Characterization of CYP71Z18 indicates a role in maize zealexin biosynthesis

Abstract

Maize (Zea mays) produces zealexins as phytoalexins, with the inducible production of these antibiotics providing biochemical protection against fungal infection. However, the biosynthesis of these sesquiterpenoids has remained unclear. In particular, it is unclear how the olefinic precursor, (S)-β-macrocarpene produced by the characterized maize sesquiterpene synthases TPS6/11, is further elaborated to form the bioactive zealexins. The first step is likely to be conversion of carbon-15 (C15) from a methyl group to a carboxylic acid by a cytochrome P450 mono-oxygenase (CYP). In this study, CYP71Z18, whose transcription is strongly induced by fungal infection, was found to catalyze oxidation of C15 in (S)-β-macrocarpene, forming zealexin A1. The inducible transcription of CYP71Z18 matches that observed for TPS6/11 and the accumulation of zealexins, which is consistent with a role for CYP71Z18 in sesquiterpenoid phytoalexin production. This completes identification of zealexin A1 biosynthesis, and represents the initial CYP identified for the production of maize terpenoid phytoalexins.

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

“…Error bars represent propagated SE values (n = 6), and letters (a-d) represent significant differences at P , 0.05 as measured using ANOVA and Tukey's tests to correct for multiple comparisons between control and treatments. the capacity of ZmCYP71Z16 and ZmCYP71Z18 (previously reported to function in zealexin biosynthesis; Mao et al, 2016) to convert both sesquiterpenoid and diterpenoid intermediates (Supplemental Fig. S6).…”

“…To analyze the catalytic specificity of ZmCYP71Z18 , the full-length construct was coexpressed with ZmCPR2 in S. cerevisiae strain BY4741 (Jensen et al, 2011;Mao et al, 2016). Whole-cell assays were conducted as reported earlier (Pompon et al, 1996).…”

“…In the absence of mutant analyses, the maize kauralexin and zealexin metabolic pathways have remained largely unproven, with the exception of the ent-CPP synthase ZmAN2, which has been empirically demonstrated to function in kauralexin formation (Harris et al, 2005;Vaughan et al, 2015). The b-macrocarpene synthases ZmTPS6, ZmTPS11, and ZmCYP71Z18 have been assigned to the zealexin pathway based on predictions from in vivo biochemical assays (Köllner et al, 2008b;Mao et al, 2016).…”

Comparative functional genomics analysis of cytochrome P450 gene superfamily in wheat and maize

“…As for production of phytoalexin, CYP79B2, CYP71A13 and CYP71B15 respectively catalyze the conversion of tryptophan to indole-3-acetaldoxime, indole-3-acetaldoxime to indole-3-acetonitrile, cysteine-indole-3-acetonitrile to camalexin in Arabidopsis [22][23][24]. In maize, the role of CYP71Z18 in antibiotic zealexin biosynthesis by catalyzing oxidation of C15 in (S)-β-macrocarpene is verified using in vitro assays [25]. And both in vitro and in vivo experiments revealed that ZmCYP79A61 accept Lphenylalanine as substrate to produce phenylacetaldoxime, and simulated herbivory on maize leaves cause an increased accumulation of ZmCYP79A61 transcripts and phenylacetaldoxime, which suggests the enzyme contributes to maize herbivore-induced aldoxime formation [26].…”

Genetic elucidation of complex biochemical traits mediating maize innate immunity

“…While correlations between ZmTPS6 ⁄ 11 transcripts, zealexin production and fungal resistance exist, all known maize lines produce zealexins and no single biosynthetic pathway node has been proven in planta . Many catalytic activities and biological roles have been assigned to the 43 TPS encoded in the maize B73 genome 34, 35 ; however, the structural diversity of zealexins, combinations of underlying TPS/P450 terpenoid-diversifying genes, and the endogenous protective function of the zealexin pathway remains unresolved 27, 36 . Recent advances in omic tools, co-regulation analyses, genetic resources, in vivo protein biochemistry and gene editing approaches now enable the critical examination and engineering of complex protective pathways underlying crop resistance.…”