Rice produces many diterpenoid phytoalexins and,reflecting the importance of these natural products in this important cereal crop plant,its genome contains three biosynthetic gene clusters(BGCs)for such metabolism.The...Rice produces many diterpenoid phytoalexins and,reflecting the importance of these natural products in this important cereal crop plant,its genome contains three biosynthetic gene clusters(BGCs)for such metabolism.The chromosome 4 BGC(c4BGC)is largely associated with momilactone production,in part due to the presence of the initiating syn-copalyl diphosphate(CPP)synthase gene(OsCPS4).Oryzalexin S is also derived from syn-CPP.However,the relevant subsequently acting syn-stemarene synthase gene(OsKSL8)is not located in the c4BGC.Production of oryzalexin S further requires hydroxylation at carbons 2 and 19(C2 and C19),presumably catalyzed by cytochrome P450(CYP)monooxygenases.Here it is reported the closely related CYP99A2 and CYP99A3,whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation,while the closely related CYP71Z21 and CYP71Z22,whose genes are found in the recently reported chromosome 7 BGC(c7BGC),catalyze subsequent hydroxylation at C2α.Thus,oryzalexin S biosynthesis utilizes two distinct BGCs,in a pathway cross-stitched together by OsKSL8.Notably,in contrast to the widely conserved c4BGC,the c7BGC is subspecies(ssp.)specific,being prevalent in ssp.japonica and only rarely found in the other major ssp.indica.Moreover,while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8,it has now been reported to be a ssp.indica derived allele at the same genetic loci.Intriguingly,more detailed analysis indicates that OsKSL8(j)is being replaced by OsKSL11(OsKSL8i),suggesting introgression from ssp.indica to(sub)tropical japonica,with concurrent disappearance of oryzalexin S production.展开更多
Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,w...Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,were only recently reported.These aromatic abietanes are presumably derived from ent-miltiradiene,but such biosynthetic capacity has not yet been reported in O.sativa.While wild rice has been reported to contain such an enzyme,specifically ent-kaurene synthase-like 10(KSL10),the only characterized ortholog from O.sativa(OsKSL10),specifically from the well-studied cultivar(cv.)Nipponbare,instead has been shown to make ent-sandaracopimaradiene,precursor to the oryzalexins.Notably,in many other cultivars,OsKSL10 is accompanied by a tandem duplicate,termed here OsKSL14.Biochemical characterization of OsKLS14 from cv.Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene.Strikingly,phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv.Nipponbare is an outlier,whereas the alleles from most other cultivars group with those from wild rice,suggesting that these also might produce ent-miltiradiene.Indeed,OsKSL10 from cv.Kitaake exhibits such activity as well,consistent with its production of abietoryzins but not oryzalexins.Similarly consistent with these results is the lack of abietoryzin production by cv.Nipponbare.Although their equivalent product outcome might suggest redundancy,OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns,indicating such differences may underlie retention of these duplicated genes.Regardless,the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.展开更多
基金The authors thank Prof.Robert Coates(Univ.Illinois,ret.)for an authentic standard of oryzalexin S.This work was supported by Grants from the NIH(GM131885)and USDA(2020-67013-32557)to R.J.P.
文摘Rice produces many diterpenoid phytoalexins and,reflecting the importance of these natural products in this important cereal crop plant,its genome contains three biosynthetic gene clusters(BGCs)for such metabolism.The chromosome 4 BGC(c4BGC)is largely associated with momilactone production,in part due to the presence of the initiating syn-copalyl diphosphate(CPP)synthase gene(OsCPS4).Oryzalexin S is also derived from syn-CPP.However,the relevant subsequently acting syn-stemarene synthase gene(OsKSL8)is not located in the c4BGC.Production of oryzalexin S further requires hydroxylation at carbons 2 and 19(C2 and C19),presumably catalyzed by cytochrome P450(CYP)monooxygenases.Here it is reported the closely related CYP99A2 and CYP99A3,whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation,while the closely related CYP71Z21 and CYP71Z22,whose genes are found in the recently reported chromosome 7 BGC(c7BGC),catalyze subsequent hydroxylation at C2α.Thus,oryzalexin S biosynthesis utilizes two distinct BGCs,in a pathway cross-stitched together by OsKSL8.Notably,in contrast to the widely conserved c4BGC,the c7BGC is subspecies(ssp.)specific,being prevalent in ssp.japonica and only rarely found in the other major ssp.indica.Moreover,while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8,it has now been reported to be a ssp.indica derived allele at the same genetic loci.Intriguingly,more detailed analysis indicates that OsKSL8(j)is being replaced by OsKSL11(OsKSL8i),suggesting introgression from ssp.indica to(sub)tropical japonica,with concurrent disappearance of oryzalexin S production.
基金supported by grants from the NIH(GM131885)and USDA(2020-67013-32557)to R.J.P.
文摘Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,were only recently reported.These aromatic abietanes are presumably derived from ent-miltiradiene,but such biosynthetic capacity has not yet been reported in O.sativa.While wild rice has been reported to contain such an enzyme,specifically ent-kaurene synthase-like 10(KSL10),the only characterized ortholog from O.sativa(OsKSL10),specifically from the well-studied cultivar(cv.)Nipponbare,instead has been shown to make ent-sandaracopimaradiene,precursor to the oryzalexins.Notably,in many other cultivars,OsKSL10 is accompanied by a tandem duplicate,termed here OsKSL14.Biochemical characterization of OsKLS14 from cv.Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene.Strikingly,phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv.Nipponbare is an outlier,whereas the alleles from most other cultivars group with those from wild rice,suggesting that these also might produce ent-miltiradiene.Indeed,OsKSL10 from cv.Kitaake exhibits such activity as well,consistent with its production of abietoryzins but not oryzalexins.Similarly consistent with these results is the lack of abietoryzin production by cv.Nipponbare.Although their equivalent product outcome might suggest redundancy,OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns,indicating such differences may underlie retention of these duplicated genes.Regardless,the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.
