Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.)

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methioni ne (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in nee dles of Scots pine seedlings upon ozone treatment, as well as in cell suspe nsion cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT p rotein was purified and partially characterised. A cDNA encoding PMT was is olated from an ozone-induced Scots pine cDNA library. Southern blot analysi s of the genomic DNA suggested the presence of a gene family. The deduced p rotein sequence showed the typical highly conserved regions of O-methyltran sferases (OMTs), and average identities of 20-56% to known OMTs. PMT expres sed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such a s astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate spec ificity. Treatment of 7-year old Scots pine trees with ozone markedly incre ased the PMT mRNA level. Our results show that PMT represents a new SAM-dep endent OMT for the methylation of stress-induced pinosylvin in Scots pine n eedles.