Isolation and characterization of two phytoalexins from rice as momilactones A and B

Two phytoalexins were isolated as chromatographically homogeneous amorphous solids from UV-irradiated, dark-grown rice coleoptiles. From their mass and ¹H NMR spectra, the compounds were characterized as the known diterpenes, momilactones A and B. The same compounds were also produced in blast-infected, WL 28325-treated rice leaves. They appear to be the first clearly identified cereal phytoalexins.     

Diterpene phytoalexins are biosynthesized in and exuded from the roots of rice seedlings

Rice (Oryza sativa L.) produces a variety of diterpene phytoalexins, such as momilactones, phytocassanes, and oryzalexins. Momilactone B was previously identified as an allelopathic substance exuded from the roots of rice. We identified in this present study momilactone A and phytocassanes A-E in extracts of, and exudates from, the roots of rice seedlings. The concentration of each compound was of the same order of magnitude as that of momilactone B. Expression analyses of the diterpene cyclase genes responsible for the biosynthesis of momilactones and phytocassanes suggest that these phytoalexins found in roots are primarily biosynthesized in those roots. None of phytocassanes B-E exhibited allelopathic activity against dicot seedling growth, whereas momilactone A showed much weaker allelopathic activity than momilactone B. The exudation of diterpene phytoalexins from the roots might be part of a system for defense against root-infecting pathogens.     

Functional analysis of eubacterial diterpene cyclases responsible for biosynthesis of a diterpene antibiotic,terpentecin

Eubacterial diterpene cyclase genes had previously been cloned from a diterpenoid antibiotic terpentecin producer (Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., and Seto, H. (2001) J. Bacteriol. 183, 6085-6094). Their products, open reading frame 11 (ORF11) and ORF12, were essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cyclized intermediate, terpentedienol diphosphate (TDP), which was then transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly catalyzed the conversion of GGDP into three olefinic compounds. Moreover, the ORF12 product utilized farnesyl diphosphate as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP with the exception of the chain lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the transformation of TDP to the olefin, terpentetriene by an ionization-initiated reaction followed by deprotonation. The kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of farnesyl diphosphate and that the relative activity of the reaction converting TDP into TTE was highest among the reactions using TDP, GGDP, or farnesyl diphosphate as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.     

Diterpene cyclases and the nature of the isoprene fold

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

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.     

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.     

Changes in the content and biosynthesis of phytoalexins in banana fruit

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-(13)C] and [2-(13)C]cinnamic acids, and [2-(13)C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4'-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.     

Convergent or parallel molecular evolution of momilactone A and B: potent allelochemicals, momilactones have been found only in rice and the moss Hypnum plumaeforme

Plant second metabolites momilactone A and B, which act as potent phytoalexins and allelochemicals, have been found thus far only in rice and the moss Hypnum plumaeforme, although both plants are taxonomically quite distinct. The concentrations of momilactone A and B, respectively, in rice plants were 4.5-140 and 2.9-85 μg/g, and those in H. plumaeforme were 8.4-58.7 and 4.2-23.4 μg/g. Momilactone A and B concentrations in rice and H. plumaeforme plants were increased by UV irradiation, elicitor and jasmonic acid treatments. Rice and H. plumaeforme plants secrete momilactone A and B into the rhizosphere, and the secretion level was also increased by UV irradiation, elicitor and jasmonic acid treatments. In addition, although endogenous concentrations of momilactone A in rice and H. plumaeforme were greater than those of momilactone B, the secretion levels of momilactone B were greater than those of momilactone A in rice and H. plumaeforme, which suggests that momilactone B may be selectively secreted by both rice and H. plumaeforme. As momilactone A and B exert potent antifungal and growth inhibitory activities, momilactone A and B may play an important role in the defense responses in H. plumaeforme and rice against pathogen infections and in allelopathy. The secretion of momilactone A and B into the rhizosphere may also prevent bacterial and fungal infections and provide a competitive advantage for nutrients through the inhibition of invading root systems of neighboring plants as allelochemicals. Therefore, both plants, despite their evolutionary distance, may use same defense strategy with respect to the momilactone A and B production and secretion, which resulting from convergent or parallel evolutionary processes. In the case of parallel evolution, there may be plant species providing the missing link in molecular evolution of momilactones between H. plumaeforme and rice.     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B.

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.     

SpaC and NisC, the cyclases involved in subtilin and nisin biosynthesis, are zinc proteins

Lantibiotics are peptide-derived antimicrobial agents that are ribosomally synthesized and posttranslationally modified by a multienzyme complex to their biologically active forms. Nisin has attracted much attention recently due to its novel mechanism of action including specific binding to the bacterial cell wall precursor lipid II, followed by membrane permeabilization. Nisin has been commercially used as a food preservative, while other lantibiotics show promising activity against bacterial infections. The posttranslational modifications are believed to be carried out by a multienzyme complex. At present the enzymes catalyzing the formation of the lantibiotic signature structural motifs, dehydroalanine (Dha), dehydrobutyrine (Dhb), lanthionine (Ln), and methyllanthionine (MeLn), are poorly characterized. In an effort to gain insight into the mechanism by which lantibiotics are biosynthesized, the cyclase enzymes involved in the synthesis of nisin and subtilin (NisC and SpaC, respectively) have been cloned, expressed, and purified. Both proteins exist as monomers in solution and contain a stoichiometric zinc atom. EXAFS data on SpaC and a C349A mutant are in line with two cysteine ligands to the metal in the wild-type enzyme with possibly two additional histidines. The two cysteine ligands are likely Cys303 and Cys349 on the basis of sequence alignments and EXAFS data. The metal may function to activate the cysteine thiol of the peptide substrate toward intramolecular Michael addition to the dehydroalanine and dehydrobutyrine residues in the peptide.     

Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice

A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a).     

Sinalbins A and B, phytoalexins from Sinapis alba: elicitation, isolation, and synthesis

The chemical structure and synthesis of sinalbin A is described. This cruciferous phytoalexin is produced by white mustard (Sinapis alba) after treatment with biotic and abiotic elicitors. In addition, a related metabolite, named sinalbin B, is present in extracts from elicited plants, but not in those from non-elicited controls. Sinalbin B, which was also synthesized, appears to be both a phytoalexin and a biosynthetic precursor of sinalbin A.