Molecular characterization and expression analysis of two distinct putrescine N-methyltransferases from roots of Anisodus acutangulus

Putrescine N-methyltransferase (PMT; EC. 2.5.1.53) catalyzes the S-adenosylmethionine-dependent N-methylation of putrescine to form N-methylputrescine, which was the first committed step in tropane alkaloid biosynthetic pathway. Two PMT cDNA clones [Anisodus acutangulus putrescine N-methyltransferase 1 (AaPMT1), GenBank Accession No. EU670745; AaPMT2, GenBank Accession No. EU670746] were obtained and characterized together from Anisodus acutangulus for the first time. The full-length cDNA of AaPMT1 was 1322 bp containing a 1014-bp open reading frame (ORF) encoding a polypeptide of 338 amino acids and AaPMT2 was 1219 bp containing a 1041-bp ORF encoding a polypeptide of 347 amino acids. Comparison of the deduced amino acid sequences of AaPMTs with those from tropane alkaloid-producing plants revealed that AaPMTs had high similarity with other plants PMT. Phylogenetic tree analysis displayed that AaPMT1 showed extensive homology with PMT from Anisodus tanguticus, and AaPMT2 had closer relationship with PMT2 from Atropa belladonna, which indicated PMTs belonged to PMT superfamily. Southern hybridization analysis of the genomic DNA revealed the occurrence of two PMT copies in A. acutangulus genome. Tissue expression pattern analysis revealed that AaPMT1 expressed strongly in roots, weakly in steams and leaves, besides, AaPMT2 presented a similar weaker trend. It indicated that AaPMTs were constitutive expression genes, which were the first reported tissue-independent PMT genes compared with other known PMT genes. AaPMT1 expression was upregulated by methyl jasmonate (MeJA) in all tissues, reaching the highest level after 24 h of the treatment. AaPMT2 also exhibited a very similar trend, whereas the expression was much weaker than that in AaPMT1. So, AaPMTs were considered to be MeJA elicitor-responsive genes and could be effectively elicited at least at the transcriptional level. The work would provide useful knowledge for tropane alkaloids biosynthesis and metabolic engineering to increase the production.