Structure and expression of the gene family encoding putrescine N-methyltransferase in Nicotiana tabacum: new clues to the evolutionary origin of cultivated tobacco

The structure and nuclear genomic organization of the gene family encoding putrescine N-methyltransferase (PMT), the key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine and related alkaloids, was examined in Nicotiana tabacum. Five genes encoding PMT are present in the N. tabacum genome and all are expressed. The complete coding region and imm ediate 5'- and 3'- flanking regions were characterized for four members of the gene family and the Exon 1 region of the fifth member of the family was determined. Comparison of the nucleotide and deduced amino acid sequences of the N. tabacum PMT genes with those of presumed progenitor species, N. s ylvestris, N. tomentosiformis and N. otophora, revealed that three members of the N. tabacum PMT gene family were most similar to the three genes pres ent in N. sylvestris, whereas the two remaining PMT genes were similar to P MT genes present in N. tomentosiformis and N. otophora genomes, respectivel y. These data are consistent with an evolutionary origin of N. tabacum resu lting from a cross involving N. sylvestris and an introgressed hybrid betwe en N. tomentosiformis and N. otophora. The five PMT genes present in N. tab acum are expressed in the roots of wild-type plants, but not in other organ s. The steady-state level of all five PMT transcripts is transiently increa sed in roots following topping (removal of the floral meristem), although t he maximum level of induction for the individual transcripts varies conside rably. In contrast to wild-type plants, no increase in PMT transcript level s was observed in a low-alkaloid (nic1nic2) mutant of Burley 21. These data support a role for nic1 and nic2 in the global regulation of alkaloid form ation in tobacco and provide for the first time molecular confirmation of t he presumed origin of cultivated tobacco.