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-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed 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 as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.
Chiron
H
H
GSF-National Research Center for Environment and Health, Institute of Biochemical Plant Pathology, Neuherberg, Germany.
Drouet
A
A
Claudot
A C
AC
Eckerskorn
C
C
Trost
M
M
Heller
W
W
Ernst
D
D
Sandermann
H
H
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D032462:Cycadopsida / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010126:Ozone / Q000494:pharmacology; D041605:Pinus sylvestris; D010940:Plant Proteins; D010944:Plants / Q000187:drug effects / Q000378:metabolism; D011497:Protein O-Methyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D013267:Stilbenes / Q000378:metabolism
D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D012333:RNA, Messenger; D013267:Stilbenes; D010126:Ozone; D008780:Methyltransferases; D011497:Protein O-Methyltransferase; C418422:pinosylvin-O-methyltransferase