SummaryWe have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspensioncultured 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 (À)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also con®rmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identi®ed ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (À)-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 biosyntheis of (À)-phytocassanes in rice.

In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors. We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor. The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h. The expression of OsDTC2 was also induced in UV-irradiated rice leaves. In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UVirradiated rice leaves.

In suspension-cultured rice (Oryza sativa L.) cells, jasmonic acid (JA) functions as a signal transducer in elicitor N-acetylchitoheptaose-induced phytoalexin production. Differential screening of a cDNA library constructed using poly(A) + RNA from suspension-cultured rice cells treated with JA (10 -4 M) for 2 h yielded a cDNA for a gene that responded to exogenous JA by an increase in mRNA level. Nucleotide sequence analysis indicated that the cDNA encodes an homologue of the yeast Old Yellow Enzyme. The deduced amino acid sequence was very similar to the sequences of 12-oxophytodienoic acid reductases (OPR) 1 and 2 from Arabidopsis thaliana (AtOPR1 and AtOPR2) and OPR1 from tomato (Lycopersicon esculentum) (LeOPR1). The cDNA-encoded protein purified from recombinant Escherichia coli cells as a hexahistidine-tagged fusion protein exhibited OPR activity similar to that of AtOPR1, AtOPR2, and LeOPR1, which catalyze reduction of (-)-cis-12-oxophytodienoic acid (OPDA) preferentially over (+)-cis-OPDA, a natural precursor of JA. Thus the rice enzyme was termed OsOPR1. The physiological roles of OsOPR1 are discussed. This is the first report of the cloning of an OPR gene from a monocot plant.

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