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.

Effects of adrenaline on circulatory dynamics and cardiac function in rats administered chlorpromazine.

We aimed to elucidate changes in circulatory dynamics and cardiac function during concomitant use of chlorpromazine (CPZ) and adrenaline (AD). An arterial line and left intraventricular pressure-volume measurement catheter were inserted in rats. CPZ 10 mg/kg was administered to the left great adductor muscle, followed by normal saline (NS) or AD 50 µg/kg through the tongue 20 min later. End-diastolic volume (V ed), end-systolic pressure (P es), stroke volume (SV), stroke work (SW), end-systolic volume elastance (E es), systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse rate (PR) were measured. Following AD administration, V ed significantly decreased at 2-4 and 10 min than that in control rats; P es significantly decreased at 1 min; E es significantly increased from 2 to 10 min; SV did not change significantly, and SW significantly reduced at 1 and 2 min; SBP and DBP were lower at 1-3 min than in the control; and PR increased at 10 min. These findings suggest that when AD-containing local anesthetics are administered during dental treatment of patients taking CPZ, there is a risk of a temporary drop in blood pressure. However, the blood pressure is recovered a few minutes later by the increase in afterload and the myocardial contractile force.

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.

Transcripts of two ent-copalyl diphosphate synthase genes differentially localize in rice plants according to their distinct biological roles.

Gibberellins (GAs) are diterpenoid phytohormones that regulate various aspects of plant growth. Tetracyclic hydrocarbon ent-kaurene is a biosynthetic intermediate of GAs, and is converted from geranylgeranyl diphosphate, a common precursor of diterpenoids, via ent-copalyl diphosphate (ent-CDP) through successive cyclization reactions catalysed by two distinct diterpene synthases, ent-CDP synthase and ent-kaurene synthase. Rice (Oryza sativa L.) has two ent-CDP synthase genes, OsCPS1 and OsCPS2. It has been thought that OsCPS1 participates in GA biosynthesis, while OsCPS2 participates in the biosynthesis of phytoalexins, phytocassanes A-E, and oryzalexins A-F. It has been shown previously that loss-of-function OsCPS1 mutants display a severe dwarf phenotype caused by GA deficiency despite possessing another ent-CDP synthase gene, OsCPS2. Here, experiments were performed to account for the non-redundant biological function of OsCPS1 and OsCPS2. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that OsCPS2 transcript levels were drastically lower than those of OsCPS1 in the basal parts, including the meristem of the second-leaf sheaths of rice seedlings. qRT-PCR results using tissue samples prepared by laser microdissection suggested that OsCPS1 transcripts mainly localized in vascular bundle tissues, similar to Arabidopsis CPS, which is responsible for GA biosynthesis, whereas OsCPS2 transcripts mainly localized in epidermal cells that address environmental stressors such as pathogen attack. Furthermore, the OsCPS2 transgene under regulation of the OsCPS1 promoter complemented the dwarf phenotype of an OsCPS1 mutant, oscps1-1. The results indicate that transcripts of the two ent-CDP synthase genes differentially localize in rice plants according to their distinct biological roles, OsCPS1 for growth and OsCPS2 for defence.