Evidence for localization of N-methyltransferase (MMT) of caffeine biosynthetic pathway in vacuolar surface of Coffea canephora endosperm elucidated through localization of GUS reporter gene driven by NMT promoter.

Abstract

Three N-methyltransferases (NMTs) are involved in the formation of caffeine from xanthosine. We have earlier cloned the promoter for one of the NMT genes. This study is focused on he localization of NMT in coffee endosperm cells. Constructs bearing the NMT promoter with and without the first exon were made in pCAMBIA 1381. These constructs and pCAMBIA 1301 bearing the intron uidA gene driven by CaMV 35S promoter were electroporated into coffee endosperm and the activity of b-glucuronidase (GUS) localized. In tissues transformed with the construct containing promoter and first exon, enzymatic activity was localized on the outer surface of the vacuole. Interestingly, antibodies to the coffee NMT also specifically localized to the same region. In tissues bearing either the NMT–GUS construct without the first exon, or pCAMBIA 1301 with intron GUS, GUS activity was spread throughout the cytoplasm. The results suggest that NMT is targetted to external surface of the vacuole. It would be interesting to discover the localization of other enzymes involved in caffeine and chlorogenic acid biosynthesis for understanding their relationship, if any.