A Rice Semi-Dwarf Gene,Tan-Ginbozu (D35), Encodes the Gibberellin Biosynthesis Enzyme,ent-Kaurene Oxidase

A rice (Oryza sativa L.) semi-dwarf cultivar, Tan-Ginbozu (d35Tan-Ginbozu), contributed to the increase in crop productivity in Japan in the 1950s. Previous studies suggested that the semi-dwarf stature of d35Tan-Ginbozu is caused by a defective early step of gibberellin biosynthesis, which is catalyzed by ent-kaurene oxidase (KO). To study the molecular characteristics of d35Tan-Ginbozu, we isolated 5 KO-like (KOL) genes from the rice genome, which encoded proteins highly homologous to Arabidopsis and pumpkin KOs. The genes (OsKOL1 to 5) were arranged as tandem repeats in the same direction within a 120 kb sequence. Expression analysis revealed that OsKOL2 and OsKOL4 were actively transcribed in various organs, while OsKOL1 and OsKOL5 were expressed only at low levels; OsKOL3 may be a pseudogene. Sequence analysis and complementation experiments demonstrated that OsKOL2 corresponds to D35. Homozygote with null alleles of D35 showed a severe dwarf phenotype; therefore, d35Tan-Ginbozu is a weak allele of D35. Introduction of OsKOL4 into d35Tan-Ginbozu did not rescue its dwarf phenotype, indicating that OsKOL4 is not involved in GA biosynthesis. OsKOL4 and OsKOL5 are likely to take part in phytoalexin biosynthesis, because their expression was promoted by UV irradiation and/or elicitor treatment. Comparing d35Tan-Ginbozu with other high yielding cultivars, we discuss strategies to produce culm architectures suitable for high crop yield by decreasing GA levels.     

The pea gene LH encodes ent-kaurene oxidase

The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants.     

The CYP701B1 of Physcomitrella patens is an ent-kaurene oxidase that resists inhibition by uniconazole-P

The moss Physcomitrella patens produces both ent-kaurene and ent-kaurenoic acid, which are intermediates of gibberellin biosynthesis in flowering plants. The CYP701 superfamily of cytochrome P450s functions as ent-kaurene oxidases in the biosynthesis of ent-kaurenoic acid. A candidate gene encoding ent-kaurene oxidase in P. patens, CYP701B1, was cloned and heterologously expressed in yeast to examine enzyme activities in vitro. The recombinant CYP701B1 protein catalyzed the oxidation reaction from ent-kaurene to ent-kaurenoic acid. CYP701B1 activity was highly resistant to the ent-kaurene oxidase inhibitor uniconazole-P (IC(50) 64 μM), even though the activity of Arabidopsis ent-kaurene oxidase (CYP701A3) was sensitive (IC(50) 0.26 μM).     

CYP701A26 is characterized as an <Emphasis Type="Italic">ent</Emphasis>-kaurene oxidase with putative involvement in maize gibberellin biosynthesis

Objectives

To characterize the ent-kaurene oxidase (KO) involved in maize (Zea mays) gibberellin (GA) biosynthesis.

Results

Two putative KO genes were identified in maize based on the homologous alignment. Biochemical characterization indicated that one of them encoded a cytochrome P450 monooxygenase (P450) CYP701A26, which reacted with ent-kaurene to form ent-kaurenoic acid, the key intermediate of GA biosynthesis. CYP701A26 showed constitutive expression in active growing tissues and no inducible expression, which led to putative designation of CYP701A26 as the ZmKO. CYP701A26 exhibited substrate promiscuity to catalyze oxidation of other labdane related diterpenes. Another maize KO homologue, CYP701A43 did not show any catalytic activities on ent-kaurene or other tested diterpenes. It exhibited inducible gene expression and might accept unknown substrates to play roles in specialized metabolism for stress response.

Conclusions

CYP701A26 was characterized to exhibit ent-kaurene oxidase activity with substrate promiscuity and might be involved in maize GA biosynthesis, and its homologue CYP701A43 did not show such function and might play roles in stress response.

     

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.     

Characterization of CYP76M5-8 indicates metabolic plasticity within a plant biosynthetic gene cluster

Recent reports have revealed genomic clustering of enzymatic genes for particular biosynthetic pathways in plant specialized/secondary metabolism. Rice (Oryza sativa) carries two such clusters for production of antimicrobial diterpenoid phytoalexins, with the cluster on chromosome 2 containing four closely related/homologous members of the cytochrome P450 CYP76M subfamily (CYP76M5-8). Notably, the underlying evolutionary expansion of these CYP appears to have occurred after assembly of the ancestral biosynthetic gene cluster, suggesting separate roles. It has been demonstrated that CYP76M7 catalyzes C11α-hydroxylation of ent-cassadiene, and presumably mediates an early step in biosynthesis of the derived phytocassane class of phytoalexins. Here we report biochemical characterization of CYP76M5, -6, and -8. Our results indicate that CYP76M8 is a multifunctional/promiscuous hydroxylase, with CYP76M5 and -7 seeming to provide only redundant activity, while CYP76M6 seems to provide both redundant and novel activity, relative to CYP76M8. RNAi-mediated double knockdown of CYP76M7 and -8 suppresses elicitor inducible phytocassane production, indicating a role for these monooxygenases in phytocassane biosynthesis. In addition, our data suggests that CYP76M5, -6, and -8 may play redundant roles in production of the oryzalexin class of phytoalexins as well. Intriguingly, the preceding diterpene synthase for oryzalexin biosynthesis, unlike that for the phytocassanes, is not found in the chromosome 2 diterpenoid biosynthetic gene cluster. Accordingly, our results not only uncover a complex evolutionary history, but also further suggest some intriguing differences between plant biosynthetic gene clusters and the seemingly similar microbial operons. The implications for the underlying metabolic evolution of plants are then discussed.     

A conformationally restricted uniconazole analogue as a specific inhibitor of rice ent-kaurene oxidase, CYP701A6

The plant growth retardant uniconazole (UNI), which has been used as an effective inhibitor of ent-kaurene oxidase (CYP701A) involved in gibberellin biosynthesis, also strongly inhibits ABA 8'-hydroxylase (CYP707A), a key enzyme in abscisic acid catabolism. Azole P450 inhibitors bind to the P450 active site by both coordinating to the heme-iron atom via an sp(2) nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from its small molecular size and flexible conformation that allows it to fit into active sites differing in size and shape. To find a selective inhibitor of CYP701A based on this hypothesis, we examined inhibitory activities of three types of UNI analogues, which were conformationally constrained, enlarged in width, and enlarged in length, against recombinant rice CYP701A6 and Arabidopsis CYP707A3. Conformationally restricted analogues, UFAP2 and UFAP2N, inhibited CYP701A6 as strongly as UNI, whereas it inhibited CYP707A3 less than UNI.     

OsLOL1, a C2C2‐type zinc finger protein,interacts with OsbZIP58 to promote seed germination through the modulation of gibberellin biosynthesis in Oryza sativa

Seed germination is a key developmental process in the plant life cycle that is influenced by various environmental cues and phytohormones through gene expression and a series of metabolism pathways. In the present study, we investigated a C2C2‐type finger protein, OsLOL1, which promotes gibberellin (GA) biosynthesis and affects seed germination in Oryza sativa (rice). We used OsLOL1 antisense and sense transgenic lines to explore OsLOL1 functions. Seed germination timing in antisense plants was restored to wild type when exogenous GA₃ was applied. The reduced expression of the GA biosynthesis gene OsKO2 and the accumulation of ent‐kaurene were observed during germination in antisense plants. Based on yeast two‐hybrid and firefly luciferase complementation analyses, OsLOL1 interacted with the basic leucine zipper protein OsbZIP58. The results from electrophoretic mobility shift and dual‐luciferase reporter assays showed that OsbZIP58 binds the G‐box cis‐element of the OsKO2 promoter and activates LUC reporter gene expression, and that interaction between OsLOL1 and OsbZIP58 activates OsKO2 gene expression. In addition, OsLOL1 decreased SOD1 gene expression and accelerated programmed cell death (PCD) in the aleurone layer of rice grains. These findings demonstrate that the interaction between OsLOL1 and OsbZIP58 influences GA biosynthesis through the activation of OsKO2 via OsbZIP58, thereby stimulating aleurone PCD and seed germination.     

The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo, leading to reduced biosynthesis of gibberellins and rice dwarf symptoms

The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development.     

The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.

The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to ent-kaurene catalyzed by ent-kaurene synthetases A and B. The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs. Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of ent-kaurene. The GA1 locus was cloned previously using the technique of genomic subtraction. Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis. This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation. In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated. This result indicates that the GA1 gene encodes the enzyme ent-kaurene synthetase A, which catalyzes the conversion of GGPP to CPP. Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts. The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.     

Biochemical and molecular analyses of gibberellin biosynthesis in fungi

The plant hormone, gibberellin (GA), regulates plant growth and development. It was first isolated as a superelongation-promoting diterpenoid from the fungus, Gibberella fujikuroi. G. fujikuroi uses different GA biosynthetic intermediates from those in plants to produce GA3. Another class of GA-producing fungus, Phaeosphaeria sp. L487, synthesizes GA1 by using the same intermediates as those in plants. A molecular analysis of GA biosynthesis in Phaeosphaeria sp. has revealed that diterpene cyclase and cytochrome P450 monooxygenases were involved in the plant-like biosynthesis of GA1. Fungal ent-kaurene synthase is a bifunctional cyclase. Subsequent oxidation steps are catalyzed by P450s, leading to biologically active GA1. GA biosynthesis in plants is divided into three steps involving soluble enzymes and membrane-bound cytochrome P450. The activation of plant GAs is catalyzed by soluble 2-oxoglutarate-dependent dioxygenases, which is in contrast to the catalysis of fungal GA biosynthesis. This difference suggests that the origin of fungal GA biosynthesis is evolutionally independent of that in plants.     

高等植物赤霉素生物合成及其调节研究进展

主要介绍近年来高等植物中生物活性GAs的生物合成,拟南芥GA生物合成途径中关键酶基因（GA1－GA5）的克隆和GA3基因CYP701A3的母（Saccharomyces cerevisiae）中的成功表达。评述了活性GAs对赤霉不生物合成的反馈抑制作用和反馈调节中信号的传递和接收问题。高等植物中光周期对GA生物合成的调节主要是在20－氧化和／或3β－羟基化步骤。     

Identification of a biosynthetic gene cluster in rice for momilactones

Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.     

Parsing a multifunctional biosynthetic gene cluster from rice: Biochemical characterization of CYP71Z6 & 7

Rice (Oryza sativa) contains a biosynthetic gene cluster associated with production of at least two groups of diterpenoid phytoalexins, the antifungal phytocassanes and antibacterial oryzalides. While cytochromes P450 (CYP) from this cluster are known to be involved in phytocassane production, such mono-oxygenase activity relevant to oryzalide biosynthesis was unknown. Here we report biochemical characterization demonstrating that CYP71Z6 from this cluster acts as an ent-isokaurene C2-hydroxylase that is presumably involved in the biosynthesis of oryzalides. Our results further suggest that the closely related and co-clustered CYP71Z7 likely acts as a C2-hydroxylase involved in a latter step of phytocassane biosynthesis. Thus, CYP71Z6 & 7 appear to have evolved distinct roles in rice diterpenoid metabolism, offering insight into plant biosynthetic gene cluster evolution.     

Production of Bioactive Diterpenoids in the Euphorbiaceae Depends on Evolutionarily Conserved Gene Clusters

The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways.     

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.     

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.     

高等植物赤霉素生物合成及其调节研究进展

主要介绍近年来高等植物中生物活性GAs的生物合成,拟南芥GA生物合成途径中关键酶基因（GA1-GA5）的克隆和GA3基因CYP701A3在酵母（Saccharomyces cerevisiae）中的成功表达。评述了活性GAs对赤霉素生物合成的反馈抑制作用和反馈调节中信号的传递和接收问题。高等植物中光周期对GA生物合成的调节主要是在20_氧化和/或3β_羟基化步骤。