title: Cytochrome P450 CYP71AT96 catalyses the final step of herbivore-induced phenylacetonitrile biosynthesis in the giant knotweed, Fallopia sachalinensis.
abstract: The giant knotweed Fallopia sachalinensis (Polygonaceae) synthesizes phenylacetonitrile (PAN) from L-phenylalanine when infested by the Japanese beetle Popillia japonica or treated with methyl jasmonate (MeJA). Here we identified (E/Z)-phenylacetaldoxime (PAOx) as the biosynthetic precursor of PAN and identified a cytochrome P450 that catalysed the conversion of (E/Z)-PAOx to PAN. Incorporation of deuterium-labelled (E/Z)-PAOx into PAN emitted from the leaves of F. sachalinensis was detected using gas chromatography-mass spectrometry. Further, using liquid chromatography-tandem mass spectrometry, we detected the accumulation of (E/Z)-PAOx in MeJA-treated leaves. These results showed that (E/Z)-PAOx is the biosynthetic precursor of PAN. MeJA-induced mRNAs were analysed by differential expression analysis using a next-generation sequencer. Of the 74,329 contigs obtained from RNA-seq and de novo assembly, 252 contigs were induced by MeJA treatment. Full-length cDNAs encoding MeJA-induced cytochrome P450s CYP71AT96, CYP82AN1, CYP82D125 and CYP715A35 were cloned using 5'- and 3'-RACE and were expressed using a baculovirus expression system. Among these cytochrome P450s, CYP71AT96 catalysed the conversion of (E/Z)-PAOx to PAN in the presence of NADPH and a cytochrome P450 reductase. It also acted on (E/Z)-4-hydroxyphenylacetaldoxime and (E/Z)-indole-3-acetaldoxime. The broad substrate specificity of CYP71AT96 was similar to that of aldoxime metabolizing cytochrome P450s. Quantitative RT-PCR analysis showed that CYP71AT96 expression was highly induced because of treatment with MeJA as well as feeding by the Japanese beetle. These results indicate that CYP71AT96 likely contributes the herbivore-induced PAN biosynthesis in F. sachalinensis.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.|Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.
authors.forename: Takuya
authors.identifier: http://orcid.org/0000-0002-7533-430X
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Department of Biological Production, Akita Prefectural University, Akita, 010-0195, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Noge
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan. asano@pu-toyama.ac.jp.|Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan. asano@pu-toyama.ac.jp.
authors.forename: Yasuhisa
authors.identifier: http://orcid.org/0000-0003-3645-3952
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000097:Acetonitriles / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D001517:Coleoptera; D003517:Cyclopentanes / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D000069263:Fallopia / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D008401:Gas Chromatography-Mass Spectrometry; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000502:physiology; D060434:Herbivory; D054883:Oxylipins / Q000494:pharmacology; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000085:Acetates; D000097:Acetonitriles; D003517:Cyclopentanes; D054883:Oxylipins; D010940:Plant Proteins; C006725:benzyl cyanide; C072239:methyl jasmonate; D003577:Cytochrome P-450 Enzyme System
keywords: Cytochrome P450; Fallopia sachalinensis; Herbivore-induced volatiles; Jasmonates; Nitrile; Popillia japonica