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.

Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha.

Gibberellins (GAs) are key phytohormones that regulate growth, development, and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthesis intermediates such as ent-kaurenoic acid (KA) are also produced by bryophytes. Here, we show that in the liverwort Marchantia polymorpha, KA and GA12 are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently exhibited altered thallus growth allometry, delayed initiation of gametogenesis, and abnormal morphology of gamete-bearing structures (gametangiophores). By chemical treatments and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, we confirmed that these phenotypes were caused by the deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism in M. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but were co-opted independently to regulate responses to light quality change in different plant lineages during the past 450 million years of evolution.

Production of desmethyl-gregatin A, a possible causative toxin of brown stem rot in adzuki bean, by Phialophora gregata f. sp. adzukicola.

To elucidate the cause of brown stem rot in the adzuki bean, we re-evaluated the phytotoxins produced in cultures of the causative agent, Phialophora gregata f. sp. adzukicola. The ethyl acetate-soluble acidic fraction of the culture, as well as the neutral fraction, inhibited the growth of alfalfa seedlings. In the neutral fraction, known phytotoxins gregatin A, B, and C or D and penicilliol A were present. Although the phytotoxins in the acidic fraction were unstable, liquid chromatography-mass spectrometry analysis of the partially purified material suggested that one phytotoxin present was the non-methylated gregatin desmethyl-gregatin A (gregatinic acid A).

Molecular characterization of a moss isoprene synthase provides insight into its evolution.

This is the first report on the molecular characterization of isoprene synthase (ISPS) from the moss Calohypnum plumiforme. After isoprene emission from C. plumiforme was confirmed, the cDNA encoding C. plumiforme ISPS (CpISPS) was narrowed down using a genome database associated with protein structure prediction, and a CpISPS gene was identified. The recombinant CpISPS, produced in Escherichia coli, converted dimethylallyl diphosphate to isoprene. Phylogenetic analysis indicated similarity between the amino acid sequences of CpISPS and moss diterpene cyclases (DTCs) but not ISPSs of higher plants, implying that CpISPS is derived from moss DTCs and is evolutionarily unrelated to canonical ISPSs of higher plants. CpISPS is a novel class I cyclase of the terpene synthase-c subfamily harboring αß domains. This study will help further study of isoprene biosynthesis and the physiological functions of isoprene in mosses.

Hormonal Diterpenoids Distinct to Gibberellins Regulate Protonema Differentiation in the Moss Physcomitrium patens.

Plants synthesize gibberellin (GA), a diterpenoid hormone, via ent-kaurenoic acid (KA) oxidation. GA has not been detected in the moss Physcomitrium patens despite its ability to synthesize KA. It was recently shown that a KA metabolite, 3OH-KA, was identified as an active regulator of protonema differentiation in P. patens. An inactive KA metabolite, 2OH-KA, was also identified in the moss, as was KA2ox, which is responsible for converting KA to 2OH-KA. In this review, we mainly discuss the GA biosynthetic gene homologs identified and characterized in bryophytes. We show the similarities and differences between the OH-KA control of moss and GA control of flowering plants. We also discuss using recent genomic studies; mosses do not contain KAO, even though other bryophytes do. This absence of KAO in mosses corresponds to the presence of KA2ox, which is absent in other vascular plants. Thus, given that 2OH-KA and 3OH-KA were isolated from ferns and flowering plants, respectively, vascular plants may have evolved from ancestral bryophytes that originally produced 3OH-KA and GA.

Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants.

Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1/HpDTC1 gene and the "dehydrogenase momilactone A synthase" gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (Oryza and Echinochloa) and C. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.

Conversion of Mevalonate 3-Kinase into 5-Phosphomevalonate 3-Kinase by Single Amino Acid Mutations.

Mevalonate 3-kinase plays a key role in a recently discovered modified mevalonate pathway specific to thermophilic archaea of the order Thermoplasmatales The enzyme is homologous to diphosphomevalonate decarboxylase, which is involved in the widely distributed classical mevalonate pathway, and to phosphomevalonate decarboxylase, which is possessed by halophilic archaea and some Chloroflexi bacteria. Mevalonate 3-kinase catalyzes the ATP-dependent 3-phosphorylation of mevalonate but does not catalyze the subsequent decarboxylation as related decarboxylases do. In this study, a substrate-interacting glutamate residue of Thermoplasma acidophilum mevalonate 3-kinase was replaced by smaller amino acids, including its counterparts in diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, with the aim of altering substrate specificity. These single amino acid mutations resulted in the conversion of mevalonate 3-kinase into 5-phosphomevalonate 3-kinase, which can synthesize 3,5-bisphosphomevalonate from 5-phosphomevalonate. The mutants catalyzing the hitherto undiscovered reaction enabled the construction of an artificial mevalonate pathway in Escherichia coli cells, as was demonstrated by the accumulation of lycopene, a red carotenoid pigment.IMPORTANCE Isoprenoid is the largest family of natural compounds, including important bioactive molecules such as vitamins, hormones, and natural medicines. The mevalonate pathway is a target for metabolic engineering because it supplies precursors for isoprenoid biosynthesis. Mevalonate 3-kinase is an enzyme involved in the modified mevalonate pathway specific to limited species of thermophilic archaea. Replacement of a single amino acid residue in the active site of the enzyme changed its substrate preference and allowed the mutant enzymes to catalyze a previously undiscovered reaction. Using the genes encoding the mutant enzymes and other archaeal enzymes, we constructed an artificial mevalonate pathway, which can produce the precursor of isoprenoid through an unexplored route, in bacterial cells.

Effect of Secondary Metabolites of Tomato ( Solanum lycopersicum) on Chemotaxis of Ralstonia solanacearum, Pathogen of Bacterial Wilt Disease.

The chemotactic activity of the pathogen of bacterial wilt disease, Ralstonia solanacearum, was tested against 30 aromatic acids and plant hormones infused on filter discs in bioassays on agar plates. 4-Hydroxycinnamic acid ( p-coumaric acid) and 4-hydroxybenzoic acid were strong chemoattractants, 3,4-dihydroxybenzoic acid (protocatechuic acid) and jasmonic acid were weak attractants, and 2-hydroxybenzoic acid (salicylic acid) showed both attracting and repelling activity depending on dose. Examination of the dose dependency revealed that the ED50 for 4-hydroxycinnamic acid and 4-hydroxybenzoic acid was 0.08 and 0.39 µmol/disc, respectively. 2-Hydroxybenzoic acid showed chemoattractant activity at 0.33 µmol/disc but chemorepellent activity at 3.3 µmol/disc, and bacterial random motility was activated at 1.0 µmol/disc and bacterial activity was suppressed at 33 µmol/disc. Although water-soluble attractants including amino acids and organic acids have been previously investigated, this is the first report of hydroxylated aromatic acids (HAAs) as chemoattractants of R. solanacearum.

Assays of Protonemal Growth Responses in Physcomitrella patens Under Blue- and Red-Light Stimuli.

The non-seed land plant Physcomitrella patens is a model species for developmental, cellular, and molecular biology studies in mosses and also for performing genetic analyses. Previously, it was shown that wild-type P. patens displays a unique photomorphogenetic behavior, in which chloronemal filaments grow in the opposite direction to a blue-light source. Here, we describe bioassay systems that can be used to study light avoidance responses as well as other aspects of photomorphogenetic regulation in P. patens grown under red- and blue-light sources.

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.

Ethyl ß-d-glucoside: a novel chemoattractant of Ralstonia solanacearum isolated from tomato root exudates by a bioassay-guided fractionation.

A chemoattractant of Ralstonia solanacearum isolated from the activated charcoal-adsorbed fraction of tomato root exudates was identified as ethyl ß-d-glucopyranoside by instrumental analyses and comparison with synthetic preparations. Ethyl ß-D-glucopyranoside showed unambiguous activity at above 1 µmol/disc. Its stereoisomers and D-glucose were inactive.

Modified mevalonate pathway of the archaeon Aeropyrum pernix proceeds via trans-anhydromevalonate 5-phosphate.

The modified mevalonate pathway is believed to be the upstream biosynthetic route for isoprenoids in general archaea. The partially identified pathway has been proposed to explain a mystery surrounding the lack of phosphomevalonate kinase and diphosphomevalonate decarboxylase by the discovery of a conserved enzyme, isopentenyl phosphate kinase. Phosphomevalonate decarboxylase was considered to be the missing link that would fill the vacancy in the pathway between mevalonate 5-phosphate and isopentenyl phosphate. This enzyme was recently discovered from haloarchaea and certain Chroloflexi bacteria, but their enzymes are close homologs of diphosphomevalonate decarboxylase, which are absent in most archaea. In this study, we used comparative genomic analysis to find two enzymes from a hyperthermophilic archaeon, Aeropyrum pernix, that can replace phosphomevalonate decarboxylase. One enzyme, which has been annotated as putative aconitase, catalyzes the dehydration of mevalonate 5-phosphate to form a previously unknown intermediate, trans-anhydromevalonate 5-phosphate. Then, another enzyme belonging to the UbiD-decarboxylase family, which likely requires a UbiX-like partner, converts the intermediate into isopentenyl phosphate. Their activities were confirmed by in vitro assay with recombinant enzymes and were also detected in cell-free extract from A. pernix These data distinguish the modified mevalonate pathway of A. pernix and likely, of the majority of archaea from all known mevalonate pathways, such as the eukaryote-type classical pathway, the haloarchaea-type modified pathway, and another modified pathway recently discovered from Thermoplasma acidophilum.

In planta functions of cytochrome P450 monooxygenase genes in the phytocassane biosynthetic gene cluster on rice chromosome 2.

In response to environmental stressors such as blast fungal infections, rice produces phytoalexins, an antimicrobial diterpenoid compound. Together with momilactones, phytocassanes are among the major diterpenoid phytoalexins. The biosynthetic genes of diterpenoid phytoalexin are organized on the chromosome in functional gene clusters, comprising diterpene cyclase, dehydrogenase, and cytochrome P450 monooxygenase genes. Their functions have been studied extensively using in vitro enzyme assay systems. Specifically, P450 genes (CYP71Z6, Z7; CYP76M5, M6, M7, M8) on rice chromosome 2 have multifunctional activities associated with ent-copalyl diphosphate-related diterpene hydrocarbons, but the in planta contribution of these genes to diterpenoid phytoalexin production remains unknown. Here, we characterized cyp71z7 T-DNA mutant and CYP76M7/M8 RNAi lines to find that potential phytoalexin intermediates accumulated in these P450-suppressed rice plants. The results suggested that in planta, CYP71Z7 is responsible for C2-hydroxylation of phytocassanes and that CYP76M7/M8 is involved in C11α-hydroxylation of 3-hydroxy-cassadiene. Based on these results, we proposed potential routes of phytocassane biosynthesis in planta.

Characterization of moss ent-kaurene oxidase (CYP701B1) using a highly purified preparation.

CYP701B1 of the moss, Physcomitrella patents, might be a unique cytochrome P450 having the ent-kaurene oxidase (KO) activity occurring in nonvascular plant. Phylogenetic analysis suggested that the gene encoding CYP701B1 was diverged from a common ancestral gene encoding KO of vascular plants. CYP701B1 expressed in Phichia yeast microsomes was purified and characterized. The purified CYP701B1 catalyzed the oxidation of ent-kaurene to ent-kaurenoic acid through three successive monooxygenations, and the rate-limiting step of this oxidation might be the initial step that forms ent-kaurenol. CYP701B1 was a typical ferric low-spin cytochrome P450 and was completely moved to high-spin state upon binding with ent-kaurene, and apparent Kd of ent-kaurene estimated by the spectral change caused by this spin-state shift was 2.5 µM. The potent KO inhibitor uniconazole, an azole compound with molecular size similar to ent-kaurene, bound CYP701B1 with high affinity. However, ketoconazole, an azole compound whose molecular size is larger than ent-kaurene could not bind to CYP701B, though it binds strongly with CYP51, lanosterol 14-demethylase. The results indicated that the active site of CYP701B1 is fitted for the molecular size of ent-kaurene. The P450 monooxygenase adapted for ent-kaurene oxidation might appear in land plants before evolutionary divergence into vascular and nonvascular plants.

Biochemical synthesis of uniformly 13C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins in planta.

Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown in planta. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly 13C-labeled ent-cassadiene and syn-pimaradiene were enzymatically synthesized with structural verification by GC-MS and 13C-NMR. Application of the 13C-labeled substrates on rice leaves led to the detection of 13C-labeled metabolites using LC-MS/MS. Further application of this method in the moss Hypnum plumaeforme and the nearest out-group of Oryza species Leersia perrieri, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-13C20] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds.

A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase.

The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of (R)-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon Sulfolobus solfataricus The crystal structures of the archaeal enzyme in complex with (R)-5-diphosphomevalonate and adenosine 5'-O-(3-thio)triphosphate or with (R)-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, (R)-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.

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.

Genome-Based Discovery of an Unprecedented Cyclization Mode in Fungal Sesterterpenoid Biosynthesis.

Sesterterpenoids are a group of terpenoid natural products that are primarily biosynthesized via cyclization of the C25 linear substrate geranylfarnesyl pyrophosphate (GFPP). Although the long carbon chain of GFPP in theory allows for many different cyclization patterns, sesterterpenoids are relatively rare species among terpenoids, suggesting that many intriguing sesterterpenoid scaffolds have been overlooked. Meanwhile, the recent identification of the first sesterterpene synthase has allowed the discovery of new sesterterpenoids by the genome mining approach. In this study, we characterized the unusual fungal sesterterpene synthase EvQS and successfully obtained the sesterterpene quiannulatene (1) with a novel and unique highly congested carbon skeleton, which is further oxidized to quiannulatic acid (2) by the cytochrome P450 Qnn-P450. A mechanistic study of its cyclization from GFPP indicated that the biosynthesis employs an unprecedented cyclization mode, which involves three rounds of hydride shifts and two successive C-C bond migrations to construct the 5-6-5-5-5 fused ring system of 1.

HpDTC1, a Stress-Inducible Bifunctional Diterpene Cyclase Involved in Momilactone Biosynthesis, Functions in Chemical Defence in the Moss Hypnum plumaeforme.

Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme.