Identification of UV-Induced Diterpenes Including a New Diterpene Phytoalexin, Phytocassane F, from Rice Leaves by Complementary GC/MS and LC/MS Approaches.

Rice phytoalexins are regarded as one of the most important weapons against pathogenic microorganisms. We attempted to identify novel phytoalexins and their derivatives using GC/MS and LC/MS analyses. Diterpene derivatives, 9ß-pimara-7,15-diene-3ß,6ß,19-triol, 1, stemar-13-en-2α-ol, 2, and 1α,2α-dihydroxy-ent-12,15-cassadiene-3,11-dione, 3, were isolated from UV-irradiated rice leaves by chromatographic methods. These structures were confirmed by 1D- and 2D-NMR and MS analyses. Interestingly, all three compounds were accumulated following an infection by the rice blast pathogen Magnaporthe oryzae. Compounds 1 and 2 exhibited weak antifungal activity and may be the biosynthetic intermediates of rice phytoalexins momilactones and oryzalexin S, respectively. Compound 3 exhibited relatively high inhibitory activity against the fungal mycelial growth of M. oryzae to the same extent as the known phytoalexin phytocassane A. We conclude that 3 is a member of the cassane-type phytoalexin family and propose the name phytocassane F.

Diterpene cyclases responsible for the biosynthesis of phytoalexins, momilactones A, B, and oryzalexins A-F in rice.

Rice (Oryza sativa L.) produces diterpene phytoalexins, such as momilactones, oryzalexins, and phytocassanes. Using rice genome information and in vitro assay with recombinant enzymes, we identified genes (OsKS4 and OsKS10) encoding the type-A diterpene cyclases 9beta-pimara-7,15-diene synthase and ent-sandaracopimaradiene synthase which are involved in the biosynthesis of momilactones A, B and oryzalexins A-F respectively. Transcript levels of these two genes increased remarkably after ultraviolet (UV) treatment, which is consistent with elevated production of phytoalexins by UV. These two genes might prove powerful tools for understanding plant defense mechanisms in rice.

Biological functions of ent- and syn-copalyl diphosphate synthases in rice: key enzymes for the branch point of gibberellin and phytoalexin biosynthesis.

Rice (Oryza sativa L.) produces ent-copalyl diphosphate (ent-CDP) and syn-CDP as precursors for several classes of phytoalexins and the phytohormones, gibberellins (GAs). It has recently been shown that a loss-of-function mutation of OsCPS1, a gene encoding a putative ent-CDP synthase, results in a severely GA-deficient dwarf phenotype in rice. To clarify the biological functions of the ent- and syn-CDP synthases involved in the biosynthesis of phytoalexins and/or GAs, we isolated two cDNAs, OsCyc1 and OsCyc2, encoding putative diterpene cyclases from ultraviolet (UV)-irradiated rice leaves (cv. Nipponbare). The production of phytoalexins in rice leaves is known to be highly induced by UV treatment. Using a bacterial expression system, we demonstrated that OsCyc1 encodes syn-CDP synthase and that OsCyc2 and OsCPS1 encode ent-CDP synthase. The level of expression of the OsCyc1 and OsCyc2 transcripts in rice leaves increased drastically in response to UV treatment, whereas expression of the OsCPS1 transcript was not induced by UV light. These results suggest that OsCyc1, OsCyc2 and OsCPS1 are responsible for the biosynthesis of momilactones A and B and oryzalexin S, oryzalexins A-F and phytocassanes A-E, and GAs, respectively. Our results strongly suggest the presence of two ent-CDP synthase isoforms in rice, one that participates in the biosynthesis of GAs and a second that is involved in the biosynthesis of phytoalexins.

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor.

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.