Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3.
ABSTRACT: The plant hormone auxin regulates many aspects of plant growth and development. Although several auxin biosynthetic pathways have been proposed, none of these pathways has been precisely defined at the molecular level. Here we provide in planta evidence that the two Arabidopsis cytochrome P450s, CYP79B2 and CYP79B3, which convert tryptophan (Trp) to indole-3-acetaldoxime (IAOx) in vitro, are critical enzymes in auxin biosynthesis in vivo. IAOx is thus implicated as an important intermediate in auxin biosynthesis. Plants overexpressing CYP79B2 contain elevated levels of free auxin and display auxin overproduction phenotypes. Conversely, cyp79B2 cyp79B3 double mutants have reduced levels of IAA and show growth defects consistent with partial auxin deficiency. Together with previous work on YUCCA, a flavin monooxygenase also implicated in IAOx production, and nitrilases that convert indole-3-acetonitrile to auxin, this work provides a framework for further dissecting auxin biosynthetic pathways and their regulation.
Project description:Early age-related developmental senescence was observed in Arabidopsis cyp79B2/cyp79B3 double mutants that cannot produce indole-3-acetaldoxime (IAOx), the precursor to indole glucosinolates (IGs), camalexin and auxin. The early senescence phenotype was not observed when senescence was induced by darkness. The cyp79B2/cyp79B3 mutants had lower auxin levels, but did not display auxin-deficient phenotypes. Camalexin biosynthesis mutants senesced normally; however, IG transport and exosome-related pen1/pen3 double mutants displayed early senescence. The early senescence in pen1/pen3 mutants depended on salicylic acid and was not observed in pen1 or pen3 single mutants. Quantitation of IGs showed reduced levels in cyp79B2/cyp79B3 mutants, but unchanged levels in pen1/pen3, even though both of these double mutants display early senescence. We discuss how these genetic data provide evidence that IAOx metabolites are playing a protective role in leaf senescence that is dependent on proper trafficking by PEN1 and PEN3, perhaps via the formation of exosomes.
Project description:Plants synthesize numerous secondary metabolites that are used as developmental signals or as defense against pathogens. Tryptophan (Trp)-derived secondary metabolites include camalexin, indole glucosinolates, and indole-3-acetic acid (IAA); however, the steps in their synthesis from Trp or its precursors remain unclear. We have identified two Arabidopsis cytochrome P450s (CYP79B2 and CYP79B3) that can convert Trp to indole-3-acetaldoxime (IAOx), a precursor to IAA and indole glucosinolates.
Project description:Plants produce diverse secondary metabolites. Although each metabolite is made through its respective biosynthetic pathway, plants coordinate multiple biosynthetic pathways simultaneously. One example is an interaction between glucosinolate and phenylpropanoid pathways in Arabidopsis thaliana. Glucosinolates are defense compounds made primarily from methionine and tryptophan, while phenylpropanoids are made from phenylalanine. Recent studies have shown that the accumulation of glucosinolate intermediate such as indole-3-acetaldoxime (IAOx) or its derivatives represses phenylpropanoid production via the degradation of phenylalanine ammonia lyase (PAL) functioning at the entry point of the phenylpropanoid pathway. Given that IAOx is a precursor of other bioactive compounds other than glucosinolates and that the phenylpropanoid pathway is present in most plants, we hypothesized that this interaction is relevant to other species. Camelina sativa is an oil crop and produces camalexin from IAOx. We enhanced IAOx production in Camelina by overexpressing Arabidopsis CYP79B2 which encodes an IAOx-producing enzyme. The overexpression of AtCYP79B2 results in increased auxin content and its associated morphological phenotypes in Camelina but indole glucosinolates were not detected in Camelina wild type as well as the overexpression lines. However, phenylpropanoid contents were reduced in AtCYP79B2 overexpression lines suggesting a link between aldoxime metabolism and phenylpropanoid production. Interestingly, the expression of PALs was not affected in the overexpression lines although PAL activity was reduced. To test if PAL degradation is involved in the crosstalk, we identified F-box genes functioning in PAL degradation through a phylogenetic study. A total of 459 transcript models encoding kelch-motifs were identified from the Camelina sativa database. Among them, the expression of CsKFBs involved in PAL degradation is up-regulated in the transgenic lines. Our results suggest a link between aldoxime metabolism and phenylpropanoid production in Camelina and that the molecular mechanism behind the crosstalk is conserved in Arabidopsis and Camelina.
Project description: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.
Project description:Auxins are hormones that regulate many aspects of plant growth and development. The main plant auxin is indole-3-acetic acid (IAA), whose biosynthetic pathway is not fully understood. Indole-3-acetaldoxime (IAOx) has been proposed to be a key intermediate in the synthesis of IAA and several other indolic compounds. Genetic studies of IAA biosynthesis in Arabidopsis have suggested that 2 distinct pathways involving the CYP79B or YUCCA (YUC) genes may contribute to IAOx synthesis and that several pathways are also involved in the conversion of IAOx to IAA. Here we report the biochemical dissection of IAOx biosynthesis and metabolism in plants by analyzing IAA biosynthesis intermediates. We demonstrated that the majority of IAOx is produced by CYP79B genes in Arabidopsis because IAOx production was abolished in CYP79B-deficient mutants. IAOx was not detected from rice, maize, and tobacco, which do not have apparent CYP79B orthologues. IAOx levels were not significantly altered in the yuc1 yuc2 yuc4 yuc6 quadruple mutants, suggesting that the YUC gene family probably does not contribute to IAOx synthesis. We determined the pathway for conversion of IAOx to IAA by identifying 2 likely intermediates, indole-3-acetamide (IAM) and indole-3-acetonitrile (IAN), in Arabidopsis. When (13)C(6)-labeled IAOx was fed to CYP79B-deficient mutants, (13)C(6) atoms were efficiently incorporated to IAM, IAN, and IAA. This biochemical evidence indicates that IAOx-dependent IAA biosynthesis, which involves IAM and IAN as intermediates, is not a common but a species-specific pathway in plants; thus IAA biosynthesis may differ among plant species.
Project description:Indole-3-acetaldoxime (IAOx) represents an early intermediate of the biosynthesis of a variety of indolic secondary metabolites including the phytoanticipin indol-3-ylmethyl glucosinolate and the phytoalexin camalexin (3-thiazol-2′-yl-indole). Arabidopsis thaliana cyp79B2 cyp79B3 double knockout plants are completely impaired in the conversion of tryptophan to indole-3-acetaldoxime and do not accumulate IAOx-derived metabolites any longer. Consequently, comparative analysis of wild-type and cyp79B2 cyp79B3 plant lines has the potential to explore the complete range of IAOx-derived indolic secondary metabolites.
Project description:The phytoalexin camalexin and indolic glucosinolates share not only a common evolutionary origin and a tightly interconnected biosynthetic pathway, but regulatory proteins controlling the shared enzymatic steps are also modulated by the same R2R3-MYB transcription factors. The indolic phytoalexin camalexin is a crucial defense metabolite in the model plant Arabidopsis. Indolic phytoalexins and glucosinolates appear to have a common evolutionary origin and are interconnected on the biosynthetic level: a key intermediate in the biosynthesis of camalexin, indole-3-acetaldoxime (IAOx), is also required for the biosynthesis of indolic glucosinolates and is under tight control by the transcription factors MYB34, MYB51, and MYB122. The abundance of camalexin was strongly reduced in myb34/51 and myb51/122 double and in triple myb mutant, suggesting that these transcription factors are important in camalexin biosynthesis. Furthermore, expression of MYB51 and MYB122 was significantly increased by biotic and abiotic camalexin-inducing agents. Feeding of the triple myb34/51/122 mutant with IAOx or indole-3-acetonitrile largely restored camalexin biosynthesis. Conversely, tryptophan could not complement the low camalexin phenotype of this mutant, which supports a role for the three MYB factors in camalexin biosynthesis upstream of IAOx. Consistently expression of the camalexin biosynthesis genes CYP71B15/PAD3 and CYP71A13 was not negatively affected in the triple myb mutant and the MYBs could not activate pCYP71B15::uidA expression in trans-activation assays with cultured Arabidopsis cells. In conclusion, this study reveals the importance of MYB factors regulating the generation of IAOx as precursor of camalexin.
Project description:Mutation of phytoglobin 2 (Pgb2) increases the number of somatic embryos in Arabidopsis. To assess the effects of the cellular localization of Pgb2 on embryo formation, an inducible system expressing a fusion protein consisting of Pgb2 linked to the steroid-binding domain of the rat glucocorticoid receptor (GR) was introduced in a pgb2 mutant line lacking the ability to express Pgb2. In this transgenic system, Pgb2 remains in the cytoplasm but migrates into the nucleus upon exposure to dexamethasone (DEX). Pgb2 retention in the cytoplasm, in the absence of DEX, increased the number of somatic embryos and reduced the expression of MYC2 - an inhibitor of the synthesis of auxin, which is the inductive signal for embryogenesis. Removal of DEX also induced the expression of several genes involved in the biosynthesis of tryptophan and the auxin, indole-3-acetic acid (IAA). These genes included: tryptophan synthase-? subunit (TSA1) and tryptophan synthase-? subunit (TSB1), which are involved in the synthesis of tryptophan, cytochrome P450 CYP79B2 (CYP79B2) and amidase 1 (AMI1), which participate in the formation of IAA via indole-3-acetaldoxime, and several members of the YUCCA family, including YUC1 and 4, which are also required for IAA synthesis. Retention of Pgb2 in the cytoplasm by removal of DEX increased the staining pattern of IAA along the cotyledons of the explants generating embryogenic tissue. Staining for IAA decreased when Pgb2 translocated into the nucleus in response to the application of DEX. Collectively, these results suggest that the presence of Pgb2 in the cytoplasm, but not in the nucleus, phenocopies the effects of Pgb2 mutation in inducing somatic embryogenesis.
Project description:Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.
Project description:Nitrilases (nitrile aminohydrolase, EC 18.104.22.168) convert nitriles to carboxylic acids. We report the cloning, characterization, and expression patterns of four Arabidopsis thaliana nitrilase genes (NIT1-4), one of which was previously described [Bartling, D., Seedorf, M., Mithöfer, A. & Weiler, E. W. (1992) Eur. J. Biochem. 205, 417-424]. The nitrilase genes encode very similar proteins that hydrolyze indole-3-acetonitrile to the phytohormone indole-3-acetic acid in vitro, and three of the four genes are tandemly arranged on chromosome III. Northern analysis using gene-specific probes and analysis of transgenic plants containing promoter-reporter gene fusions indicate that the four genes are differentially regulated. NIT2 expression is specifically induced around lesions caused by bacterial pathogen infiltration. The sites of nitrilase expression may represent sites of auxin biosynthesis in A. thaliana.