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.

Potential of Oryza officinalis to augment the cold tolerance genetic mechanisms of Oryza sativa by network complementation.

Oryza officinalis is an accessible alien donor for genetic improvement of rice. Comparison across a representative panel of Oryza species showed that the wild O. officinalis and cultivated O. sativa ssp. japonica have similar cold tolerance potentials. The possibility that either distinct or similar genetic mechanisms are involved in the low temperature responses of each species was addressed by comparing their transcriptional networks. General similarities were supported by shared transcriptomic signatures indicative of equivalent metabolic, hormonal, and defense status. However, O. officinalis has maintained an elaborate cold-responsive brassinosteroid-regulated BES1-network that appeared to have been fragmented in O. sativa. BES1-network is potentially important for integrating growth-related responses with physiological adjustments and defenses through the protection of photosynthetic machinery and maintenance of stomatal aperture, oxidative defenses, and osmotic adjustment. Equivalent physiological processes are functional in O. sativa but their genetic mechanisms are under the direct control of ABA-dependent, DREB-dependent and/or oxidative-mediated networks uncoupled to BES1. While O. officinalis and O. sativa represent long periods of speciation and domestication, their comparable cold tolerance potentials involve equivalent physiological processes but distinct genetic networks. BES1-network represents a novel attribute of O. officinalis with potential applications in diversifying or complementing other mechanisms in the cultivated germplasm.

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.

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.

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.

Rapid turnover of antimicrobial-type cysteine-rich protein genes in closely related Oryza genomes.

Defensive and reproductive protein genes undergo rapid evolution. Small, cysteine-rich secreted peptides (CRPs) act as antimicrobial agents and function in plant intercellular signaling and are over-represented among reproductively expressed proteins. Because of their roles in defense, reproduction and development and their presence in multigene families, CRP variation can have major consequences for plant phenotypic and functional diversification. We surveyed the CRP genes of six closely related Oryza genomes comprising Oryza sativa ssp. japonica and ssp. indica, Oryza glaberrima and three accessions of Oryza rufipogon to observe patterns of evolution in these gene families and the effects of variation on their gene expression. These Oryza genomes, like other plant genomes, have accumulated large reservoirs of CRP sequences, comprising 26 groups totaling between 676 and 843 genes, in contrast to antimicrobial CRPs in animal genomes. Despite the close evolutionary relationships between the genomes, we observed rapid changes in number and structure among CRP gene families. Many CRP sequences are in gene clusters generated by local duplications, have undergone rapid turnover and are more likely to be silent or specifically expressed. By contrast, conserved CRP genes are more likely to be highly and broadly expressed. Variable CRP genes created by repeated duplication, gene modification and inactivation can gain new functions and expression patterns in newly evolved gene copies. For the CRP proteins, the process of gain/loss by deletion or duplication at gene clusters seems to be an important mechanism in evolution of the gene families, which also contributes to their expression evolution.

Use of intercellular washing fluid to investigate the secreted proteome of the rice-Magnaporthe interaction.

Early interactions between invading penetration hyphae of the pathogenic fungus Magnaporthe oryzae and rice cells occur at the apoplast, the free diffusional space outside the plasma membrane of leaves. After initial colonization, intercellular hyphae are again in intimate contact with the rice apoplast. While several studies have looked at proteomics in rice-Magnaporthe interactions, none have focused on apoplast localized proteins. We adjusted a protocol for intercellular washing fluids (IWF) to rice leaves infected with Magnaporthe oryzae for proteomic analysis. In our IWF extract, we identified several proteins associated with compatible or incompatible pathogen interactions. Three DUF26 domain proteins were identified as changing in abundance 12 h after inoculation, confirming DUF26 domain-containing proteins are among early, pathogen stress-responsive proteins induced by infection with Magnaporthe oryzae. A Magnaporthe cyclophilin, previously identified as a virulence factor was also identified in the intercellular washing fluid.

UV and blue light signalling: pathways regulating chalcone synthase gene expression in Arabidopsis.

UV-B, UV-A and blue light control a variety of aspects of plant development via distinct photoreceptors and signalling pathways. The known photoreceptors for UV-A/blue light are cryptochrome (cry)1 and cry2, and the phototropism photoreceptor, phototropin. Redox processes are important in cry and phototropin signal transduction. A specific photoreceptor for UV-B has not been identified and there appear to be several possible UV-B signalling pathways. We are investigating the UV and blue light regulation of transcription of the chalcone synthase gene (CHS) in Arabidopsis. Experiments with photoreceptor mutants show that distinct UV-A/blue (cry mediated) and UV-B photoreception systems control CHS expression. Experiments with an Arabidopsis cell suspension culture show that the UV-B and cry1 signalling pathways differ kinetically and pharmacologically. In contrast to some other UV-B responses, the UV-B induction of CHS does not appear to involve oxidative stress signalling. Promoter elements and candidate transcription factors that effect CHS induction have been identified. Interactions within a network of UV-B, cry and phytochrome signalling pathways regulate CHS expression. Synergistic interactions between the UV-B pathway and distinct UV-A and blue-light pathways maximize the response. In addition, specific phytochromes positively control the cry1 pathway via distinct potentiation and coaction effects, and negatively regulate the UV-B pathway.