Cloning and functional analysis of caffeic acid 3-O-methyltransferase from rice (Oryza sativa)
Journal of pesticide science
Rice plants produce diterpene and flavanone phytoalexins which are involved in the defense of the plant. 1-4) Rakwal et al. reported the purification and partial amino acid sequence of naringenin 7-O-methyltransferase (NOMT) which is involved in the biosynthesis of a flavanone phytoalexin, sakuranetin. 5) We have attempted to clone and characterize an NOMT gene from rice. However, we found that the NOMT protein reported by Rakwal et al. has not NOMT activity but caffeic acid
... e (COMT) activity. COMT is widespread throughout the plant kingdom and is found in all lignin-producing plants. 6) COMT cDNAs or genes have been isolated from a number of species including aspen, 7) common zinnia, 8) wheat, 9) maize, 10) etc. Here, we report the cloning of a gene encoding COMT (OsCOMT1) from rice and the characterization of the recombinant protein, though our objective was the cloning of an NOMT gene. To our knowledge, this is the first report on the cloning and characterization of a rice COMT cDNA. Materials and Methods Plant materials Rice plants (Oryza sativa L. cv. Nipponbare) were cultivated in a greenhouse. Chemicals Eriodictyol, homoeriodictyol, hesperetin, kaempferol, myricetin, isorhametin, luteolin and chrysoeriol were purchased from Extrasynthese. p-Coumaric acid, quercetin and apigenin were obtained from Sigma Chemical. Caffeic acid was from Katayama Chemical. (Ϯ)-Naringenin was from Aldrich Chemical. The configurations of eriodictyol, homoeriodictyol and hesperetin are not determined. UV irradiation, CuCl 2 and jasmonic acid (JA) treatment The second leaves of 5-week-old plants were used for all treatments. UV irradiation was applied as described previously. 2) The rice leaves on which brown spots appeared were harvested and stored at Ϫ80°C prior to use. The treatments with CuCl 2 , jasmonic acid (JA) and H 2 O (control) were carried out as follows. The rice leaves (0.5 g) were cut into 1-2 cm long sections. The sections were then floated in 100 ml of A putative O-methyltransferase cDNA was cloned from UV-irradiated rice leaves based on an amino acid sequence reported as that of naringenin 7-O-methyltransferase, which is involved in the biosynthesis of a rice phytoalexin, sakuranetin. However, the recombinant protein (approximately 41 kDa) expressed in Escherichia coli showed not naringenin 7-O-methyltransferase activity but caffeic acid 3-O-methyltransferase activity. Semiquantitative RT-PCR revealed that the mRNA was expressed in all tissues tested and was not affected by CuCl 2 , jasmonic acid or UV treatment. The enzyme also methylated flavonoids which have two adjacent hydroxyl groups in the B ring. Caffeic acid 3-O-methyltransferase from rice leaves 53 Fig. 4. COMT activities of proteins (A) from aerial parts and roots at different growth stages, (B) from different leaf positions of 5week-old plants, and (C) from leaves treated with CuCl 2 , UV and JA. L1 to L4, first (youngest) to fourth (oldest) leaf of 5-week-old plants.