Diterpene synthesis in Stevia rebaudiana: recruitment and up-regulation of key enzymes from the gibberellin biosynthetic pathway

Stevia rebaudiana Bertoni leaves accumulate a mixture of at least eight different glycosides derived from the tetracyclic diterpene steviol. These natural products taste intensely sweet and have similar biosynthetic origins to those of gibberellic acid (GA). The initial steps leading to the formation of GA result from the two-step cyclization of geranylgeranyl diphosphate (GGDP) to (-)-kaurene via the action of two terpene cyclases (-)-copalyl diphosphate synthase (CPS) and (-)-kaurene synthase (KS). Steviol biosynthesis probably uses the same mechanism although the genes and enzymes from S. rebaudiana that are involved in the cyclization of GGDP have not been characterized. We have isolated both the CPS and KS genes from S. rebaudiana and found that recombinant CPS and KS were catalytically active, suggesting that the CPS and KS genes participate in steviol biosynthesis. The genes coding for CPS and KS are usually present in single copies in most plant species and their expression is normally low and limited to rapidly growing tissues. The KS gene has been duplicated in the S. rebaudiana genome and both the KS and CPS genes are highly expressed in mature leaves, a pattern opposite to that found with GA biosynthesis. This pattern may, at least in part, lead to temporal and spatial separation of GA and steviol biosynthesis and probably helps to prevent over-expression from interfering with normal GA metabolism. Our results show that CPS and KS are part of the steviol glycoside biosynthetic pathway and that Stevia rebaudiana has recruited two genes to secondary metabolism from a highly regulated pathway involved in hormone biosynthesis.     

Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens

ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16alpha-hydroxykaurene).     

Functional Conservation of the Capacity for ent-Kaurene Biosynthesis and an Associated Operon in Certain Rhizobia

Bacterial interactions with plants are accompanied by complex signal exchange processes. Previously, the nitrogen-fixing symbiotic (rhizo)bacterium Bradyrhizobium japonicum was found to carry adjacent genes encoding two sequentially acting diterpene cyclases that together transform geranylgeranyl diphosphate to ent-kaurene, the olefin precursor to the gibberellin plant hormones. Species from the three other major genera of rhizobia were found to have homologous terpene synthase genes. Cloning and functional characterization of a representative set of these enzymes confirmed the capacity of each genus to produce ent-kaurene. Moreover, comparison of their genomic context revealed that these diterpene synthases are found in a conserved operon which includes an adjacent isoprenyl diphosphate synthase, shown here to produce the geranylgeranyl diphosphate precursor, providing a critical link to central metabolism. In addition, the rest of the operon consists of enzymatic genes that presumably lead to a more elaborated diterpenoid, although the production of gibberellins was not observed. Nevertheless, it has previously been shown that the operon is selectively expressed during nodulation, and the scattered distribution of the operon via independent horizontal gene transfer within the symbiotic plasmid or genomic island shown here suggests that such diterpenoid production may modulate the interaction of these particular symbionts with their host plants.     

Functional analysis of the two interacting cyclase domains in ent-kaurene synthase from the fungus Phaeosphaeria sp. L487 and a comparison with cyclases from higher plants

We report here kinetic analysis and identification of the two cyclase domains in a bifunctional diterpene cyclase, Phaeosphaeria ent-kaurene synthase (FCPS/KS). Kinetics of a recombinant FCPS/KS protein indicated that the affinity for copalyl diphosphate is higher than that for geranylgeranyl diphosphate (GGDP). ent-Kaurene production from GGDP by FCPS/KS was enhanced by the addition of a plant ent-kaurene synthase (KS) but not by plant CDP synthase (CPS), suggesting that the rate of ent-kaurene production of FCPS/KS may be limited by the KS activity. Site-directed mutagenesis of aspartate-rich motifs in FCPS/KS indicated that the (318)DVDD motif near the N terminus and the (656)DEFFE motif near the C terminus may be part of the active site for the CPS and KS reactions, respectively. The other aspartate-rich (132)DDVLD motif near the N terminus is thought to be involved in both reactions. Functional analysis of the N- and C-terminal truncated mutants revealed that a N-terminal 59-kDa polypeptide catalyzed the CPS reaction and a C-terminal 66-kDa polypeptide showed KS activity. A 101-kDa polypeptide lacking the first 43 amino acids of the N terminus reduced KS activity severely without CPS activity. These results indicate that there are two separate interacting domains in the 106-kDa polypeptide of FCPS/KS.     

Identification of the single amino acid involved in quenching the ent-kauranyl cation by a water molecule in ent-kaurene synthase of Physcomitrella patens

ent-Kaurene is a tetracyclic diterpene hydrocarbon and a biosynthetic intermediate of the plant hormone gibberellins. In flowering plants, ent-kaurene is biosynthesized from geranylgeranyl diphosphate (GGDP) by two distinct cyclases, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Recently, the moss Physcomitrella patens ent-kaurene biosynthetic gene was cloned and functionally characterized. The bifunctional ent-kaurene synthase [P. patens CPS/KS (PpCPS/KS)] produces both ent-kaurene and 16α-hydroxy-ent-kaurane from GGDP via ent-copalyl diphosphate. Here, we cloned and analyzed the function of a cDNA encoding bifunctional ent-kaurene synthase from the liverwort Jungermannia subulata [J. subulata CPS/KS (JsCPS/KS)]. JsCPS/KS catalyzes the cyclization reaction of GGDP to produce ent-kaurene but not 16α-hydroxy-ent-kaurane, even though the PpCPS/KS (881 amino acids) and JsCPS/KS (886 amino acids) sequences share 60% identity. To determine the regions and amino acids involved in 16α-hydroxy-ent-kaurane formation, we analyzed the enzymic functions of JsCPS/KS and PpCPS/KS chimeric proteins. When the C-terminal region of PpCPS/KS was exchanged with the JsCPS/KS C-terminal region, the chimeric cyclases produced only ent-kaurene. The replacement of PpCPS/KS Ala710 with Met or Phe produced a JsCPS/KS-type cyclase that converted GGDP to ent-kaurene as the sole product. In contrast, replacing Ala710 with Gly, Cys or Ser did not affect the PpCPS/KS product profile as much as replacement of Cys of JsCPS/KS by Ala. Thus, the hydrophobicity and size of the side chain residue at the PpCPS/KS amino acid 710 is responsible for quenching the ent-kauranyl cation by the addition of a water molecule.     

A plastid envelope location of Arabidopsis ent-kaurene oxidase links the plastid and endoplasmic reticulum steps of the gibberellin biosynthesis pathway

We have used fusions of gibberellin biosynthesis enzymes to green fluorescent protein (GFP) to determine the subcellular localization of the early steps of the pathway. Gibberellin biosynthesis from geranylgeranyl diphosphate is catalysed by enzymes of the terpene cyclase, cytochrome P450 mono-oxygenase and 2-oxoglutarate-dependent dioxygenase classes. We show that the N-terminal pre-sequences of the Arabidopsis thaliana terpene cyclases copalyl diphosphate synthase (AtCPS1) and ent-kaurene synthase (AtKS1) direct GFP to chloroplasts in transient assays following microprojectile bombardment of tobacco leaves. The AtKS1-GFP fusion is also imported by isolated pea chloroplasts. The N-terminal portion of the cytochrome P450 protein ent-kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. Chloroplast import assays with 35S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. The leader sequences of the two ent-kaurenoic acid oxidases (AtKAO1 and AtKAO2) from Arabidopsis direct GFP to the endoplasmic reticulum. These data suggest that the AtKO1 protein links the plastid- and endoplasmic reticulum-located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid.     

Diterpene cyclases and the nature of the isoprene fold

The structures and mechanism of action of many terpene cyclases are known, but no structures of diterpene cyclases have yet been reported. Here, we propose structural models based on bioinformatics, site‐directed mutagenesis, domain swapping, enzyme inhibition, and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ～20 α‐helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ‐domain structure together with a third, α‐domain, forming an αβγ structure, and in H⁺‐initiated cyclases, there is an EDxxD‐like Mg²⁺/diphosphate binding motif located in the γ‐domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (βγ) bacterial triterpene cyclases to (βγ) bacterial and thence to (αβγ) plant diterpene cyclases. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Mutational analysis of white spruce (Picea glauca) ent-kaurene synthase (PgKS) reveals common and distinct mechanisms of conifer diterpene synthases of general and specialized metabolism

Conifer diterpene synthases (diTPSs) catalyze the multi-step cycloisomerization of geranylgeranyl diphosphate, or copalyl diphosphate, to a variety of diterpenes in general (i.e., primary) and specialized (i.e., secondary) metabolism. Despite their functional diversity, the known conifer diTPSs are structurally closely related, with variations in three conserved domains, α, β and γ. The catalytic specificity of conifer class I and class I/II diTPSs is predominantly determined by the protein environment of the C-terminal class I active site through stabilization of common and unique carbocation intermediates. Using the crystal structure of Taxus brevifolia taxadiene synthase as template, comparative modeling and mutagenesis of the class I diTPS ent-kaurene synthase from Picea glauca (PgKS) was performed to elucidate the catalytic specificity of PgKS relative to spruce diTPSs of specialized metabolism. N-terminal truncations demonstrated a role for the βγ domain in class I enzyme activity for PgKS, facilitating the closure of the class I active site upon substrate binding. Based on position, Arg476 and Asp736 in the C-terminal α domain of PgKS may contribute to this conformational transition and appear critical for catalysis. Consistent with the mechanism of other diTPSs, the subsequent ionization of a copalyl diphosphate substrate and coordination of the diphosphate group is controlled by strictly conserved residues in the DDxxD and NDIQGCKRE motif of PgKS, such as Asn656 and Arg653. Furthermore, Lys478, Trp502, Met588, Ala615 and Ile619 control the enzymatic activity and specificity of PgKS via carbocation stabilization en route to ent-kaurene. These positions show a high level of amino acid variation, consistent with functional plasticity among conifer diTPSs of different functions in general or specialized metabolism.     

X-ray crystal structure of aristolochene synthase from Aspergillus terreus and evolution of templates for the cyclization of farnesyl diphosphate

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 A resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the alpha-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. Intriguingly, the 2.15 A resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved alpha-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.     

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

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 (Fig. 1; 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 windowFigure 1.Comparison 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 (Fig. 1), 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.     

Genomic organization of plant terpene synthases and molecular evolutionary implications

Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.     

Gibberellin precursor is involved in spore germination in the moss Physcomitrella patens

Gibberellins are ent-kaurene derived phytohormones that are involved in seed germination, stem elongation, and flower induction in seed plants, as well as in antheridia formation and spore germination in ferns. Although ubiquitous in vascular plants, the occurrence and potential function(s) of gibberellins in bryophytes have not yet been resolved. To determine the potential role of gibberellin and/or gibberellin-like compounds in mosses, the effect of AMO-1618 on spores of Physcomitrella patens (Hedw.) B.S.G. was tested. AMO-1618, which inhibited ent-kaurene and gibberellin biosynthesis in angiosperms, also inhibited the bifunctional copalyl diphosphate synthase (E.C. 5.5.1.13)/ent-kaurene synthase (E.C. 4.2.3.19) of P. patens. AMO-1618 also caused a decrease in spore germination rates of P. patens, and this inhibitory effect was less pronounced in the presence of ent-kaurene. These results suggest that ent-kaurene biosynthesis is required by P. patens spores to germinate, implying the presence of gibberellin-like phytohormones in mosses.     

Structure and evolution of linalool synthase

Plant terpene synthases constitute a group of evolutionarily related enzymes. Within this group, however, enzymes that employ two different catalytic mechanisms, and their associated unique domains, are known. We investigated the structure of the gene encoding linalool synthase (LIS), an enzyme that uses geranyl pyrophosphate as a substrate and catalyzes the formation of linalool, an acyclic monoterpene found in the floral scents of many plants. Although LIS employs one catalytic mechanism (exemplified by limonene synthase [LMS]), it has sequence motifs indicative of both LMS-type synthases and the terpene synthases employing a different mechanism (exemplified by copalyl diphosphate synthase [CPS]). Here, we report that LIS genes analyzed from several species encode proteins that have overall 40%-96% identity to each other and have 11 introns in identical positions. Only the region encoding roughly the last half of the LIS gene (exons 9-12) has a gene structure similar to that of the LMS-type genes. On the other hand, in the first part of the LIS gene (exons 1-8), LIS gene structure is essentially identical to that found in the first half of the gene encoding CPS. In addition, the level of similarity in the coding information of this region between the LIS and CPS genes is also significant, whereas the second half of the LIS protein is most similar to LMS-type synthases. Thus, LIS appears to be a composite gene which might have evolved from a recombination event between two different types of terpene synthases. The combined evolutionary mechanisms of duplication followed by divergence and/or "domain swapping" may explain the extraordinarily large diversity of proteins found in the plant terpene synthase family.     

Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins

The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.     

Emission of ent-kaurene, a diterpenoid hydrocarbon precursor for gibberellins, into the headspace from plants

ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. To address whether fungal GA biosynthesis enzymes function in plants, we generated transgenic Arabidopsis plants overexpressing ent-kaurene synthase (GfCPS/KS) from a GA-producing fungus Gibberella fujikuroi. GfCPS/KS catalyzes a two-step reaction corresponding to ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) activities in plants. When GfCPS/KS was overexpressed and targeted to plastids, a range of GA-deficient phenotypes of the ga1-3 and ga2-1 mutants (defective in CPS and KS, respectively) were restored to wild type. Unexpectedly, the transgenic lines overproducing GfCPS/KS emitted the GA precursor ent-kaurene into the headspace besides its accumulation in the plant body. When co-cultivated with the ent-kaurene overproducers in a closed environment, the airborne ent-kaurene was able to fully complement the dwarf phenotype of ga1-3 and ga2-1 mutants, but not that of the ga3-1 mutant (defective in ent-kaurene oxidase). These results suggest that ent-kaurene may be efficiently metabolized into bioactive GAs in Arabidopsis when supplied as a volatile. We also provide evidence that ent-kaurene is released in the headspace of wild-type Chamaecyparis obtusa and Cryptomeria japonica plants, suggesting the occurrence of this hydrocarbon GA precursor as a volatile in nature.