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
ent-kaurene 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.Conifers (Coniferophyta) are well known for producing an abundant and diverse assortment of oleoresin diterpenoids, predominantly in the form of diterpene resin acids from specialized (or secondary) metabolism, that play roles in conifer defense (
Trapp and Croteau, 2001a;
Keeling and Bohlmann, 2006a;
Bohlmann, 2008) and are an important source of biomaterials (
Bohlmann and Keeling, 2008). Several conifer diterpene synthases (diTPSs) that biosynthesize these compounds have been functionally characterized (
Stofer Vogel et al., 1996;
Peters et al., 2000;
Martin et al., 2004;
Keeling and Bohlmann, 2006b;
Ro and Bohlmann, 2006). The formation of diterpene resin acids of conifer specialized metabolism parallels the formation of
ent-kaurenoic acid in the biosynthesis of the gibberellin diterpenoid phytohormones (;
Keeling and Bohlmann, 2006a;
Yamaguchi, 2008). In gibberellin biosynthesis, geranylgeranyl diphosphate (GGPP) is cyclized by diTPS activity to
ent-copalyl diphosphate (
ent-CPP), and the
ent-CPP is further cyclized by diTPS activity to
ent-kaurene. A cytochrome P450 (P450)-dependent monooxygenase (CYP701) oxidizes
ent-kaurene to
ent-kaurenoic acid (
Davidson et al., 2006), paralleling the activity of a P450 (CYP720B1) that oxidizes abietadiene to abietic acid in conifer diterpene resin acid biosynthesis (
Ro et al., 2005). Other P450s further functionalize
ent-kaurenoic acid to form the biologically active gibberellins. Surprisingly, no conifer diTPS involved in the general (or primary) metabolism of gibberellins has been reported to date, while metabolite profiles of gibberellins have been well characterized in conifers for their role in flowering (
Moritz et al., 1990).
Open in a separate windowComparison of the biosynthesis of gibberellins, as it is known in angiosperm and lower plants, with the biosynthesis of diterpene resin acids in conifers, a large group of gymnosperm trees. In conifers, the formation of diterpene resin acids involves bifunctional diTPS (e.g. abietadiene synthase) for the stepwise cyclization of GGPP into diterpenes such as abietadiene via a copalyl diphosphate intermediate that moves between the two active sites of the bifunctional diTPS (
Peters et al., 2001). The products of the diTPS are subsequently oxidized by P450 to the resin acids. In contrast, gibberellin biosynthesis in angiosperms requires two monofunctional diTPSs to convert GGPP into
ent-kaurene, which is subsequently modified by P450s. The two monofunctional diTPSs in angiosperm gibberellin biosynthesis are CPS and KS. In the lower plant
P. patens, the CPS and KS activities are combined in a bifunctional diTPS similar to the bifunctional diTPS in conifer diterpene resin acid biosynthesis. Prior to this work, to our knowledge, it was not known if the formation of gibberellins in a gymnosperm involves two monofunctional diTPSs, as in angiosperms, or a bifunctional diTPS, as in gymnosperm diterpene resin acid biosynthesis and in
P. patens gibberellin biosynthesis. (Figure adapted from
Keeling and Bohlmann [2006a].)In the fungi
Gibberella fujikuroi (
Toyomasu et al., 2000) and
Phaeosphaeria species L487 (
Kawaide et al., 1997) and in the primitive land plant
Physcomitrella patens (Bryophyta;
Hayashi et al., 2006;
Anterola and Shanle, 2008), the formation of
ent-kaurene from GGPP is catalyzed by bifunctional diTPS enzymes. These enzymes contain two active sites. The N-terminal active site domain harbors a conserved DXDD motif and catalyzes the protonation-initiated cyclization of GGPP to
ent-CPP (
Prisic et al., 2007). In the C-terminal active site domain, a conserved DDXXD motif is essential for the diphosphate ionization-initiated cyclization of
ent-CPP to
ent-kaurene (
Christianson, 2006). The presence of two active sites with their characteristic DXDD and DDXXD motifs resembles the structure of conifer bifunctional diTPSs in specialized metabolism of diterpene resin acid biosynthesis (), such as the grand fir (
Abies grandis) abietadiene synthase (AgAS) and Norway spruce (
Picea abies) levopimaradiene/abietadiene synthases (PaLAS;
Peters et al., 2001;
Martin et al., 2004;
Keeling and Bohlmann, 2006a). In contrast, the formation of
ent-kaurene from GGPP in angiosperms is catalyzed by two separate monofunctional enzymes, one with only the DXDD motif and having
ent-copalyl diphosphate synthase (
ent-CPS) activity and the other with only the DDXXD motif and having
ent-kaurene synthase (
ent-KS) activity (
Yamaguchi, 2008).A previously published model for the evolution of plant diTPS (
Trapp and Croteau, 2001b) suggests that genes encoding the monofunctional CPS and KS enzymes known in angiosperms originated by gene duplication and subfunctionalization (
Lynch and Force, 2000) of an ancestral bifunctional
CPS/KS gene that may have been similar to the gene for the CPS/KS enzyme of the moss
P. patens. The same model also suggests that genes for diTPSs of gymnosperm specialized diterpene resin acid metabolism arose from duplication and subsequent neofunctionalization of an ancestral bifunctional diTPS of the gibberellin pathway (
Trapp and Croteau, 2001b). The pathways to specialized oleoresin diterpenes existed in ancient plants prior to the differentiation of gymnosperms and angiosperms (
Bray and Anderson, 2009). Vascular plants split from nonvascular plants approximately 500 million years ago, and angiosperms split from gymnosperms approximately 300 million years ago (
Palmer et al., 2004). As there has been no report to date of genes involved in gibberellin biosynthesis in gymnosperms, it remains unresolved and cannot be predicted whether conifers have a bifunctional CPS/KS for the formation of
ent-kaurene similar to the primitive land plant
P. patens and paralleling the diTPSs for conifer specialized diterpene resin acid biosynthesis or whether they have separate monofunctional CPS and KS enzymes, as is the case in angiosperms.In this study, we made use of the extensive EST resources for spruce species (
Pavy et al., 2005;
Ralph et al., 2008), combined with isolation and sequencing of full-length cDNAs, genomic (g)DNA, and targeted bacterial artificial chromosome (BAC) clones, as well as enzyme assays with recombinant proteins to search for, and functionally characterize, possible monofunctional or bifunctional diTPS for
ent-kaurene biosynthesis in a gymnosperm. In summary, we successfully isolated and characterized monofunctional
ent-CPS (PgCPS) and
ent-KS (PgKS) from white spruce (
Picea glauca) and isolated orthologous cDNAs from Sitka spruce (
Picea sitchensis). Comparison of enzyme functions and gene structures support common ancestry but different routes of evolution of monofunctional and bifunctional diTPS in conifer general and specialized metabolism, respectively.
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