both 50 and 100µM promoted adventitious root formation at the base of the hypocotyl of cucumber seedlings. At these concentrations, SA could induce the expression of CYCLIN and Cyclin-dependent Kinase (CDK) genes during adventitious rooting. IAA was shown to be involved in SA-induced adventitious root formation in cucumber hypocotyls. Exposure to exogenous SA led to a slight increase in the free IAA content, and pre-treatment with the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) almost completely abolished the inducible effects of SA on adventitious root number. SA-induced IAA accumulation was also associated with the enhanced expression of Gretchen Hagen3.5 (CsGH3.5). The in vitro enzymatic assay indicated that CsGH3.5 has both IAA- and SA-amido synthetase activity and prefers aspartate (Asp) as the amino acid conjugate. The Asp concentration dictated the functional activity of CsGH3.5 on IAA. Both affinity and catalytic efficiency (Kcat/Km) increased when the Asp concentration increased from 0.3 to 1mM. In contrast, CsGH3.5 showed equal catalytic efficiency for SA at low and high Asp concentrations. Furthermore, SA functioned as a competitive inhibitor of the IAA-Asp synthetase activity of CsGH3.5. During adventitious formation, SA application indeed repressed the IAA-Asp levels in the rooting zone. These data show that SA plays an inducible role in adventitious root formation in cucumber through competitive inhibition of the auxin conjugation enzyme CsGH3.5. SA reduces the IAA conjugate levels, thereby increasing the local free IAA level and ultimately enhancing adventitious root formation.

Chlorinated Auxins-How Does Arabidopsis Thaliana Deal with Them?

Plant hormones have various functions in plants and play crucial roles in all developmental and differentiation stages. Auxins constitute one of the most important groups with the major representative indole-3-acetic acid (IAA). A halogenated derivate of IAA, 4-chloro-indole-3-acetic acid (4-Cl-IAA), has previously been identified in Pisum sativum and other legumes. While the enzymes responsible for the halogenation of compounds in bacteria and fungi are well studied, the metabolic pathways leading to the production of 4-Cl-IAA in plants, especially the halogenating reaction, are still unknown. Therefore, bacterial flavin-dependent tryptophan-halogenase genes were transformed into the model organism Arabidopsis thaliana. The type of chlorinated indole derivatives that could be expected was determined by incubating wild type A. thaliana with different Cl-tryptophan derivatives. We showed that, in addition to chlorinated IAA, chlorinated IAA conjugates were synthesized. Concomitantly, we found that an auxin conjugate synthetase (GH3.3 protein) from A. thaliana was able to convert chlorinated IAAs to amino acid conjugates in vitro. In addition, we showed that the production of halogenated tryptophan (Trp), indole-3-acetonitrile (IAN) and IAA is possible in transgenic A. thaliana in planta with the help of the bacterial halogenating enzymes. Furthermore, it was investigated if there is an effect (i) of exogenously applied Cl-IAA and Cl-Trp and (ii) of endogenously chlorinated substances on the growth phenotype of the plants.

A transcriptome analysis reveals a role for the indole GLS-linked auxin biosynthesis in secondary dormancy in rapeseed (Brassica napus L.)

BackgroundBrassica napus L. has little or no primary dormancy, but exhibits great variation in secondary dormancy. Secondary dormancy potential in oilseed rape can lead to the emergence of volunteer plants that cause genetic contamination, reduced quality and biosafety issues. However, the mechanisms underlying secondary dormancy are poorly understood. In this study, cultivars Huaiyou-WSD-H2 (H) and Huaiyou-SSD-V1 (V), which exhibit low (approximately 5%) and high (approximately 95%) secondary dormancy rate, respectively, were identified. Four samples, before (Hb and Vb) and after (Ha and Va) secondary dormancy induction by polyethylene glycol (PEG), were collected to identify the candidate genes involved in secondary dormancy via comparative transcriptome profile analysis.ResultsA total of 998 differentially expressed genes (DEGs), which are mainly involved in secondary metabolism, transcriptional regulation, protein modification and signaling pathways, were then detected. Among these DEGs, the expression levels of those involved in the sulfur-rich indole glucosinolate (GLS)-linked auxin biosynthesis pathway were markedly upregulated in the dormant seeds (Va), which were validated by qRT-PCR and subsequently confirmed via detection of altered concentrations of indole-3-acetic acid (IAA), IAA conjugates and precursors. Furthermore, exogenous IAA applications to cultivar H enhanced secondary dormancy.ConclusionThis study first (to our knowledge) elucidated that indole GLS-linked auxin biosynthesis is enhanced during secondary dormancy induced by PEG, which provides valuable information concerning secondary dormancy and expands the current understanding of the role of auxin in rapeseed.

Preparation of synthetic auxin-amino acid conjugates

Auxin amide conjugates are regulators of the most important auxin, indole-3-acetic acid (IAA), which is considered responsible for many important processes within the plants. Herein, amide conjugates of IAA were synthesized employing a simple and efficient coupling method with WSCI·HCl, a water-soluble condensing reagent, in the presence of 1-hydroxybenzotriazole. IAA conjugates with 10 amino acids along with their corresponding methyl esters were prepared in excellent yields, up to 95%, aiming to facilitate their identification in plant species. Eight IAA-amino acid methyl ester conjugates are characterized here for the first time.

Expression of two indole-3-acetic acid (IAA)-amido synthetase (GH3) genes during fruit development of raspberry (Rubus idaeus Heritage)

The conjugation of indole-3-acetic acid (IAA) to amino acids by indole-3-acetic acid (IAA)-amido synthetases (GH3) is an important part of auxin level regulation. However, the auxin conjugation during development of soft fruits such as raspberry is poorly understood. In this study, indole-3-acetic acid (IAA)-amido synthetases in raspberry, designated as RiGH3 (RiGH3.1, RiGH3.5 transcripts) were evaluated during fruit development of raspberry cultivar Rubus idaeus Heritage, and under IAA treatment. The results showed that before to the onset of ripening the fruit size, weight and the expression of IAA-amido synthetase RiGH3.1 transcript levels increased. Then when the fruits attain full development, fruit firmness and titratable acidity decreased, in the contrast to ethylene production and total soluble solids content increasing. However, the RiGH3.5 transcript was found to be expressed primarily in flowers. When compared to untreated control fruit, fruit treated with 1 mM of IAA at white stage, showed an increase of RiGH3.1 transcript during in-vitro assay (10 °C by 18 h). However, no significant change in the levels of RiGH3.5 was observed during IAA treatment. Multiple alignments of the full-length predicted RiGH3.1 protein sequences revealed a high sequence homology with proteins deduced sequences described for other fruit of Rosaceae species. The RiGH3.1 deduced sequence showed the presence of binding motives for IAA and aspartic acid, and indicate that the isolated sequence have the typical motives of GH3.1 protein family. These findings give new insights into the possible role of RiGH3.1 transcripts, and the IAA conjugation (in maintaining the low concentration of free IAA) during raspberry fruit ripening.

New insights into indole-3-acetic acid metabolism in Azospirillum brasilense.

The aim of this research was to analyse the global indole-3-acetic acid (IAA) metabolism in three commercially used strains of Azospirillum brasilense. Azospirillum brasilense Sp245, Az39 and Cd, containing a plasmid with the ipdC-gusA fusion (pFAJ64), were cultured in minimal medium MMAB with or without 10mgl-1 of l-trp till exponential or stationary growth phase. The cultures were then split into 10ml tubes and individually treated with 10mgml-1 IAA, IBA or NAA (auxin catabolism and homeostasis); IAPhe, IALeu, IAA-ala, IAA-glucose (IAA conjugate hydrolysis); or l-lys, l-leu, l-ileu, l-phe, l-ala, l-val, l-arg, l-glu, l-his, l-met, l-asp, l-cys, l-ser, l-pro, l-thr and l-trp (regulation of IAA biosynthesis and IAA conjugation). Bacterial growth, IAA production and ipdC expression were evaluated. None of the A. brasilense strains were able to hydrolyse IAA conjugates, catabolize auxins, or conjugate IAA with amino acids or glucose. l-amino acids l-met, l-val, l-cys and l-ser inhibited bacterial growth and decreased IAA biosynthesis. The expression of ipdC and IAA biosynthesis but not bacterial growth was affected by l-leu, l-phe, l-ala, l-ile, l-pro. l-arg, l-glu, l-his, l-lys, l-asp and l-thr did not affect any of the measured parameters. In this paper, we confirmed that A. brasilense produces IAA only in presence of l-trp is not able to degrade auxins, conjugate IAA with sugars and/or l-amino acids, or hydrolyse such conjugates to release free IAA. Finally, we found that bacterial growth and/or IAA biosynthesis were inhibited by the presence of several l-amino acids probably by diversion of the cellular metabolism. We propose a renewed model to explain IAA metabolism in A. brasilense, one of the most studied phytostimulatory bacteria.

Involvement of Indole-3-Acetic Acid Metabolism in the Early Fruit Development of the Parthenocarpic Tomato Cultivar, MPK-1

Parthenocarpy, or fruit set and growth without fertilization, is a desirable trait in tomato cultivation as it reduces the cost of tomato production. MPK-1 is a Japanese parthenocarpic tomato cultivar, and the gene responsible for parthenocarpy of MPK-1 is Pat-k. As MPK-1 is a stable parthenocarpic tomato cultivar, we investigated the physiological mechanism of parthenocarpy in this cultivar. Indole-3-acetic acid (IAA) is considered as the main factor contributing to parthenocarpy, as its exogenous application to unpollinated ovaries triggers parthenocarpic fruit development in tomato. In this study, we investigated the level of IAA and its metabolites and the expression of genes involved in IAA metabolism in unpollinated ovaries of MPK-1. We observed an increase in the level of IAA accompanied by an elevated level of expression of an IAA biosynthesis gene, ToFZY5 in parthenocarpic ovaries of MPK-1. Simultaneously, the level of IAA-glutamate (IAA-Glu), one of the IAA conjugates comprising a potential IAA inactivation pathway, was also increased. These results suggest that the increase in IAA levels, driven by the up-regulation of IAA biosynthesis genes, promotes the growth of parthenocarpic fruits in MPK-1, and that the IAA synthesized in parthenocarpic ovaries is primarily metabolized to IAA-Glu. In addition, expression profiles of some genes involved in IAA metabolism were different between pollinated and parthenocarpic ovaries, suggesting that the specific transcriptional regulation of IAA metabolism in parthenocarpic ovaries of MPK-1 differs from that in pollinated ovaries.

Regulation of polar auxin transport in grapevine fruitlets (Vitis vinifera L.) and the proposed role of auxin homeostasis during fruit abscission.

BackgroundIndole-3-acetic acid (IAA), the most abundant auxin, is a growth promoter hormone involved in several developmental processes. Auxin homeostasis is very important to its function and this is achieved through the regulation of IAA biosynthesis, conjugation, degradation and transport. In grapevine, IAA plays an essential role during initial stages of berry development, since it delays fruitlet abscission by reducing the ethylene sensitivity in the abscission zone. For this reason, Continuous polar IAA transport to the pedicel is required. This kind of transport is controlled by IAA, which regulates its own movement by modifying the expression and localization of PIN-FORMED (PIN) auxin efflux facilitators that localize asymmetrically within the cell. On the other hand, the hormone gibberellin (GA) also activates the polar auxin transport by increasing PIN stability. In Vitis vinifera, fruitlet abscission occurs during the first two to three weeks after flowering. During this time, IAA and GA are present, however the role of these hormones in the control of polar auxin transport is unknown.ResultsIn this work, the use of radiolabeled IAA showed that auxin is basipetally transported during grapevine fruitlet abscission. This observation was further supported by immunolocalization of putative VvPIN proteins that display a basipetal distribution in pericarp cells. Polar auxin transport and transcripts of four putative VvPIN genes decreased in conjunction with increased abscission, and the inhibition of polar auxin transport resulted in fruit drop. GA3 and IAA treatments reduced polar auxin transport, but only GA3 treatment decreased VvPIN transcript abundance. When GA biosynthesis was blocked, IAA was capable to increase polar auxin transport, suggesting that its effect depends on GA content. Finally, we observed significant changes in the content of several IAA-related compounds during the abscission period.ConclusionsThese results provide evidence that auxin homeostasis plays a central role during grapevine initial fruit development and that GA and IAA controls auxin homeostasis by reducing polar auxin transport.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0914-1) contains supplementary material, which is available to authorized users.

Hydrolases of the ILR1-like family of Arabidopsis thaliana modulate auxin response by regulating auxin homeostasis in the endoplasmic reticulum.

Amide-linked conjugates of indole-3-acetic acid (IAA) have been identified in most plant species. They function in storage, inactivation or inhibition of the growth regulator auxin. We investigated how the major known endogenous amide-linked IAA conjugates with auxin-like activity act in auxin signaling and what role ILR1-like proteins play in this process in Arabidopsis. We used a genetically encoded auxin sensor to show that IAA-Leu, IAA-Ala and IAA-Phe act through the TIR1-dependent signaling pathway. Furthermore, by using the sensor as a free IAA reporter, we followed conjugate hydrolysis mediated by ILR1, ILL2 and IAR3 in plant cells and correlated the activity of the hydrolases with a modulation of auxin response. The conjugate preferences that we observed are in agreement with available in vitro data for ILR1. Moreover, we identified IAA-Leu as an additional substrate for IAR3 and showed that ILL2 has a more moderate kinetic performance than observed in vitro. Finally, we proved that IAR3, ILL2 and ILR1 reside in the endoplasmic reticulum, indicating that in this compartment the hydrolases regulate the rates of amido-IAA hydrolysis which results in activation of auxin signaling.

Hormone crosstalk in wound stress response: wound-inducible amidohydrolases can simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana.

Jasmonate (JA) and auxin are essential hormones in plant development and stress responses. While the two govern distinct physiological processes, their signaling pathways interact at various levels. Recently, members of the Arabidopsis indole-3-acetic acid (IAA) amidohydrolase (IAH) family were reported to metabolize jasmonoyl-isoleucine (JA-Ile), a bioactive form of JA. Here, we characterized three IAH members, ILR1, ILL6, and IAR3, for their function in JA and IAA metabolism and signaling. Expression of all three genes in leaves was up-regulated by wounding or JA, but not by IAA. Purified recombinant proteins showed overlapping but distinct substrate specificities for diverse amino acid conjugates of JA and IAA. Perturbed patterns of the endogenous JA profile in plants overexpressing or knocked-out for the three genes were consistent with ILL6 and IAR3, but not ILR1, being the JA amidohydrolases. Increased turnover of JA-Ile in the ILL6- and IAR3-overexpressing plants created symptoms of JA deficiency whereas increased free IAA by overexpression of ILR1 and IAR3 made plants hypersensitive to exogenous IAA conjugates. Surprisingly, ILL6 overexpression rendered plants highly resistant to exogenous IAA conjugates, indicating its interference with IAA conjugate hydrolysis. Fluorescent protein-tagged IAR3 and ILL6 co-localized with the endoplasmic reticulum-localized JA-Ile 12-hydroxylase, CYP94B3. Together, these results demonstrate that in wounded leaves JA-inducible amidohydrolases contribute to regulate active IAA and JA-Ile levels, promoting auxin signaling while attenuating JA signaling. This mechanism represents an example of a metabolic-level crosstalk between the auxin and JA signaling pathways.

Transcription factor WRKY46 modulates the development of Arabidopsis lateral roots in osmotic/salt stress conditions via regulation of ABA signaling and auxin homeostasis.

The development of lateral roots (LR) is known to be severely inhibited by salt or osmotic stress. However, the molecular mechanisms underlying LR development in osmotic/salt stress conditions are poorly understood. Here we show that the gene encoding the WRKY transcription factor WRKY46 (WRKY46) is expressed throughout lateral root primordia (LRP) during early LR development and that expression is subsequently restricted to the stele of the mature LR. In osmotic/salt stress conditions, lack of WRKY46 (in loss-of-function wrky46 mutants) significantly reduces, while overexpression of WRKY46 enhances, LR development. We also show that exogenous auxin largely restores LR development in wrky46 mutants, and that the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits LR development in both wild-type (WT; Col-0) and in a line overexpressing WRKY46 (OV46). Subsequent analysis of abscisic acid (ABA)-related mutants indicated that WRKY46 expression is down-regulated by ABA signaling, and up-regulated by an ABA-independent signal induced by osmotic/salt stress. Next, we show that expression of the DR5:GUS auxin response reporter is reduced in roots of wrky46 mutants, and that both wrky46 mutants and OV46 display altered root levels of free indole-3-acetic acid (IAA) and IAA conjugates. Subsequent RT-qPCR and ChIP-qPCR experiments indicated that WRKY46 directly regulates the expression of ABI4 and of genes regulating auxin conjugation. Finally, analysis of wrky46 abi4 double mutant plants confirms that ABI4 acts downstream of WRKY46. In summary, our results demonstrate that WRKY46 contributes to the feedforward inhibition of osmotic/salt stress-dependent LR inhibition via regulation of ABA signaling and auxin homeostasis.