Isolation of the cDNAs encoding (+)6a-hydroxymaackiain 3-O-methyltransferase, the terminal step for the synthesis of the phytoalexin pisatin in Pisum sativum.
Pisatin is the major phytoalexin produced by pea upon microbial infection. The enzyme that catalyzes the terminal step in the pisatin biosynthetic pathway is (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM). We report here the isolation and characterization of two HMM cDNA clones (pHMM1 and pHMM2) made from RNA obtained from Nectria haematococca-infected pea tissue. The two clones were confirmed to encode HMM activity by heterologous expression in Escherichia coli. The substrate specificity of the methyltransferases in E. coli was similar to the activity detected in CuCl2-treated pea tissue. Nucleotide sequence analysis of Hmm1 and Hmm2 revealed an open reading frame of 1080 bp and 360 amino acid residues which would encode 40.36 kda and 40.41 kDa polypeptides, respectively. The deduced amino acid sequence of HMM1 has 95.8% identity to HMM2, 40.6% identity to Zrp4, a putative O-methyltransferase (OMT) in maize root, and 39.1% to pBH72-F1, a putative OMT induced in barley by fungal pathogens or UV light. Comparison of the deduced amino acid sequences of the cDNA clones to OMTs from other higher plants identified the binding sites of S-adenosylmethionine (AdoMet). Southern blot analysis showed two closely linked genes with strong homology to Hmm in the pea genome.
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Department of Plant Pathology, University of Arizona, Tucson 85721, USA.
Preisig
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VanEtten
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001578:Benzopyrans / Q000378:metabolism*; D003001:Cloning, Molecular; D003300:Copper / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D004790:Enzyme Induction; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics*; D010936:Plant Extracts / Q000096:biosynthesis; D036343:Pterocarpans; D012333:RNA, Messenger / Q000032:analysis; D018749:RNA, Plant / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes; D013379:Substrate Specificity; D013729:Terpenes; D000094162:Phytoalexins
D001578:Benzopyrans; D018076:DNA, Complementary; D018744:DNA, Plant; D010936:Plant Extracts; D036343:Pterocarpans; D012333:RNA, Messenger; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; D013729:Terpenes; D003300:Copper; C108854:(+)6a-hydroxymaackiain 3-O-methyltransferase; D008780:Methyltransferases; C029892:cupric chloride; C003161:pisatin; D000094162:Phytoalexins