Free Indole-3-acetic Acid Research Articles

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.

YUCCA-Mediated Biosynthesis of the Auxin IAA Is Required during the Somatic Embryogenic Induction Process in Coffea canephora.

Despite the existence of considerable research on somatic embryogenesis (SE), the molecular mechanism that regulates the biosynthesis of auxins during the SE induction process remains unknown. Indole-3-acetic acid (IAA) is an auxin that is synthesized in plants through five pathways. The biosynthetic pathway most frequently used in this synthesis is the conversion of tryptophan to indol-3-pyruvic acid (IPA) by tryptophan aminotransferase of Arabidopsis (TAA) followed by the conversion of IPA to IAA by enzymes encoded by YUCCA (YUC) genes of the flavin monooxygenase family; however, it is unclear whether YUC-mediated IAA biosynthesis is involved in SE induction. In this study, we report that the increase of IAA observed during SE pre-treatment (plants in MS medium supplemented with 1-naphthaleneacetic acid (NAA) 0.54 µM and kinetin (Kin) 2.32 µM for 14 days) was due to its de novo biosynthesis. By qRT-PCR, we demonstrated that YUC gene expression was consistent with the free IAA signal found in the explants during the induction of SE. In addition, the use of yucasin to inhibit the activity of YUC enzymes reduced the signal of free IAA in the leaf explants and dramatically decreased the induction of SE. The exogenous addition of IAA restored the SE process in explants treated with yucasin. Our findings suggest that the biosynthesis and localization of IAA play an essential role during the induction process of SE in Coffea canephora.

Overexpression of MsGH3.5 inhibits shoot and root development through the auxin and cytokinin pathways in apple plants.

Phytohormonal interactions are crucial for plant development. Auxin and cytokinin (CK) both play critical roles in regulating plant growth and development; however, the interaction between these two phytohormones is complex and not fully understood. Here, we isolated a wild apple (Malus sieversii Roem) GRETCHEN HAGEN3 (GH3) gene, MsGH3.5, encoding an indole-3-acetic acid (IAA)-amido synthetase. Overexpression of MsGH3.5 significantly reduced the free IAA content and increased the content of some IAA-amino acid conjugates, and MsGH3.5-overexpressing lines were dwarfed and produced fewer adventitious roots (ARs) than the control. This phenotype is consistent with the role of GH3 in conjugating excess free active IAA to amino acids in auxin homeostasis. Surprisingly, overexpression of MsGH3.5 significantly increased CK concentrations in the whole plant, and altered the expression of genes involved in CK biosynthesis, metabolism and signaling. Furthermore, exogenous CK application induced MsGH3.5 expression through the activity of the CK type-B response regulator, MsRR1a, which mediates the CK primary response. MsRR1a activated MsGH3.5 expression by directly binding to its promoter, linking auxin and CK signaling. Plants overexpressing MsRR1a also displayed fewer ARs, in agreement with the regulation of MsGH3.5 expression by MsRR1a. Taken together, we reveal that MsGH3.5 affects apple growth and development by modulating auxin and CK levels and signaling pathways. These findings provide insight into the interaction between the auxin and CK pathways, and might have substantial implications for efforts to improve apple architecture.

Indole-3-acetate beta-glucosyltransferase OsIAGLU regulates seed vigour through mediating crosstalk between auxin and abscisic acid in rice.

SummarySeed vigour is an important trait for direct seeding in rice. In this study, indole‐3‐acetate beta‐glucosyltransferase OsIAGLU was cloned in rice, and its roles on seed vigour were mainly investigated. Disruption of OsIAGLU resulted in low seed vigour in rice. Quantitative RT‐PCR analysis showed that the expressions of OsIAGLU were relatively higher in the late developing and the early germinating seeds and were significantly induced by indole‐3‐acetic acid (IAA) and abscisic acid (ABA). Transcriptome analysis revealed that the IAA‐ and ABA‐related genes were involved in the OsIAGLU regulation of seed vigour in rice. The higher levels of free IAA and ABA were identified in germinating seeds of osiaglu mutants compared to wild‐type (WT) plants. When treated with exogenous IAA and ABA, the osiaglu mutants and WT plants showed sensitivity to ABA while not IAA, but the exogenous IAA amplified ABA‐induced reduction of seed vigour in rice. The continuously higher expressions of ABA‐INSENSITIVE 3 (OsABI3) and OsABI5 occurred in germinating seeds of osiaglu mutants compared to WT plants. The regulation of seed vigour by OsIAGLU might be through modulating IAA and ABA levels to alert OsABIs expression in germinating seeds in rice. Based on analysis of single‐nucleotide polymorphism data of rice accessions, two haplotypes of OsIAGLU that positively correlated with seed vigour were identified in indica accessions. This study provides important insights into the roles of OsIAGLU on seed vigour and facilitates the practical use of OsIAGLU in rice breeding.

A bacterial assay for rapid screening of IAA catabolic enzymes

BackgroundPlants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays.ResultsIn this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein.ConclusionsThis new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

Overexpression of OsIAAGLU reveals a role for IAA-glucose conjugation in modulating rice plant architecture.

OsIAAGLU could catalyze the reaction of IAA with glucose to generate IAA-glucose. Overexpression of OsIAAGLU in rice resulted in altered rice shoot architecture and root gravitropism. The distribution and levels of indole-3-acetic acid (IAA) within plant tissues are well known to play vital roles in plant growth and development. An important mechanism of regulating free IAA levels in monocots is formation of IAA ester conjugates. In this study, a cytosol-localized protein encoded by the rice gene of indole-3-acetic acid glucosyltransferase (OsIAAGLU) was found to catalyze the reaction of free IAA with glucose to generate IAA-glucose. Expression of OsIAAGLU could be induced by IAA and NAA. The number of tillers and leaf angle was significantly increased with a concomitant decrease in plant height and panicle length in the transgenic rice lines overexpressing OsIAAGLU compared to the wild-type (WT) plants. Phenotypes of iaaglu mutants constructed using the CRISPR/Cas9 system had no obvious differences with WT plants. Furthermore, overexpression of OsIAAGLU resulted in reduced sensitivity to IAA/NAA and altered gravitropic response of the roots in the transgenic plants. Free IAA contents in the leaves, root tips, and lamina joint of OsIAAGLU-overexpressing transgenic lines were lower than those of WT plants. These results support that OsIAAGLU could play a regulatory role in IAA homeostasis and rice architecture.

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 auxin in the regulation of ammonium tolerance in rice (Oryza sativa L.)

Ammonium (NH4+) is an important nitrogen source and is widely used as a fertilizer in agricultural systems. However, excess NH4+ inhibits root growth, and, subsequently, vegetative shoot growth and yield. This study examines whether auxin is involved in differential NH4+ tolerance in rice (Oryza sativa L.), and how auxin is regulated under high-NH4+ conditions in rice. An NH4+-sensitive (Kasalath, Kas) and an NH4+-insensitive (Koshihikari, Kos) rive cultivar were cultured hydroponically with or without exogenous indole-3-acetic acid (IAA) and auxin biosynthesis inhibitors. Root growth, root area, tissue IAA content, and transcription of genes involved in auxin biosynthesis, conjugation and degradation were determined. pDR5::GUS staining and auxin measurement show that high NH4+ can decrease free IAA content in roots. In addition, quantitative RT-PCR, pharmacology, and genetics analysis suggest that Kos possesses a higher capacity for auxin biosynthesis and a weaker capacity for auxin metabolism compared to Kas under high-NH4+ stress. We conclude that the NH4+-tolerant cultivar possesses a higher capacity to maintain auxin homeostasis under high-NH4+ stress, and that this advantage is incurred by promotion of auxin biosynthesis and a suppression of auxin metabolism.

Deciphering the growth pattern and phytohormonal content in Saskatoon berry (Amelanchier alnifolia) in response to in vitro cytokinin application

Clonal propagation plays a critical integral role in the growth and success of a global multi-billion dollar horticulture industry through a constant supply of healthy stock plants. The supply chain depends on continuously improving the micropropagation process, thus, understanding the physiology of in vitro plants remains a core component. We evaluated the influence of exogenously applied cytokinins (CKs, N6-benzyladenine = BA, isopentenyladenine = iP, meta-topolin = mT, 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine = mTTHP) in Murashige and Skoog (MS)-supplemented media on organogenic response and accumulation of endogenous CK and indole-3-acetic acid (IAA) metabolites. The highest shoot proliferation (30 shoots/explant) was obtained with 20 μM mT treatment. However, the best quality regenerants were produced in 10 μM mT treatment. Rooting of Amelanchier alnifolia in vitro plantlets was observed at the lowest CK concentrations, with the highest root proliferation (3 roots/explant) in 1 μM mTTHP regenerants. Similar to the organogenic response, high levels of endogenous bioactive CK metabolites (free bases, ribosides, and nucleotides) were detected in mT and mTTHP-derived regenerants. The level of O-glucosides was also comparatively high in these cultures. All CK-treated plants had high levels of endogenous free IAA compared to the control. This may suggest an influence of CKs on biosynthesis of IAA.

Indole-3-acetic acid metabolism and growth in young kiwifruit berry

The patterns of auxin concentration and metabolism were investigated in distinct kiwifruit portions and compared with the rate of fruit growth during early developmental stages. Indole-3-acetic acid (IAA) level was higher in inner fruit tissues, particularly in younger fruit, while the hormone was barely detectable in outer tissues. Modulation of free IAA concentration did not appear to depend tightly on conjugation of the hormone. Despite the lack of a strong correlation between the levels of IAA and enzymes involved in its catabolism, in some portions of the fruit a low hormone level corresponded to a higher IAA degradation activity. An inverse correlation was also observed between hormone levels and the appearance/increase in some bands with high mobility in peroxidase gel activity assay. Phenols, compounds with a potential auxin-protecting activity, appeared to be involved mostly in photoprotection of the fruit than in the regulation of IAA levels. Beyond catabolism and conjugation, other metabolic pathways, particularly those occurring in the developing seeds, may have decisively influenced auxin levels in fruit tissues, as well as the amount of the hormone exported from the fruit. The latter, estimated by analyzing the concentration of IAA in the sap exuded from the pedicel, showed a time course which was similar to that displayed by inner fruit tissues. Furthermore, similarities were found between the pattern of IAA concentration in inner fruit tissues and fruit growth rate. The possible role of IAA in promoting growth during early fruit development is discussed.

Arabidopsis NITRILASE 1 Contributes to the Regulation of Root Growth and Development through Modulation of Auxin Biosynthesis in Seedlings.

Nitrilases consist of a group of enzymes that catalyze the hydrolysis of organic cyanides. They are found ubiquitously distributed in the plant kingdom. Plant nitrilases are mainly involved in the detoxification of ß-cyanoalanine, a side-product of ethylene biosynthesis. In the model plant Arabidopsis thaliana a second group of Brassicaceae-specific nitrilases (NIT1-3) has been found. This so-called NIT1-subfamily has been associated with the conversion of indole-3-acetonitrile (IAN) into the major plant growth hormone, indole-3-acetic acid (IAA). However, apart of reported functions in defense responses to pathogens and in responses to sulfur depletion, conclusive insight into the general physiological function of the NIT-subfamily nitrilases remains elusive. In this report, we test both the contribution of the indole-3-acetaldoxime (IAOx) pathway to general auxin biosynthesis and the influence of altered nitrilase expression on plant development. Apart of a comprehensive transcriptomics approach to explore the role of the IAOx route in auxin formation, we took a genetic approach to disclose the function of NITRILASE 1 (NIT1) of A. thaliana. We show that NIT1 over-expression (NIT1ox) results in seedlings with shorter primary roots, and an increased number of lateral roots. In addition, NIT1ox plants exhibit drastic changes of both free IAA and IAN levels, which are suggested to be the reason for the observed phenotype. On the other hand, NIT2RNAi knockdown lines, capable of suppressing the expression of all members of the NIT1-subfamily, were generated and characterized to substantiate the above-mentioned findings. Our results demonstrate for the first time that Arabidopsis NIT1 has profound effects on root morphogenesis in early seedling development.

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.

Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants

In plants, indole-3-acetic acid (IAA) amido hydrolases (AHs) participate in auxin homeostasis by releasing free IAA from IAA-amino acid conjugates. We investigated the role of IAR3, a member of the IAA amido hydrolase family, in the response of Solanaceous plants challenged by biotrophic and hemi-biotrophic pathogens. By means of genome inspection and phylogenic analysis we firstly identified IAA-AH sequences and putative IAR3 orthologs in Nicotiana benthamiana, tomato and potato. We evaluated the involvement of IAR3 genes in defense responses by using virus-induced gene silencing. We observed that N. benthamiana and tomato plants with knocked-down expression of IAR3 genes contained lower levels of free IAA and presented altered responses to pathogen attack, including enhanced basal defenses and higher tolerance to infection in susceptible plants. We showed that IAR3 genes are consistently up-regulated in N. benthamiana and tomato upon inoculation with Phytophthora infestans and Cladosporium fulvum respectively. However, IAR3 expression decreased significantly when hypersensitive response was triggered in transgenic tomato plants coexpressing the Cf-4 resistance gene and the avirulence factor Avr4. Altogether, our results indicate that changes in IAR3 expression lead to alteration in auxin homeostasis that ultimately affects plant defense responses.

Indole-3-acetic acid in plant–pathogen interactions: a key molecule for in planta bacterial virulence and fitness

The plant pathogenic bacterium Pseudomonas savastanoi, the causal agent of olive and oleander knot disease, uses the so-called “indole-3-acetamide pathway” to convert tryptophan to indole-3-acetic acid (IAA) via a two-step pathway catalyzed by enzymes encoded by the genes in the iaaM/iaaH operon. Moreover, pathovar nerii of P. savastanoi is able to conjugate IAA to lysine to generate the less biologically active compound IAA-Lys via the enzyme IAA-lysine synthase encoded by the iaaL gene. Interestingly, iaaL is now known to be widespread in many Pseudomonas syringae pathovars, even in the absence of the iaaM and iaaH genes for IAA biosynthesis.Here, two knockout mutants, ΔiaaL and ΔiaaM, of strain Psn23 of P. savastanoi pv. nerii were produced. Pathogenicity tests using the host plant Nerium oleander showed that ΔiaaL and ΔiaaM were hypervirulent and hypovirulent, respectively and these features appeared to be related to their differential production of free IAA. Using the Phenotype Microarray approach, the chemical sensitivity of these mutants was shown to be comparable to that of wild-type Psn23. The main exception was 8 hydroxyquinoline, a toxic compound that is naturally present in plant exudates and is used as a biocide, which severely impaired the growth of ΔiaaL and ΔiaaM, as well as growth of the non-pathogenic mutant ΔhrpA, which lacks a functional Type Three Secretion System (TTSS). According to bioinformatics analysis of the Psn23 genome, a gene encoding a putative Multidrug and Toxic compound Extrusion (MATE) transporter, was found upstream of iaaL. Similarly to iaaL and iaaM, its expression appeared to be TTSS-dependent. Moreover, auxin-responsive elements were identified for the first time in the modular promoters of both the iaaL gene and the iaaM/iaaH operon of P. savastanoi, suggesting their IAA-inducible transcription. Gene expression analysis of several genes related to TTSS, IAA metabolism and drug resistance confirmed the presence of a concerted regulatory network in this phytopathogen among virulence, fitness and drug efflux.

Influence of Different Yeast Strains on Metabolism of Tryptophan and Indole-3-Acetic Acid During Fermentation

The effect of supplementation of Riesling (Vitis vinifera L.) must with ammonium sulphate (AS) on tryptophan (Trp) and indole-3-acetic acid (IAA) metabolism during alcoholic fermentation with four commercial yeast strains (Uvaferm CEG, Lalvin Cross Evolution, Anchor VIN 13 and Anchor exotic SPH) were examined. AS addition reduced Trp uptake by yeasts from 1% to 15%. Wines produced by Anchor VIN 13 and Uvaferm CEG yeasts with AS addition had the highest concentrations of Trp (0.60 and 0.59 mg/L respectively). Concentrations of total and bound IAA decreased during all treatments. At the end of fermentation the highest concentration of free IAA was measured in the control must inoculated with Anchor VIN 13 yeast (35.83 μg/L) and the lowest in the sample inoculated with Uvaferm CEG yeast (13.08 μg/L). Anchor VIN 13 yeast showed the strongest change in metabolism due to AS addition during fermentation. 2-Aminoacetophenone (2-AAP) formation was not observed in all the studied wines.

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.

Tracking biochemical changes during adventitious root formation in olive (Olea europaea L.)

The activity of oxidative enzymes and the levels of free auxins were determined during adventitious root formation in olive explants. Rooting trials were performed both with in vitro-cultured microshoots of the cultivar ‘Galega Vulgar’, treated with indole-3-butyric acid (IBA) and with salicylhydroxamic acid (SHAM)+IBA, as well as with semi-hardwood cuttings of the cultivars ‘Galega Vulgar’ (difficult-to-root) and ‘Cobrançosa’ (easy-to-root), treated with IBA. The auxin (IBA) was used in all experiments as a rooting promoter, while SHAM was used in micropropagation trials as rooting inhibitor, providing a negative control. Free indole-3-acetic acid (IAA) and IBA concentrations were determined in microshoots, as well as in semi-hardwood cuttings, throughout the rooting period at pre-established time-points. At the same time-points, the enzymatic activity of polyphenol oxidases (PPO), peroxidases (POX), and IAA oxidase (IAAox) was evaluated in the microshoots. Microshoots treated with SHAM+IBA revealed higher POX and IAAox activity, as well as lower PPO activity, than those treated only with IBA. IAA levels were higher in IBA-treated microshoots during induction phase, but lower during early initiation phase. In contrast, free IBA levels were higher in microshoots treated with SHAM+IBA during induction, but lower during initiation. A similar pattern of free auxin levels was observed in semi-hardwood cuttings of the two contrasting cultivars under evaluation. The similarities found on the auxin patterns of microshoots treated with SHAM and those of semi-hardwood cuttings of the difficult-to-root olive cultivar allow considering SHAM a reliable control for when simulation of a difficult-to-root behavior is necessary. The inhibitory effect of SHAM in root formation could be related with 1) the inhibition of alternative oxidase (AOX), leading to a downregulation of phenylpropanoid biosynthetic pathways, which would decrease the concentration of phenolic substrates for PPO; 2) an increase in IAAox activity resulting in lower free IAA levels or; 3) a defective conversion of IBA into IAA.

Local auxin metabolism regulates environment-induced hypocotyl elongation

A hallmark of plants is their adaptability of size and form in response to widely fluctuating environments. The metabolism and redistribution of the phytohormone auxin play pivotal roles in establishing active auxin gradients and resulting cellular differentiation. In Arabidopsis thaliana, cotyledons and leaves synthesize indole-3-acetic acid (IAA) from tryptophan through indole-3-pyruvic acid (3-IPA) in response to vegetational shade. This newly synthesized auxin moves to the hypocotyl where it induces elongation of hypocotyl cells. Here we show that loss of function of VAS2 (IAA-amido synthetase Gretchen Hagen 3 (GH3).17) leads to increases in free IAA at the expense of IAA-Glu (IAA-glutamate) in the hypocotyl epidermis. This active IAA elicits shade- and high temperature-induced hypocotyl elongation largely independently of 3-IPA-mediated IAA biosynthesis in cotyledons. Our results reveal an unexpected capacity of local auxin metabolism to modulate the homeostasis and spatial distribution of free auxin in specialized organs such as hypocotyls in response to shade and high temperature.