Indole-3-acetic Acid Conjugates Research Articles

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.

Development of the Poplar-Laccaria bicolor Ectomycorrhiza Modifies Root Auxin Metabolism, Signaling, and Response.

Root systems of host trees are known to establish ectomycorrhizae (ECM) interactions with rhizospheric fungi. This mutualistic association leads to dramatic developmental modifications in root architecture, with the formation of numerous short and swollen lateral roots ensheathed by a fungal mantle. Knowing that auxin plays a crucial role in root development, we investigated how auxin metabolism, signaling, and response are affected in poplar (Populus spp.)-Laccaria bicolor ECM roots. The plant-fungus interaction leads to the arrest of lateral root growth with simultaneous attenuation of the synthetic auxin response element DR5. Measurement of auxin-related metabolites in the free-living partners revealed that the mycelium of L. bicolor produces high concentrations of the auxin indole-3-acetic acid (IAA). Metabolic profiling showed an accumulation of IAA and changes in the indol-3-pyruvic acid-dependent IAA biosynthesis and IAA conjugation and degradation pathways during ECM formation. The global analysis of auxin response gene expression and the regulation of AUXIN SIGNALING F-BOX PROTEIN5, AUXIN/IAA, and AUXIN RESPONSE FACTOR expression in ECM roots suggested that symbiosis-dependent auxin signaling is activated during the colonization by L. bicolor. Taking all this evidence into account, we propose a model in which auxin signaling plays a crucial role in the modification of root growth during ECM formation.

Abscission research: what we know and what we still need to study

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

Quantitative evaluation of IAA conjugate pools in Arabidopsis thaliana.

This work has demonstrated that the major method of estimating the amount of unknown IAA conjugates-base hydrolysis-can be significantly complicated by chemical artifacts such as glucobrassicin or protein degradation. The concept of 'bound auxin' traces its origin back to more than 80 years ago and has driven research on the sources and forms of these plant hormones since. Indeed, analytical studies have demonstrated that the majority of cellular auxin is conjugated to simple sugars, cyclitols, glycans, amino acids, and other biomolecules. A number of studies have confirmed the enzymatic systems responsible for the synthesis and hydrolysis of a number of such conjugates in Arabidopsis thaliana and some of these compounds have been identified in situ. However, the amount of indole-3-acetic acid (IAA) released upon treating Arabidopsis tissue extracts with base, a commonly employed technique for estimating the amount of IAA conjugates, greatly exceeded the summation of all the IAA conjugates known individually to be present in Arabidopsis. This discrepancy has remained as an unsolved question. In this study, however, we found that a significant portion of the IAA found after base treatment could be attributed to chemical conversions other than conjugate hydrolysis. Specifically, we showed that glucobrassicin conversion, previously thought to occur at insignificant levels, actually accounted for the majority of solvent soluble IAA released and that proteinaceous tryptophan degradation accounted for a large portion of solvent insoluble IAA. These studies clearly demonstrated the limits associated with using a harsh technique like base hydrolysis in determining IAA conjugates and support using more direct approaches such as mass spectrometry-based strategies for unambiguous characterizations of the total complement of IAA conjugates in new plant materials under study.

IAA–amido synthetase activity and GH3 expression during development of pea seedlings

Indole-3-acetic acid (IAA) conjugation is an integral component of a sensitive mechanism for the regulation of free auxin level in the cell, in addition to hormone synthesis, degradation and transport. One mechanism of regulating both the level and biological activity of hormone involves the conjugation of IAA to amino acids through IAA–amido synthetases. These enzymes belong to the family of GH3 proteins encoded by early-auxin responsive genes, which are conserved in monocots and dicots. In this investigation, we identified pea IAA–Asp synthetase as a member of the GH3 family proteins based on amino acid sequence determined by tandem mass spectrometry (LC–MS/MS). IAA–Asp synthetase was highly homologous (54–84 % amino acid identity) to the GH3 proteins from Populus trichocarpa, Glycine max, Riccinus communis, Nicotiana tabacum, and Arabidopsis thaliana. Moreover, we studied the subcellular distribution and activity of IAA amido synthetase in developing pea seedlings. The cytoplasmic localization of GH3 IAA amido synthetase was indicated in 12-day-old pea seedlings by immunofluorescence method using anti-AtGH3.5 antibodies. This finding was confirmed by the measurement of the enzyme activity in the subcellular fractions collected by differential centrifugation. The most of IAA–Asp synthetase activity (88 % of the total activity) was detected in the 100,000×g supernatant fluid fraction containing soluble cytoplasmic proteins. 3- and 6-day-old pea seedlings showed a weak IAA–Asp synthetase activity and GH3 mRNA level. Twelve-day-old pea stems exhibited 19-fold, and 4-fold higher levels of transcript and the enzyme activity, respectively than 3-day-old plants. We hypothesized that IAA–amido synthetase expression and activity depend on developmental stage of vegetative tissues and this is associated with changes in free IAA level.

A facile means for the identification of indolic compounds from plant tissues.

The bulk of indole-3-acetic acid (IAA) in plants is found in the form of conjugated molecules, yet past research on identifying these compounds has largely relied on methods that were both laborious and inefficient. Using recent advances in analytical instrumentation, we have developed a simple yet powerful liquid chromatography-mass spectrometry (LC-MS)-based method for the facile characterization of the small IAA conjugate profile of plants. The method uses the well-known quinolinium ion (m/z130.0651) generated in MS processes as a signature with high mass accuracy that can be used to screen plant extracts for indolic compounds, including IAA conjugates. We reinvestigated Glycine max (soybean) for its indoles and found indole-3-acetyl-trytophan (IA-Trp) in addition to the already known indole-3-acetyl-aspartic acid (IA-Asp) and indole-3-acetyl-glutamic acid (IA-Glu) conjugates. Surprisingly, several organic acid conjugates of tryptophan were also discovered, many of which have not been reported in planta before. These compounds may have important physiological roles in tryptophan metabolism, which in turn can affect human nutrition. We also demonstrated the general applicability of this method by identifying indolic compounds in different plant tissues of diverse phylogenetic origins. It involves minimal sample preparation but can work in conjunction with sample enrichment techniques. This method enables quick screening of IAA conjugates in both previously characterized as well as uncharacterized species, and facilitates the identification of indolic compounds in general.

Evidence that Indole-3-Acetic Acid is Not Synthesized Via the Indole-3-Acetamide Pathway in Pea Roots

The biosynthetic route of the key plant hormone, indole-3-acetic acid (IAA) has confounded generations of biologists. Evidence in higher plants has implicated two auxin intermediates with roles established in bacteria: indole-3-acetamide (IAM) and indole-3-pyruvic acid. Herein, the IAM pathway is investigated in pea (Pisum sativum), a model legume. The compound was not detected in pea tissue, although evidence was obtained for its presence in Arabidopsis, tobacco, and maize. Deuterium-labeled tryptophan was not converted to IAM in pea roots, despite being converted to IAA. After feeds of deuterium-labeled IAM, label was recovered in the IAA conjugate IAA-aspartate (IAAsp), although there was little or no labeling of IAA itself. Plants treated with IAM did not exhibit high-IAA phenotypes, and did not accumulate IAA. This evidence, taken together, indicates that although exogenous IAM may be converted to IAA (and further to IAAsp), the IAM pathway does not operate naturally in pea roots.

Auxin Input Pathway Disruptions Are Mitigated by Changes in Auxin Biosynthetic Gene Expression in Arabidopsis.

Auxin is a phytohormone involved in cell elongation and division. Levels of indole-3-acetic acid (IAA), the primary auxin, are tightly regulated through biosynthesis, degradation, sequestration, and transport. IAA is sequestered in reversible processes by adding amino acids, polyol or simple alcohols, or sugars, forming IAA conjugates, or through a two-carbon elongation forming indole-3-butyric acid. These sequestered forms of IAA alter hormone activity. To gain a better understanding of how auxin homeostasis is maintained, we have generated Arabidopsis (Arabidopsis thaliana) mutants that combine disruptions in the pathways, converting IAA conjugates and indole-3-butyric acid to free IAA. These mutants show phenotypes indicative of low auxin levels, including delayed germination, abnormal vein patterning, and decreased apical dominance. Root phenotypes include changes in root length, root branching, and root hair growth. IAA levels are reduced in the cotyledon tissue but not meristems or hypocotyls. In the combination mutants, auxin biosynthetic gene expression is increased, particularly in the YUCCA/Tryptophan Aminotransferase of Arabidopsis1 pathway, providing a feedback mechanism that allows the plant to compensate for changes in IAA input pathways and maintain cellular homeostasis.

Auxin production by the plant trypanosomatid Phytomonas serpens and auxin homoeostasis in infected tomato fruits.

Previously we have characterized the complete gene encoding a pyruvate decarboxylase (PDC)/indolepyruvate decarboxylase (IPDC) of Phytomonas serpens, a trypanosomatid highly abundant in tomato fruits. Phylogenetic analyses indicated that the clade that contains the trypanosomatid protein behaves as a sister group of IPDCs of γ-proteobacteria. Since IPDCs are key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid (IAA), the ability for IAA production by P. serpens was investigated. Similar to many microorganisms, the production of IAA and related indolic compounds, quantified by high performance liquid chromatography, increased in P. serpens media in response to amounts of tryptophan. The auxin functionality was confirmed in the hypocotyl elongation assay. In tomato fruits inoculated with P. serpens the concentration of free IAA had no significant variation, whereas increased levels of IAA-amide and IAA-ester conjugates were observed. The data suggest that the auxin produced by the flagellate is converted to IAA conjugates, keeping unaltered the concentration of free IAA. Ethanol also accumulated in P. serpens-conditioned media, as the result of a PDC activity. In the article we discuss the hypothesis of the bifunctionality of P. serpens PDC/IPDC and provide a three-dimensional model of the enzyme.

Cambium reactivation independent of bud unfolding involves de novo IAA biosynthesis in cambium regions in Populus tomentosa Carr.

Cambium reactivation of temperate trees in spring is important for adaptation and growth. In some trees, the resumption of cambium cell division occurs before bud flush. However, knowledge about the source of the internal stimuli that promote cambium cell division during reactivation in these trees is limited. Here, the anatomical changes of cambium during reactivation along with the unfolding of vegetative buds in Populus tomentosa were examined under natural and water-culture conditions. Meanwhile, the levels of free indole-3-acetic acid (IAA) and transcripts of enzymes involved in its production were measured during cambium reactivation. The results showed that cambium cell division occurred before bud unfolding during cambium reactivation in P. tomentosa. Interestingly, the levels of IAA and the transcripts of poplar homologs of aldehyde oxidase 2 and nitrilases 2 encoding enzymes functioning in de novo IAA biosynthesis increased before bud unfolding, while the transcript levels of IAA conjugate hydrolases almost showed no increase. These results suggest that free IAA from de novo biosynthesis in cambium regions promotes cambium reactivation before bud unfolding, providing new insights into the regulatory mechanism of cambium reactivation.

Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis

Levels of the phytohormone indole-3-acetic acid (IAA) can be altered by the formation and hydrolysis of IAA conjugates. The isolation and characterization of Arabidopsis thaliana mutants with reduced IAA-conjugate sensitivity and wild-type IAA responses is advancing the understanding of auxin homeostasis by uncovering the factors needed for conjugate metabolism. For example, the discovery that the IAA-Ala-resistant mutant iar1 is defective in a protein in the ZIP family of metal transporters uncovered a link between metal homeostasis and IAA-conjugate sensitivity. To uncover additional factors impacting auxin conjugate metabolism, we conducted a genetic modifier screen and isolated extragenic mutations that restored IAA-amino acid conjugate sensitivity to the iar1 mutant. One of these suppressor mutants is defective in a putative cation diffusion facilitator, MTP5 (At3g12100; formerly known as MTPc2). Loss of MTP5 function restored IAA conjugate sensitivity to iar1 but not to mutants defective in IAA-amino acid conjugate amidohydrolases. Our results are consistent with a model in which MTP5 and IAR1 transport metals in an antagonistic fashion to regulate metal homeostasis within the subcellular compartment in which the IAA-conjugate amidohydrolases reside, and support previous suggestions that the ion composition in this compartment influences hydrolase activity.

Studies on the Rice LEAF INCLINATION1 (LC1), an IAA–amido Synthetase, Reveal the Effects of Auxin in Leaf Inclination Control

The angle of rice leaf inclination is an important agronomic trait and closely related to the yields and architecture of crops. Although few mutants with altered leaf angles have been reported, the molecular mechanism remains to be elucidated, especially whether hormones are involved in this process. Through genetic screening, a rice gain-of-function mutant leaf inclination1, lc1-D, was identified from the Shanghai T-DNA Insertion Population (SHIP). Phenotypic analysis confirmed the exaggerated leaf angles of lc1-D due to the stimulated cell elongation at the lamina joint. LC1 is transcribed in various tissues and encodes OsGH3-1, an indole-3-acetic acid (IAA) amido synthetase, whose homolog of Arabidopsis functions in maintaining the auxin homeostasis by conjugating excess IAA to various amino acids. Indeed, recombinant LC1 can catalyze the conjugation of IAA to Ala, Asp, and Asn in vitro, which is consistent with the decreased free IAA amount in lc1-D mutant. lc1-D is insensitive to IAA and hypersensitive to exogenous BR, in agreement with the microarray analysis that reveals the altered transcriptions of genes involved in auxin signaling and BR biosynthesis. These results indicate the crucial roles of auxin homeostasis in the leaf inclination control.

A GH3 family member, OsGH3-2, modulates auxin and abscisic acid levels and differentially affects drought and cold tolerance in rice.

Plant responses to abiotic stresses are coordinated by arrays of growth and developmental processes. Indole-3-acetic acid (IAA) and abscisic acid (ABA) play critical roles in developmental programmes and environmental responses, respectively, through complex signalling and metabolism networks. However, crosstalk between the two phytohormones in the stress responses remains largely unknown. Here, it is reported that a GH3 family gene, OsGH3-2, encoding an enzyme catalysing IAA conjugation to amino acids, is involved in the modulation of ABA level and stress tolerance. Expression of OsGH3-2 was induced by drought but was suppressed by cold. Overexpression of OsGH3-2 in rice caused significant morphological aberrations related to IAA deficiency, such as dwarfism, smaller leaves, and fewer crown roots and root hairs. The overexpressing line showed significantly reduced carotene, ABA, and free IAA levels, greater stomata aperture, and faster water loss, and was hypersensitive to drought stress. However, the overexpressing line showed increased cold tolerance, which was due to the combined effects of reduced free IAA content, alleviated oxidative damage, and decreased membrane penetrability. Furthermore, expression levels of some ABA synthesis- and stress-related genes were significantly changed in the overexpression line. It was conclude that OsGH3-2 modulates both endogenous free IAA and ABA homeostasis and differentially affects drought and cold tolerance in rice.

Transcript profiling reveals complex auxin signalling pathway and transcription regulation involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton.

BackgroundSomatic embryogenesis (SE), by which somatic cells of higher plants can dedifferentiate and reorganize into new plants, is a notable illustration of cell totipotency. However, the precise molecular mechanisms regulating SE remain unclear. To characterize the molecular events of this unique process, transcriptome analysis, in combination with biochemical and histological approaches, were conducted in cotton, a typical plant species in SE. Genome-wide profiling of gene expression allowed the identification of novel molecular markers characteristic of this developmental process.ResultsRNA-Seq was used to identify 5,076 differentially expressed genes during cotton SE. Expression profile and functional assignments of these genes indicated significant transcriptional complexity during this process, associated with morphological, histological changes and endogenous indole-3-acetic acid (IAA) alteration. Bioinformatics analysis showed that the genes were enriched for basic processes such as metabolic pathways and biosynthesis of secondary metabolites. Unigenes were abundant for the functions of protein binding and hydrolase activity. Transcription factor–encoding genes were found to be differentially regulated during SE. The complex pathways of auxin abundance, transport and response with differentially regulated genes revealed that the auxin-related transcripts belonged to IAA biosynthesis, indole-3-butyric acid (IBA) metabolism, IAA conjugate metabolism, auxin transport, auxin-responsive protein/indoleacetic acid-induced protein (Aux/IAA), auxin response factor (ARF), small auxin-up RNA (SAUR), Aux/IAA degradation, and other auxin-related proteins, which allow an intricate system of auxin utilization to achieve multiple purposes in SE. Quantitative real-time PCR (qRT-PCR) was performed on selected genes with different expression patterns and functional assignments were made to demonstrate the utility of RNA-Seq for gene expression profiles during cotton SE.ConclusionWe report here the first comprehensive analysis of transcriptome dynamics that may serve as a gene expression profile blueprint in cotton SE. Our main goal was to adapt the RNA-Seq technology to this notable development process and to analyse the gene expression profile. Complex auxin signalling pathway and transcription regulation were highlighted. Together with biochemical and histological approaches, this study provides comprehensive gene expression data sets for cotton SE that serve as an important platform resource for further functional studies in plant embryogenesis.

Auxin conjugates: their role for plant development and in the evolution of land plants

Auxin conjugates are thought to play important roles as storage forms for the active plant hormone indole-3-acetic acid (IAA). In its free form, IAA comprises only up to 25% of the total amount of IAA, depending on the tissue and the plant species studied. The major forms of IAA conjugate are low molecular weight ester or amide forms, but there is increasing evidence of the occurrence of peptides and proteins modified by IAA. Since the discovery of genes and enzymes involved in synthesis and hydrolysis of auxin conjugates, much knowledge has been gained on the biochemistry and function of these compounds, but there is still much to discover. For example, recent work has shown that some auxin conjugate hydrolases prefer conjugates with longer-chain auxins such as indole-3-propionic acid and indole-3-butyric acid as substrate. Also, the compartmentation of these reactions in the cell or in tissues has not been resolved in great detail. The function of auxin conjugates has been mainly elucidated by mutant analysis in genes for synthesis or hydrolysis and a possible function for conjugates inferred from these results. In the evolution of land plants auxin conjugates seem to be connected with the development of certain traits such as embryo, shoot, and vasculature. Most likely, the synthesis of auxin conjugates was developed first, since it has been already detected in moss, whereas sequences typical of auxin conjugate hydrolases were found according to database entries first in moss ferns. The implications for the regulation of auxin levels in different species will be discussed.

Biomimetic fabrication of gold nanoparticles on templated indole-3-acetic acid based nanofibers

Self-assembly of the phytohormone indole-3-acetic acid (IAA) was examined in aqueous solutions of varying pH values. Further, it was observed that IAA biomimetically reduced Au ions leading to the formation of Au nanoparticles at a pH range of 4–9. It was found that at a pH range of 4–7, uniform spherical gold nanoparticles in the size ranges of 20–30nm were obtained, while under basic conditions, a high degree of aggregation was observed. In addition, we conjugated IAA with the organic linker 1,4-diaminobutane in order to enhance the attachment of Au ions to the IAA moiety via the free amino group and examined its self-assembly. In general, higher yields of nanofibers were formed upon self-assembly of the amide conjugate, with lengths in the micron range. Further, it was observed that the Au nanoparticles formed in the presence of the self-assembled amide conjugates were uniformly coated, leading to the formation of uniform gold nanowires. Thus, a new class of materials based on IAA and IAA-amide conjugates could be efficiently used for formation of gold nanoparticles using environmentally friendly mild synthetic methods in the absence of harsh reducing agents. Such materials may have potential applications in optoelectronics, bioimaging and sensing.

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

Indole-3-acetic acid (IAA) amidosynthetases catalyzing the ATP-dependent conjugation of IAA and amino acids play an important role in the maintenance of auxin homeostasis in plant cells. A new amidosynthetase, indole-3-acetic acid:l-aspartic acid ligase (IAA-Asp synthetase) involved in IAA-amino acid biosynthesis, was isolated via a biochemical approach from immature seeds of the pea (Pisum sativum L). The enzyme was purified to homogeneity by a three-step procedure, involving PEG 6000 fractionation, DEAE-Sephacel anion-exchange chromatography, and preparative PAGE, and characterized as a 70-kDa monomeric protein by analytical gel filtration and SDS-PAGE. Rabbit antiserum against recombinant AtGH3.5 cross-reacted with the pea IAA-Asp synthetase, and a single immunoreactive polypeptide band was observed at 70 kDa. The purified enzyme had an apparent isoelectric point at pH 4.7, the highest activity at pH 8.2, preferred Mg2+ as a cofactor, and was strongly activated by reducing agents. Similar to known recombinant GH3 enzymes, an IAA-Asp synthetase from pea catalyzes the conjugation of phytohormone acyl substrates to amino acids. The enzyme had the highest synthesizing activity on IAA, followed by 1-NAA, SA, 2,4-D, and IBA, whereas activities on l-Trp, IPA, PAA, (±)JA, and 2-NAA were not significant or not detected. Of 14 amino acids tested, the enzyme had the highest activity on Asp and lower activity on Ala and Lys. Glutamate was found to be a very poor substrate and no conjugating activity was observed on the rest of the amino acids. Steady-state kinetic analysis indicated that IAA and aspartate were preferred substrates for the pea IAA-Asp synthetase. The enzyme exhibited both higher affinities for IAA and Asp (K m = 0.2 and 2.5 mM, respectively) and catalytic efficiencies (k cat/K m = 682,608.7 and 5080 s−1 M−1, respectively) compared with other auxins and amino acids examined. This study describes the first amidosynthetase isolated and purified from plant tissue and provides the foundation for future genetic approaches to explain the role of IAA-Asp in Pisum sativum physiology.

Altered Architecture and Enhanced Drought Tolerance in Rice via the Down-Regulation of Indole-3-Acetic Acid by TLD1/OsGH3.13 Activation

Plant architecture is determined by genetic and developmental programs as well as by environmental factors. Sessile plants have evolved a subtle adaptive mechanism that allows them to alter their growth and development during periods of stress. Phytohormones play a central role in this process; however, the molecules responsible for integrating growth- and stress-related signals are unknown. Here, we report a gain-of-function rice (Oryza sativa) mutant, tld1-D, characterized by (and named for) an increased number of tillers, enlarged leaf angles, and dwarfism. TLD1 is a rice GH3.13 gene that encodes indole-3-acetic acid (IAA)-amido synthetase, which is suppressed in aboveground tissues under normal conditions but which is dramatically induced by drought stress. The activation of TLD1 reduced the IAA maxima at the lamina joint, shoot base, and nodes, resulting in subsequent alterations in plant architecture and tissue patterning but enhancing drought tolerance. Accordingly, the decreased level of free IAA in tld1-D due to the conjugation of IAA with amino acids greatly facilitated the accumulation of late-embryogenesis abundant mRNA compared with the wild type. The direct regulation of such drought-inducible genes by changes in the concentration of IAA provides a model for changes in plant architecture via the process of drought adaptation, which occurs frequently in nature.

Isolation of novel indole-3-acetic acid conjugates by immunoaffinity extraction

An analytical protocol for the isolation and quantification of indole-3-acetic acid (IAA) and its amino acid conjugates was developed. IAA is an important phytohormone and formation of its conjugates plays a crucial role in regulating IAA levels in plants. The developed protocol combines a highly specific immunoaffinity extraction with a sensitive and selective LC–MS/MS analysis. By using internal standards for each of the studied compounds, IAA and seven amino acid conjugates were analyzed in quantities of fresh plant material as low as 30 mg. In seeds of Helleborus niger, physiological levels of these compounds were found to range from 7.5 nmol g −1 fresh weight (IAA) to 0.44 pmol g −1 fresh weight (conjugate with Ala). To our knowledge, the identification of IAA conjugates with Gly, Phe and Val from higher plants is reported here for the first time.

Dead end for auxin conjugates in Physcomitrella?

For proper development of plants auxin levels need to be tightly controlled. For this, several routes have evolved and it is plausible that different organisms use these differently. To determine whether members of the family of GH3 proteins, which partially act as auxin conjugate synthetases in Arabidopsis thaliana, have similar roles in the moss Physcomitrella patens, we have investigated the in vitro activity of the two GH3 members in moss. We showed that both proteins can form amino acid conjugates with indole-3-acetic acid (IAA) but also with jasmonic acid (JA). Confirming these findings, single and double knockout-mutants showed lower levels of IAA conjugates than wild type. We discuss the results in light of the possible functions of IAA conjugate formation in lower land plants.