camalexin and indole-3-acetaldoxime

In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO3-challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism.

Topics: Arabidopsis; Arabidopsis Proteins; Base Sequence; Cytochrome P-450 Enzyme System; Deoxyribonucleases; Gene Knockout Techniques; Indoles; Inheritance Patterns; Metabolomics; Models, Biological; Molecular Sequence Data; Mutagenesis; Mutation; Oximes; Secondary Metabolism; Thiazoles; Trans-Activators

Indole-3-acetaldoxime (IAOx) is a key branching point between primary and secondary metabolism. IAOx serves as an intermediate in the biosynthesis of indole glucosinolates (I-GLSs), camalexin and the plant hormone indole-3-acetic acid (IAA). The cytochrome P450s CYP79B2 and CYP79B3 catalyze the conversion of tryptophan to IAOx. CYP83B1 channels IAOx into I-GLS biosynthesis, CYP71A13 channels IAOx into camalexin biosynthesis, whereas the IAOx-metabolizing enzyme in IAA biosynthesis is not known. In this report, we demonstrate controlled production of I-GLSs by introducing an ethanol (EtOH)-inducible CYP79B2 construct into double (cyp79b2 cyp79b3) or triple (cyp79b2 cyp79b3 cyp83b1) mutant lines. We show EtOH-dependent induction of camalexin and identify a number of candidate IAA homeostasis- or defense-related genes by clustered microarray analysis. The transgenic mutant lines are thus promising tools for elucidating the interplay between primary and secondary metabolism.

Topics: Arabidopsis; Arabidopsis Proteins; Biosynthetic Pathways; Chromatography, Liquid; Cluster Analysis; Cytochrome P-450 Enzyme System; Ethanol; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Glucosinolates; Indoleacetic Acids; Indoles; Mass Spectrometry; Molecular Structure; Mutation; Oligonucleotide Array Sequence Analysis; Oximes; Plants, Genetically Modified; Thiazoles

Camalexin (3-thiazol-2-yl-indole) is an indole alkaloid phytoalexin produced by Arabidopsis thaliana that is thought to be important for resistance to necrotrophic fungal pathogens, such as Alternaria brassicicola and Botrytis cinerea. It is produced from Trp, which is converted to indole acetaldoxime (IAOx) by the action of cytochrome P450 monooxygenases CYP79B2 and CYP79B3. The remaining biosynthetic steps are unknown except for the last step, which is conversion of dihydrocamalexic acid to camalexin by CYP71B15 (PAD3). This article reports characterization of CYP71A13. Plants carrying cyp71A13 mutations produce greatly reduced amounts of camalexin after infection by Pseudomonas syringae or A. brassicicola and are susceptible to A. brassicicola, as are pad3 and cyp79B2 cyp79B3 mutants. Expression levels of CYP71A13 and PAD3 are coregulated. CYP71A13 expressed in Escherichia coli converted IAOx to indole-3-acetonitrile (IAN). Expression of CYP79B2 and CYP71A13 in Nicotiana benthamiana resulted in conversion of Trp to IAN. Exogenously supplied IAN restored camalexin production in cyp71A13 mutant plants. Together, these results lead to the conclusion that CYP71A13 catalyzes the conversion of IAOx to IAN in camalexin synthesis and provide further support for the role of camalexin in resistance to A. brassicicola.

Topics: Alternaria; Arabidopsis; Arabidopsis Proteins; Carbon Monoxide; Catalysis; Cytochrome P-450 Enzyme System; DNA, Bacterial; Gene Expression Regulation, Plant; Genes, Plant; Immunity, Innate; Indoles; Mutagenesis, Insertional; Nicotiana; Oximes; Plant Diseases; Plant Leaves; Pseudomonas syringae; Recombinant Proteins; RNA, Messenger; Substrate Specificity; Thiazoles

Characteristic for cruciferous plants is their production of N- and S-containing indole phytoalexins with disease resistance and cancer-preventive properties, previously proposed to be synthesized from indole independently of tryptophan. We show that camalexin, the indole phytoalexin of Arabidopsis thaliana, is synthesized from tryptophan via indole-3-acetaldoxime (IAOx) in a reaction catalyzed by CYP79B2 and CYP79B3. Cyp79B2/cyp79B3 double knockout mutant is devoid of camalexin, as it is also devoid of indole glucosinolates [Zhao, Y., Hull, A. K., Gupta, N. R., Goss, K. A., Alonso, J., Ecker, J. R., Normanly, J., Chory, J. & Celenza, J. L. (2002) Genes Dev. 16, 3100-3112], and isotope-labeled IAOx is incorporated into camalexin. These results demonstrate that only CYP79B2 and CYP79B3 contribute significantly to the IAOx pool from which camalexin and indole glucosinolates are synthesized. Furthermore, production of camalexin in the sur1 mutant devoid of glucosinolates excludes the possibility that camalexin is derived from indole glucosinolates. CYP79B2 plays an important role in camalexin biosynthesis in that the transcript level of CYP79B2, but not CYP79B3, is increased upon induction of camalexin by silver nitrate as evidenced by microarray analysis and promoter-beta-glucuronidase data. The structural similarity between cruciferous indole phytoalexins suggests that these compounds are biogenetically related and synthesized from tryptophan via IAOx by CYP79B homologues. The data show that IAOx is a key branching point between several secondary metabolic pathways as well as primary metabolism, where IAOx has been shown to play a critical role in IAA homeostasis.

Topics: Arabidopsis; Cytochrome P-450 Enzyme System; Enzyme Induction; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Plant; Indoles; Oligonucleotide Array Sequence Analysis; Oximes; Plant Leaves; Promoter Regions, Genetic; Silver Nitrate; Thiazoles