An overview of gibberellin metabolism enzyme genes and their related mutants in rice

To enhance our understanding of GA metabolism in rice (Oryza sativa), we intensively screened and identified 29 candidate genes encoding the following GA metabolic enzymes using all available rice DNA databases: ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox). In contrast to the Arabidopsis genome, multiple CPS-like, KS-like, and KO-like genes were identified in the rice genome, most of which are contiguously arranged. We also identified 18 GA-deficient rice mutants at six different loci from rice mutant collections. Based on the mutant and expression analyses, we demonstrated that the enzymes catalyzing the early steps in the GA biosynthetic pathway (i.e. CPS, KS, KO, and KAO) are mainly encoded by single genes, while those for later steps (i.e. GA20ox, GA3ox, and GA2ox) are encoded by gene families. The remaining CPS-like, KS-like, and KO-like genes were likely to be involved in the biosynthesis of diterpene phytoalexins rather than GAs because the expression of two CPS-like and three KS-like genes (OsCPS2, OsCPS4, OsKS4, OsKS7, and OsKS8) were increased by UV irradiation, and four of these genes (OsCPS2, OsCPS4, OsKS4, and OsKS7) were also induced by an elicitor treatment.     

Comparison of the enzymatic properties of ent-copalyl diphosphate synthases in the biosynthesis of phytoalexins and gibberellins in rice

The rice genome contains two ent-copalyl diphosphate synthase genes: OsCPS1 acts in gibberellin (phytohormone) biosynthesis, and OsCPS2/OsCyc2 acts in the synthesis of oryzalexins A-F and phytocassanes A-E (phytoalexins). We characterized the enzymatic properties of recombinant OsCPS2/OsCyc2 fused with a tag-protein at the N-terminus, and compared them to those of OsCPS1. Several enzymatic properties of OsCPS2/OsCyc2, including the optimal pH, optimal temperature, divalent cation requirement, and kinetic values for the geranylgeranyl diphosphate (GGDP) substrate, were almost the same as those of OsCPS1. However, OsCPS2/OsCyc2 activity was not inhibited by 50-60 muM GGDP substrate, by which the OsCPS1 activity was inhibited. Furthermore, the OsCPS1 activity exhibited approximately 70% inhibition by 100 muM Amo-1618 (a gibberellin biosynthetic inhibitor), whereas the OsCPS2/OsCyc2 activity exhibited approximately 10% inhibition. These results indicate that the properties of OsCPS2/OsCyc2 were partially different from those of OsCPS1, although OsCPS2/OsCyc2 catalyzes the same reaction step as OsCPS1.     

Characterization of a rice gene family encoding type-A diterpene cyclases

We have previously isolated and characterized the rice (Oryza sativa) cDNAs, OsCyc1/OsCPS4, OsCyc2/OsCPS2, OsKS4, OsDTC1/OsKS7, OsDTC2/OsKS8 and OsKS10, which encode cyclases that are responsible for diterpene phytoalexin biosynthesis. Among the other members of this gene family, OsCPS1 and OsKS1 have been suggested as being responsible for gibberellin biosynthesis, OsKSL11 has recently been shown to encode stemodene synthase, and the functions of the three other diterpene cyclase genes in the rice genome, OsKS3, OsKS5 and OsKS6, have not yet been determined. In this study, we show that recombinant OsKS5 and OsKS6 expressed in E. coli converted ent-copalyl diphosphate into ent-pimara-8(14),15-diene and ent-kaur-15-ene, respectively. Neither product is a hydrocarbon precursor required in the biosynthesis of either gibberellins or phytoalexins. OsKS3 may be a pseudogene from which the translated product is a truncated enzyme. These results suggest that the diterpene cyclase genes responsible for gibberellin and phytoalexin biosynthesis are not functionally redundant.     

gid1, a gibberellin-insensitive dwarf mutant, shows altered regulation of probenazole-inducible protein (PBZ1) in response to cold stress and pathogen attack

A recessive gibberellin (GA)-insensitive dwarf mutant of rice, gibberellin-insensitive dwarf1 (gid1), has been identified, which shows a severe dwarf phenotype and contains high concentrations of endogenous GA. To elucidate the function of gid1, proteins regulated downstream of gid1 were analysed using a proteomic approach. Proteins extracted from suspension-cultured cells of gid1 and its wild type were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of a total of 962 proteins identified from the suspension-cultured cells, 16 were increased and 14 were decreased in gid1 compared with its wild type. Among the proteins hyper-accumulated in gid1 were osmotin, triosephosphate isomerase, probenazole inducible protein (PBZ1) and pathogenesis-related protein 10. Of these four genes, only the expression of PBZ1 was increased by exogenous GA3 application. Expression of this gene was also enhanced in shoots of the wild type by cold stress or by rice blast fungus infection. Under normal growth conditions, there was more PBZ1 protein in gid1 than in the wild type. In addition, gid1 showed increased tolerance to cold stress and resistance to blast fungus infection. The entcopalyl diphosphate synthase (OsCPS) genes, which encode enzymes at the branch point between GA and phytoalexin biosynthesis, were expressed differentially in gid1 relative to the wild type. Specifically, OsCPS1, which encodes an enzyme in the GA biosynthesis pathway, was down-regulated and OsCPS2 and OsCPS4, which encode enzymes in phytoalexin biosynthesis, were up-regulated in gid1. These results suggest that the expression of PBZ1 is regulated by GA signalling and stress stimuli, and that gid1 is involved in tolerance to cold stress and resistance to blast fungus.     

The gibberellin 20-oxidase of Gibberella fujikuroi is a multifunctional monooxygenase

The genes for gibberellin (GA) biosynthesis are clustered in the fungus Gibberella fujikuroi. In addition to genes encoding a GA-specific geranylgeranyl diphosphate synthase and a bifunctional ent-copalyl diphosphate/ent-kaurene synthase, the cluster contains four cytochrome P450 monooxygenase genes (P450-1, -2, -3, -4). Recently it was shown that P450-4 and P450-1 encode multifunctional enzymes catalyzing the three oxidation steps from ent-kaurene to ent-kaurenoic acid and the four oxidation steps from ent-kaurenoic acid to GA14, respectively. Here we describe the functional analysis of the P450-2 gene by gene disruption and by expressing the gene in a mutant that lacks the entire GA biosynthesis gene cluster. Mutants in which P450-2 is inactivated by the insertion of a large piece of DNA accumulated GA14 and lacked biosynthetically more advanced metabolites, indicating that the gene encodes a 20-oxidase. This was confirmed by incubating lines containing P450-2 in the absence of the other GA biosynthesis genes with isotopically labeled substrates. The P450-2 gene product oxidized the 3beta-hydroxylated intermediate, GA14, and its non-hydroxylated analogue GA12 to GA4 and GA9, respectively. Expression of P450-2 is repressed by high amounts of nitrogen in the culture medium but is not affected by the presence of biosynthetically advanced GAs, i.e. there is no evidence for feedback regulation. The fact that the GA 20-oxidase is a cytochrome P450 monooxygenase in G. fujikuroi and not a 2-oxoglutarate-dependent dioxygenase as in plants, together with the significant differences in regulation of gene expression, are further evidence for independent evolution of the GA biosynthetic pathways in plants and fungi.     

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.     

Expression pattern of the coparyl diphosphate synthase gene in developing rice anthers

Rice anthers contain high concentrations of gibberellins A(4) and A(7). To understand their physiological roles, we examined the site of their biosynthesis by analyzing the expression pattern of a gene (OsCPS) encoding coparyl diphosphate synthase in developing rice flowers. Expression was apparent in the anthers 1-2 days before flowering, and CPS mRNA accumulated in the maturing pollen.     

Synergistic substrate inhibition of ent-copalyl diphosphate synthase: a potential feed-forward inhibition mechanism limiting gibberellin metabolism

Gibberellins (GAs) or gibberellic acids are ubiquitous diterpenoid phytohormones required for many aspects of plant growth and development, including repression of photosynthetic pigment production (i.e. deetiolation) in the absence of light. The committed step in GA biosynthesis is catalyzed in plastids by ent-copalyl diphosphate synthase (CPS), whose substrate, (E,E,E,)-geranylgeranyl diphosphate (GGPP), is also a direct precursor of carotenoids and the phytol side chain of chlorophyll. Accordingly, during deetiolation, GA production is repressed, whereas flux toward these photosynthetic pigments through their common GGPP precursor is dramatically increased. How this is accomplished has been unclear because no mechanism for regulation of CPS activity has been reported. We present here kinetic analysis of recombinant pseudomature CPS from Arabidopsis (Arabidopsis thaliana; rAtCPS) demonstrating that Mg(2+) and GGPP exert synergistic substrate inhibition effects on CPS activity. These results suggest that GA metabolism may be limited by feed-forward inhibition of CPS; in particular, the effect of Mg(2+) because light induces increases in plastid Mg(2+) levels over a similar range as that observed here to affect rAtCPS activity. Notably, this effect is most pronounced in the GA-specific AtCPS because the corresponding activity of the resin acid biosynthetic enzyme abietadiene synthase is 100-fold less sensitive to [Mg(2+)]. Furthermore, Mg(2+) allosterically activates the plant porphobilinogen synthase involved in chlorophyll production. Hence, Mg(2+) may have a broad role in regulating plastidial metabolic flux during deetiolation. Finally, the observed synergistic substrate/feed-forward inhibition of CPS also seems to provide a novel example of direct regulation of enzymatic activity in hormone biosynthesis.     

The P450-4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthesis pathway

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA(4). The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3' consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.     

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.     

Tissue-specific localization of gibberellins and expression of gibberellin-biosynthetic and signaling genes in wood-forming tissues in aspen

Bioactive gibberellins (GAs) are known regulators of shoot growth and development in plants. In an attempt to identify where GAs are formed, we have analyzed the expression patterns of six GA biosynthesis genes and two genes with predicted roles in GA signaling and responses in relation to measured levels of GAs. The analysis was based on tangential sections, giving tissue-specific resolution across the cambial region of aspen trees (Populus tremula). Gibberellin quantification by GC/MS-SRM showed that the bioactive GA1 and GA4 were predominantly located in the zone of expansion of xylem cells. Based on co-localization of the expression of the late GA biosynthesis gene GA 20-oxidase 1 and bioactive GAs, we suggest that de novo GA biosynthesis occurs in the expanding xylem. However, expression levels of the first committed GA biosynthesis enzyme, ent-copalyl diphosphate synthase, were high in the phloem, suggesting that a GA precursor(s) may be transported to the xylem. The expression of the GA signaling and response genes DELLA-like1 and GIP-like1 coincided well with sites of high bioactive GA levels. We therefore suggest that the main role of GA during wood formation is to regulate early stages of xylem differentiation, including cell elongation.     

Characterization of a Rice Gene Family Encoding Type-A Diterpene Cyclases

We have previously isolated and characterized the rice (Oryza sativa) cDNAs, OsCyc1/OsCPS4, OsCyc2/OsCPS2, OsKS4, OsDTC1/OsKS7, OsDTC2/OsKS8 and OsKS10, which encode cyclases that are responsible for diterpene phytoalexin biosynthesis. Among the other members of this gene family, OsCPS1 and OsKS1 have been suggested as being responsible for gibberellin biosynthesis, OsKSL11 has recently been shown to encode stemodene synthase, and the functions of the three other diterpene cyclase genes in the rice genome, OsKS3, OsKS5 and OsKS6, have not yet been determined. In this study, we show that recombinant OsKS5 and OsKS6 expressed in E. coli converted ent-copalyl diphosphate into ent-pimara-8(14),15-diene and ent-kaur-15-ene, respectively. Neither product is a hydrocarbon precursor required in the biosynthesis of either gibberellins or phytoalexins. OsKS3 may be a pseudogene from which the translated product is a truncated enzyme. These results suggest that the diterpene cyclase genes responsible for gibberellin and phytoalexin biosynthesis are not functionally redundant.     

Functional characterization of wheat copalyl diphosphate synthases sheds light on the early evolution of labdane-related diterpenoid metabolism in the cereals

Two of the most agriculturally important cereal crop plants are wheat (Triticum aestivum) and rice (Oryza sativa). Rice has been shown to produce a number of diterpenoid natural products as phytoalexins and/or allelochemicals – specifically, labdane-related diterpenoids, whose biosynthesis proceeds via formation of an eponymous labdadienyl/copalyl diphosphate (CPP) intermediate (e.g., the ent-CPP of gibberellin phytohormone biosynthesis). Similar to rice, wheat encodes a number of CPP synthases (CPS), and the three CPS characterized to date (TaCPS1–3) all have been suggested to produce ent-CPP. However, several of the downstream diterpene synthases will only react with CPP intermediate of normal or syn, but not ent, stereochemistry, as described in the accompanying report. Investigation of additional CPS did not resolve this issue, as the only other functional synthase (TaCPS4) also produced ent-CPP. Chiral product characterization of all the TaCPS then established that TaCPS2 uniquely produces normal, rather than ent-, CPP, thus, providing a suitable substrate source for the downstream diterpene synthases. Notably, TaCPS2 is most homologous to the similarly stereochemically differentiated syn-CPP synthase from rice (OsCPS4), while the non-inducible TaCPS3 and TaCPS4 cluster with the rice OsCPS1 required for gibberellin phytohormone biosynthesis, as well as with a barley (Hordeum vulgare) CPS (HvCPS1) that also is characterized here as similarly producing ent-CPP. These results suggest that diversification of labdane-related diterpenoid metabolism beyond the ancestral gibberellins occurred early in cereal evolution, and included the type of stereochemical variation demonstrated here.