Cloning and Characterization of cDNAs Encoding<i>ent</i>-Copalyl Diphosphate Synthases in Wheat: Insight into the Evolution of Rice Phytoalexin Biosynthetic Genes

Toyomasu, Tomonobu; Kagahara, Takuma; Hirose, Yuko; Usui, Masami; Abe, Shiho; Okada, Kazunori; Koga, Jinichiro; Mitsuhashi, Wataru; Yamane, Hisakazu

doi:10.1271/bbb.80781

Cited by 27 publications

(31 citation statements)

References 12 publications

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“…All of these enzymes are monofunctional. A similar pattern of all monofunctional diTPSs is found in general and specialized metabolism of other members of the grass (Poacea) family (Toyomasu et al, 2009) and in Stevia rebaudina, a dicot species that produces several specialized diterpene glycosides (Richman et al, 1999). In contrast to the conifer system described here, and to the best of our knowledge, there is no report of both monofunctional and bifunctional diTPS in any angiosperm system.…”

Section: Discussionsupporting

confidence: 80%

Identification and Functional Characterization of Monofunctionalent-Copalyl Diphosphate andent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

et al. 2009

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The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for entkaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

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Section: Discussionsupporting

confidence: 80%

Identification and Functional Characterization of Monofunctionalent-Copalyl Diphosphate andent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

et al. 2009

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“…Previously, it has been shown that conifer diTPSs involved in specialized metabolism of DRAs are bifunctional class I/II enzymes that catalyze the conversion of GGPP, through the stable intermediate (+)-CPP, to produce diterpene olefins or alcohols (Vogel et Ro and Bohlmann, 2006;Keeling et al, 2011b;Zerbe et al, 2012a). Conversely, the general GA metabolism in gymnosperms and angiosperms, and likewise the specialized metabolism of labdane-related diterpenoids in angiosperms, involve separate consecutively acting monofunctional class I and class II enzymes (Richman et al, 1999;Xu et al, 2004;Harris et al, 2005;Prisic and Peters, 2007;Xu et al, 2007a;Gao et al, 2009;Toyomasu et al, 2009;Keeling et al, 2010;Falara et al, 2010;Caniard et al, 2012).…”

Section: Monofunctional Ditpss Are New Modules In the Biosynthesis Ofmentioning

confidence: 99%

“…diTPSs involved in general metabolism of GA biosynthesis in angiosperms and gymnosperms, as well as those involved in the biosynthesis of the large class of labdane-related specialized diterpenes in angiosperms, are monofunctional class II and class I enzymes Peters, 2010). The class II diTPSs contain a DxDD signature motif in the gb-domain relevant for catalyzing the protonation-initiated cyclization of GGPP to a bicyclic diphosphate intermediate (Harris et al, 2005;Prisic and Peters, 2007;Xu et al, 2007a;Gao et al, 2009;Toyomasu et al, 2009;Falara et al, 2010;Keeling et al, 2010;Caniard et al, 2012;Sallaud et al, 2012). Class I diTPSs, which harbor DDxxD and NSE/DTE functional motifs in the a-domain, then catalyze the ionization of the diphosphate ester and subsequent rearrangement reactions (Xu et al, 2007a;Gao et al, 2009;Keeling et al, 2010;Caniard et al, 2012;Sallaud et al, 2012).…”

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confidence: 99%

Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases

et al. 2012

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Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs.Conifer trees, including lodgepole pine (Pinus contorta) and jack pine (Pinus banksiana), produce complex mixtures of mono-, sesqui-, and diterpenoid specialized (i.e. secondary) metabolites, most prominently in the form of oleoresin, that can act as a physical and chemical defense against insect and pathogen attack

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“…Hence, the upstream enzymes, ent-CPP synthase (CPS), KS, and kaurene oxidase (KO)/CYP701, can be diverted to alternative labdane-related diterpenoid biosynthesis. Many examples of CPS and KS-like (KSL) diterpene synthases that operate in more specialized metabolism are known Nemoto et al, 2004;Otomo et al, 2004aOtomo et al, , 2004bPrisic et al, 2004;Wilderman et al, 2004;Xu et al, 2004Xu et al, , 2007Harris et al, 2005;Kanno et al, 2006;Morrone et al, 2006;Gao et al, 2009;Toyomasu et al, 2009;Falara et al, 2010). By contrast, no KO/ CYP701 family member has been shown to act as anything other than a KO-i.e.…”

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confidence: 99%

CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism

et al. 2012

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All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4a-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3a position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3a-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice.

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Cloning and Characterization of cDNAs Encodingent-Copalyl Diphosphate Synthases in Wheat: Insight into the Evolution of Rice Phytoalexin Biosynthetic Genes

Cited by 27 publications

References 12 publications

Identification and Functional Characterization of Monofunctionalent-Copalyl Diphosphate andent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

Identification and Functional Characterization of Monofunctionalent-Copalyl Diphosphate andent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases

CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism

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