Gibberellin-biosynthetic ent-kaurene synthases in higher plants do not require their non-catalytic domains for the catalysis.

ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αßγ domains. Active sites of ent-CPS exist at the interface of ß and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or ßγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its ßγ domains. Domain-swapping experiments showed that replacing ßγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of ßγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without ßγ domains.

Characterization of diterpene synthase genes in Brachypodium distachyon, a monocotyledonous model plant, provides evolutionary insight into their multiple homologs in cereals.

Gibberellins are diterpenoid phytohormones that regulate plant growth, and are biosynthesized from a diterpene intermediate, ent-kaurene, which is produced from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive 2 cyclization reactions are catalyzed by 2 distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Various diterpene synthase genes involved in specialized metabolism were likely created through duplication and neofunctionalization of gibberellin-biosynthetic ent-CPS and KS genes in crops. Brachypodium distachyon is a monocotyledonous species that is a model plant in grasses. We herein found 1 ent-CPS gene homolog BdCPS and 4 tandemly arrayed KS-like genes BdKS1, KSL2, KSL3, and KSL4 in the B. distachyon genome, a simpler collection of paralogs than in crops. Phylogenetic and biochemical analyses showed that BdCPS and BdKS1 are responsible for gibberellin biosynthesis. BdKSL2 and BdKSL3 are suggested to be involved in specialized diterpenoid metabolism. Moreover, we restored KS activity of BdKSL2 through amino acid substitution.

Functional kaurene-synthase-like diterpene synthases lacking a gamma domain are widely present in Oryza and related species.

Various diterpene synthases have been functionally identified in cultivated rice (Oryza sativa). These are the homologs of ent-copalyl diphosphate (ent-CDP) synthase and ent-kaurene synthase (KS) that are responsible for the biosynthesis of gibberellins, diterpenoid phytohormones. We isolated a cDNA encoding full-length OsKSL12, a previously uncharacterized KS like (KSL) enzyme that consists of a ß-domain and an α-domain with an active center, but lacks an N-terminal γ-domain. Functional analysis using a bacterial expression system showed that recombinant OsKSL12 converted ent-CDP into ent-manool or ent-13-epi-manool. Comparative genomics revealed that functional OsKSL12 homologs exist in diverse wild species in the Oryzeae-Oryza nivara (Oryza rufipogon), Oryza coarctata, Oryza granulata, Leersia perrieri, and Leersia tisseranti. KSL12 homologs in O. granulata, L. perrieri, and L. tisseranti preferentially reacted with geranylgeranyl diphosphate rather than ent-CDP, resulting in geranyllinalool rather than ent-manool or ent-13-epi-manool as the main product, meaning that KSL12 functionally diversified during evolution in the Oryzeae.

Evolution of Labdane-Related Diterpene Synthases in Cereals.

Gibberellins (GAs) are labdane-related diterpenoid phytohormones that regulate various aspects of higher plant growth. A biosynthetic intermediate of GAs is ent-kaurene, a tetra-cyclic diterpene that is produced through successive cyclization of geranylgeranyl diphosphate catalyzed by the two distinct monofunctional diterpene synthases-ent-copalyl diphosphate synthase (ent-CPS) and ent-kaurene synthase (KS). Various homologous genes of the two diterpene synthases have been identified in cereals, including rice (Oryza sativa), wheat (Triticum aestivum) and maize (Zea mays), and are believed to have been derived from GA biosynthetic ent-CPS and KS genes through duplication and neofunctionalization. They play roles in specialized metabolism, giving rise to diverse labdane-related diterpenoids for defense because a variety of diterpene synthases generate diverse carbon-skeleton structures. This review mainly describes the diterpene synthase homologs that have been identified and characterized in rice, wheat and maize and shows the evolutionary history of various homologs in rice inferred by comparative genomics studies using wild rice species, such as Oryza rufipogon and Oryza brachyantha. In addition, we introduce labdane-related diterpene synthases in bryophytes and gymnosperms to illuminate the macroscopic evolutionary history of diterpene synthases in the plant kingdom-bifunctional enzymes possessing both CPS and KS activities are present in bryophytes; gymnosperms possess monofunctional CPS and KS responsible for GA biosynthesis and also possess bifunctional diterpene synthases facilitating specialized metabolism for defense.

Biosynthetic study of conidiation-inducing factor conidiogenone: heterologous production and cyclization mechanism of a key bifunctional diterpene synthase.

Conidiogenone, a diterpene with a unique structure, is known to induce the conidiation of Penicillium cyclopium. The biosynthetic pathway of (-)-conidiogenone has been fully elucidated by the heterologous expression of biosynthetic genes in Aspergillus oryzae and by in vitro enzyme assay with 13C-labeled substrates. After construction of deoxyconidiogenol by the action of bifunctional terpene synthase, one cytochrome P450 catalyzes two rounds of oxidation to furnish conidiogenone. Notably, similar biosynthetic genes are conserved among more than 10 Penicillium sp., suggesting that conidiogenone is a common conidiation inducer in this genus. The cyclization mechanism catalyzed by terpene synthase, which involves successive 1,2-alkyl shifts, was fully elucidated using 13C-labeled geranylgeranyl pyrophosphate (GGPP) as substrate. During the structural analysis of deoxyconidiogenol, we observed broadening of some of the 13C signals measured at room temperature, which has not been observed with other structurally related compounds. Careful examination using techniques including 13C NMR studies at -80 °C, conformational analysis and prediction of the 13C chemical shifts using density functional theory gave insights into this intriguing phenomenon.

Characterization of diterpene synthase genes in the wild rice species Oryza brachyatha provides evolutionary insight into rice phytoalexin biosynthesis.

Cultivated rice (Oryza sativa; Os) produces a variety of labdane-related diterpenoids; not only phytohormone gibberellins (GAs) but also phytoalexins for defense including phytocassanes, momilactones and oryzalexins. Their carbon skeleton diterpenes are constructed from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP) or its diastereomer syn-CDP. These two-step reactions are successively catalyzed by homologs of the two diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS) that are responsible for the biosynthesis of GAs; e.g. OsCPS4 and OsKSL8 that are involved in the biosynthesis of oryzalexin S, a rice phytoalexin. Oryza brachyantha (Ob) is the most distant wild rice species from Os among the Oryza genus. We previously reported that the Ob genome contains ObCPS_11g, ObKSL8-a, ObKSL8-b and ObKSL8-c for specialized metabolism at a locus similar to the OsKSL8 locus on chromosome 11. These Ob genes are closely related to OsCPS4 and OsKSL8, respectively. We herein characterize the diterpene synthase genes in Ob, using functional analyses and expression analysis. Recombinant OsKSL8 and ObKSL8-a showed the same in vitro function when syn-CDP or normal-CDP were used as substrates. Nonetheless, our results suggest that Ob produces normal-CDP-related diterpenoid phytoalexins, presumably via ObKSL8-a, while Os produces a syn-CDP-related phytoalexin, oryzalexin S, via OsKSL8. This difference must be due to the kinds of CPS that are present in each species; Os has OsCPS4 encoding syn-CPS, while Ob has ObCPS_11g encoding normal-CPS. Thus, we propose the evolutionary history underlying oryzalexin S biosynthesis: the gain of a syn-CPS was a critical event allowing the biosynthesis of oryzalexin S.

Dark conditions enhance aluminum tolerance in several rice cultivars via multiple modulations of membrane sterols.

Aluminum-sensitive rice (Oryza sativa L.) cultivars showed increased Al tolerance under dark conditions, because less Al accumulated in the root tips (1 cm) under dark than under light conditions. Under dark conditions, the root tip concentration of total sterols, which generally reduce plasma membrane permeabilization, was higher in the most Al-sensitive japonica cultivar, Koshihikari (Ko), than in the most Al-tolerant cultivar, Rikuu-132 (R132), but the phospholipid content did not differ between the two. The Al treatment increased the proportion of stigmasterol (which has no ability to reduce membrane permeabilization) out of total sterols similarly in both cultivars under light conditions, but it decreased more in Ko under dark conditions. The carotenoid content in the root tip of Al-treated Ko was significantly lower under dark than under light conditions, indicating that isopentenyl diphosphate transport from the cytosol to plastids was decreased under dark conditions. HMG2 and HMG3 (encoding the key sterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase) transcript levels in the root tips were enhanced under dark conditions. We suggest that the following mechanisms contribute to the increase in Al tolerance under dark conditions: inhibition of stigmasterol formation to retain membrane integrity; greater partitioning of isopentenyl diphosphate for sterol biosynthesis; and enhanced expression of HMGs to increase sterol biosynthesis.

Evolutionary trajectory of phytoalexin biosynthetic gene clusters in rice.

Plants frequently possess operon-like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdane-related diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate or syn-copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical-modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane-related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon-like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes.

Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogon.

Cultivated rice (Oryza sativa) possesses various labdane-related diterpene synthase genes, homologs of ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins.

The rice ent-KAURENE SYNTHASE LIKE 2 encodes a functional ent-beyerene synthase.

The rice genome contains a family of kaurene synthase-like (OsKSL) genes that are responsible for the biosynthesis of various diterpenoids, including gibberellins and phytoalexins. While many OsKSL genes have been functionally characterized, the functionality of OsKSL2 is still unclear and it has been proposed to be a pseudogene. Here, we found that OsKSL2 is drastically induced in roots by methyl jasmonate treatment and we successfully isolated a full-length cDNA for OsKSL2. Sequence analysis of the OsKSL2 cDNA revealed that the open reading frame of OsKSL2 is mispredicted in the two major rice genome databases, IRGSP-RAP and MSU-RGAP. In vitro conversion assay indicated that recombinant OsKSL2 catalyzes the cyclization of ent-CDP into ent-beyerene as a major and ent-kaurene as a minor product. ent-Beyerene is an antimicrobial compound and OsKSL2 is induced by methyl jasmonate; these data suggest that OsKSL2 is a functional ent-beyerene synthase that is involved in defense mechanisms in rice roots.

Higher sterol content regulated by CYP51 with concomitant lower phospholipid content in membranes is a common strategy for aluminium tolerance in several plant species.

Several studies have shown that differences in lipid composition and in the lipid biosynthetic pathway affect the aluminium (Al) tolerance of plants, but little is known about the molecular mechanisms underlying these differences. Phospholipids create a negative charge at the surface of the plasma membrane and enhance Al sensitivity as a result of the accumulation of positively charged Al(3+) ions. The phospholipids will be balanced by other electrically neutral lipids, such as sterols. In the present research, Al tolerance was compared among pea (Pisum sativum) genotypes. Compared with Al-tolerant genotypes, the Al-sensitive genotype accumulated more Al in the root tip, had a less intact plasma membrane, and showed a lower expression level of PsCYP51, which encodes obtusifoliol-14α-demethylase (OBT 14DM), a key sterol biosynthetic enzyme. The ratio of phospholipids to sterols was higher in the sensitive genotype than in the tolerant genotypes, suggesting that the sterol biosynthetic pathway plays an important role in Al tolerance. Consistent with this idea, a transgenic Arabidopsis thaliana line with knocked-down AtCYP51 expression showed an Al-sensitive phenotype. Uniconazole-P, an inhibitor of OBT 14DM, suppressed the Al tolerance of Al-tolerant genotypes of maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa), wheat (Triticum aestivum), and triticale (×Triticosecale Wittmark cv. Currency). These results suggest that increased sterol content, regulated by CYP51, with concomitant lower phospholipid content in the root tip, results in lower negativity of the plasma membrane. This appears to be a common strategy for Al tolerance among several plant species.

Transcripts of two ent-copalyl diphosphate synthase genes differentially localize in rice plants according to their distinct biological roles.

Gibberellins (GAs) are diterpenoid phytohormones that regulate various aspects of plant growth. Tetracyclic hydrocarbon ent-kaurene is a biosynthetic intermediate of GAs, and is converted from geranylgeranyl diphosphate, a common precursor of diterpenoids, via ent-copalyl diphosphate (ent-CDP) through successive cyclization reactions catalysed by two distinct diterpene synthases, ent-CDP synthase and ent-kaurene synthase. Rice (Oryza sativa L.) has two ent-CDP synthase genes, OsCPS1 and OsCPS2. It has been thought that OsCPS1 participates in GA biosynthesis, while OsCPS2 participates in the biosynthesis of phytoalexins, phytocassanes A-E, and oryzalexins A-F. It has been shown previously that loss-of-function OsCPS1 mutants display a severe dwarf phenotype caused by GA deficiency despite possessing another ent-CDP synthase gene, OsCPS2. Here, experiments were performed to account for the non-redundant biological function of OsCPS1 and OsCPS2. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that OsCPS2 transcript levels were drastically lower than those of OsCPS1 in the basal parts, including the meristem of the second-leaf sheaths of rice seedlings. qRT-PCR results using tissue samples prepared by laser microdissection suggested that OsCPS1 transcripts mainly localized in vascular bundle tissues, similar to Arabidopsis CPS, which is responsible for GA biosynthesis, whereas OsCPS2 transcripts mainly localized in epidermal cells that address environmental stressors such as pathogen attack. Furthermore, the OsCPS2 transgene under regulation of the OsCPS1 promoter complemented the dwarf phenotype of an OsCPS1 mutant, oscps1-1. The results indicate that transcripts of the two ent-CDP synthase genes differentially localize in rice plants according to their distinct biological roles, OsCPS1 for growth and OsCPS2 for defence.

Reverse-genetic approach to verify physiological roles of rice phytoalexins: characterization of a knockdown mutant of OsCPS4 phytoalexin biosynthetic gene in rice.

A variety of labdane-related diterpenoids, including phytocassanes, oryzalexins and momilactones, were identified as phytoalexins in rice (Oryza sativa L.). Momilactone B was also isolated as an allelochemical exuded from rice roots. The biosynthetic genes of these phytoalexins have been identified, including six labdane-related diterpene cyclase genes such as OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8 and OsKSL10. Here we identified an OsCPS4 knockdown mutant, cps4-tos, by screening Tos17 mutant lines using polymerase chain reaction. OsCPS4 encodes a syn-copalyl diphosphate synthase responsible for momilactones and oryzalexin S biosynthesis. Because Tos17 was inserted into the third intron of OsCPS4, the mature OsCPS4 mRNA was detected in the cps4-tos mutant as well as the wild type. Nevertheless, mature OsCPS4 transcript levels in the cps4-tos mutant were about one sixth those in the wild type. The cps4-tos mutant was more susceptible to rice blast fungus than the wild type, possibly due to lower levels of momilactones and oryzalexin S in the mutant. Moreover, co-cultivation experiments suggested that the allelopathic effect of cps4-tos against some kinds of lowland weeds was significantly lower than that of the wild type, probably because of lower momilactone content exuded from cps4-tos roots. A reverse-genetic strategy using the cps4-tos mutant showed the possible roles of momilactones not only as phytoalexins but also as allelopathic substances.

Essential residues in diterpene synthases for biosynthesis of oryzalexins A-F in rice phytoalexin.

Cultivated rice (Oryza sativa) produces a variety of diterpenoid-type phytoalexins. Diterpene synthase genes that are responsible for the biosynthesis of momilactones, phytocassanes, and oryzalexins have been identified in O. sativa cv. Nipponbare. OsKSL10 (Os12t0491800 in RAP and LOC_Os12g30824 in MSU) was previously identified as an enzyme catalyzing the conversion of ent-copalyl diphosphate to ent-sandaracopimaradiene for the production of oryzalexins A to F. Our previous study on Oryza rufipogon, a wild progenitor of Asian cultivated rice, showed that both OrKSL10 and OrKSL10ind from O. rufipogon accessions W1943 and W0106, respectively, closely related to the japonica and indica subspecies, converted ent-copalyl diphosphate to ent-miltiradiene. Thus, the functional conversion of ent-miltiradiene synthase into ent-sandaracopimaradiene synthase is implied to have occurred through natural amino acid mutations, the details of which have not been elucidated. In this study, we show that introduction of A654G substitution into OrKSL10 significantly alters its function into more closely resembling that of OsKSL10. Moreover, double substitution V546I/A654G almost completely converts the function of OrKSL10 into that of OsKSL10. On the other hand, the reversed substitution I546V/G654A was insufficient to convert the function of OsKSL10 into OrKSL10, indicating the introduction of additional substitution S522I is required for the functionality of OsKSL10. Lastly, point mutations at the 654A residue in OrKSL10 suggest that hydrophobic side chains at this position have a negative influence on the production of ent-sandaracopimaradiene.

Enzymatic (13)C labeling and multidimensional NMR analysis of miltiradiene synthesized by bifunctional diterpene cyclase in Selaginella moellendorffii.

Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-(13)C(6)]mevalonate, all carbons were labeled with (13)C stable isotope (>99%). The fully (13)C-labeled product was subjected to (13)C-(13)C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and multidimensional (13)C NMR analyses of completely (13)C-labeled compound are powerful methods for biosynthetic studies.

Domain loss has independently occurred multiple times in plant terpene synthase evolution.

The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these synthases have evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a approximately 220 amino acid 'internal sequence element' (now recognized as the γ domain) gave rise to the precursor of the modern mono- and sesqui-TPSs found in all higher plants. Intriguingly, TPSs are conserved by taxonomic relationships rather than function. This relationship demonstrates that such functional radiation has occurred both repeatedly and relatively recently, yet phylogenetic analyses assume that the 'internal/γ' domain loss represents a single evolutionary event. Here we provide evidence that such a loss was not a singular event, but rather has occurred multiple times. Specifically, we provide an example of a bi-domain diterpene synthase from Salvia miltiorrhiza, along with a sesquiterpene synthase from Triticum aestivum (wheat) that is not only closely related to diterpene synthases, but retains the ent-kaurene synthase activity relevant to the ancestral gibberellin metabolic function. Indeed, while the wheat sesquiterpene synthase clearly no longer contains the 'internal/γ' domain, it is closely related to rice diterpene synthase genes that retain the ancestral tri-domain structure. Thus, these findings provide examples of key evolutionary intermediates that underlie the bi-domain structure observed in the expansive plant TPS gene family, as well as indicating that 'internal/γ' domain loss has occurred independently multiple times, highlighting the complex evolutionary history of this important enzymatic family.

An enzyme catalyzing O-prenylation of the glucose moiety of fusicoccin A, a diterpene glucoside produced by the fungus Phomopsis amygdali.

Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane-bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant-pathogenic fungus Phomopsis amygdali and has a unique O-prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6'-hydroxy group of the glucose moiety of FC A to yield an O-prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 °C, respectively. K(m) values were calculated as 0.49±0.037 µM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3±0.63 µM for DMAPP, with a k(cat) of 55.3±3.3×10⁻³ s. The enzyme did not act on representative substrates of the above-mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.

An unusual spliced variant of DELLA protein, a negative regulator of gibberellin signaling, in lettuce.

DELLA proteins are negative regulators of the signaling of gibberellin (GA), a phytohormone regulating plant growth. DELLA degradation is triggered by its interaction with GID1, a soluble GA receptor, in the presence of bioactive GA. We isolated cDNA from a spliced variant of LsDELLA1 mRNA in lettuce, and named it LsDELLA1sv. It was deduced that LsDELLA1sv encodes truncated LsDELLA1, which has DELLA and VHYNP motifs at the N terminus but lacks part of the C-terminal GRAS domain. The recombinant LsDELLA1sv protein interacted with both Arabidopsis GID1 and lettuce GID1s in the presence of GA. A yeast two-hybrid assay suggested that LsDELLA1sv interacted with LsDELLA1. The ratio of LsDELLA1sv to LsDELLA1 transcripts was higher in flower samples at the late reproductive stage and seed samples (dry seeds and imbibed seeds) than in the other organ samples examined. This study suggests that LsDELLA1sv is a possible modulator of GA signaling in lettuce.

Dioxygenases, key enzymes to determine the aglycon structures of fusicoccin and brassicicene, diterpene compounds produced by fungi.

Fusicoccin A and cotylenin A are structurally related diterpene glucosides and show a phytohormone-like activity. However, only cotylenin A induces the differentiation of human myeloid leukemia cells. Since the cotylenin A producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out and a modified fusicoccin A with hydroxyl group at the 3-position showed a similar biological activity with that of cotylenin A. We then searched for an enzyme source that catalyzes the introduction of a hydroxyl group into the 3-position and found that brassicicene C, which is structurally related to fusicoccin A with hydroxyl group at the 3-position, was produced by Alternaria brassicicola ATCC96836. We recently cloned a brassicicene C biosynthetic gene cluster including the genes encoding fusicocca-2,10(14)-diene synthase and two cytochrome P450s, which were responsible for the formation of fusicocca-2,10(14)-diene-8ß,16-diol. In this study, we report that a α-ketoglutarate dependent dioxygenase, the gene coding for which was located in the cluster, catalyzed a hydroxylation at the 3-position of fusicocca-2,10(14)-diene-8ß,16-diol. On the other hand, a α-ketoglutarate-dependent dioxygenase, which had been identified in a fusicoccin A biosynthetic gene cluster, catalyzed the 16-oxidation of fusicocca-2,10(14)-diene-8ß,16-diol to yield an aldehyde (8ß-hydroxyfusicocca-1,10(14)-dien-16-al), although both dioxygenases had 51% amino acid sequence identity. These findings suggested that the dioxygenases played critical roles for the formation of the fusicoccin A-type and cotylenin A-/brassicicene C-type aglycons. Moreover, we showed that short-chain dehydrogenase/reductase located in the fusicoccin A biosynthetic gene cluster catalyzed the reduction of the aldehyde to yield fusicocca-1,10(14)-diene-8ß,16-diol.

Characterization of a loss-of-function mutant of gibberellin biosynthetic gene LsGA3ox1 in lettuce.

A previous study generated lettuce (Lactuca sativa) mutant lines tagged by retrotransposon Tnt1 from tobacco (Nicotiana tabacum) and identified a homozygous mutant, Tnt6a, that exhibited severe dwarf phenotype. Here we show that Tnt1 is inserted into the intron of gibberellin biosynthetic gene LsGA3ox1 in Tnt6a mutants. Expression analysis suggests that LsGA3ox1 is nearly knocked out in the Tnt6a mutants.