camalexin and indole-3-acetonitrile

The mycotoxin fumonisin B1 (FB1) causes the accumulation of reactive oxygen species (ROS) which then leads to programmed cell death (PCD) in Arabidopsis. In the process of studying FB1-induced biosynthesis of glucosinolates, we found that indole glucosinolate (IGS) is involved in attenuating FB1-induced PCD. Treatment with FB1 elevates the expression of genes related to the biosynthesis of camalexin and IGS. Mutants deficient in aliphatic glucosinolate (AGS) or camalexin biosynthesis display similar lesions to Col-0 upon FB1 infiltration; however, the cyp79B2 cyp79B3 double mutant, which lacks induction of both IGS and camalexin, displays more severe lesions. Based on the fact that the classic myrosinase β-thioglucoside glucohydrolase (TGG)-deficient double mutant tgg1 tgg2, rather than atypical myrosinase-deficient mutant pen2-2, is more sensitive to FB1 than Col-0, and the elevated expression of TGG1, but not of PEN2, correlates with the decrease in IGS, we conclude that TGG-dependent IGS hydrolysis is involved in FB1-induced PCD. Indole-3-acetonitrile (IAN) and indole-3-carbinol (I3C), the common derivatives of IGS, were used in feeding experiments, and this rescued the severe cell death phenotype, which is associated with reduced accumulation of ROS as well as increased activity of antioxidant enzymes and ROS-scavenging ability. Despite the involvement of indole-3-acetic acid (IAA) in restricting FB1-induced PCD, feeding of IAN and I3C attenuated FB1-induced PCD in the IAA receptor mutant tir1-1 just as in Col-0. Taken together, our results indicate that TGG-catalyzed breakdown products of IGS decrease the accumulation of ROS by their antioxidant behavior, and attenuate FB1 induced PCD in an IAA-independent way.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Death; Cytochrome P-450 Enzyme System; Fumonisins; Glucosinolates; Glycoside Hydrolases; Indoleacetic Acids; Indoles; Mutation; Thiazoles

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

Camalexin, a major phytoalexin in Arabidopsis thaliana, consists of an indole ring and a thiazole ring. The indole ring is produced from Trp, which is converted to indole-3-acetonitrile (IAN) by CYP79B2/CYP79B3 and CYP71A13. Conversion of Cys(IAN) to dihydrocamalexic acid and subsequently to camalexin is catalyzed by CYP71B15. Recent studies proposed that Cys derivative, not Cys itself, is the precursor of the thiazole ring that conjugates with IAN. The nature of the Cys derivative and how it conjugates to IAN and subsequently forms Cys(IAN) remain obscure. We found that protein accumulation of multiple glutathione S-transferases (GSTs), elevation of GST activity, and consumption of glutathione (GSH) coincided with camalexin production. GSTF6 overexpression increased and GSTF6-knockout reduced camalexin production. Arabidopsis GSTF6 expressed in yeast cells catalyzed GSH(IAN) formation. GSH(IAN), (IAN)CysGly, and γGluCys(IAN) were determined to be intermediates within the camalexin biosynthetic pathway. Inhibitor treatments and mutant analyses revealed the involvement of γ-glutamyl transpeptidases (GGTs) and phytochelatin synthase (PCS) in the catabolism of GSH(IAN). The expression of GSTF6, GGT1, GGT2, and PCS1 was coordinately upregulated during camalexin biosynthesis. These results suggest that GSH is the Cys derivative used during camalexin biosynthesis, that the conjugation of GSH with IAN is catalyzed by GSTF6, and that GGTs and PCS are involved in camalexin biosynthesis.

Topics: Arabidopsis; Arabidopsis Proteins; Gene Expression Regulation, Plant; Gene Knockout Techniques; Glutathione; Glutathione Transferase; Indoles; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mutagenesis, Insertional; Plants, Genetically Modified; Thiazoles

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, gamma-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate-treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.

Topics: Arabidopsis; Arabidopsis Proteins; Cyanides; Cysteine; Cytochrome P-450 Enzyme System; Indoles; Mass Spectrometry; Metabolomics; NADP; 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