Molecular cloning and characterization of coumarin glucosyltransferase in hairy roots of Pharbitis nil (Ipomoea nil)

Abstract

The hairy roots induced from Pharbitis nil (Ipomoea nil) by infection of the plant pathogen Agrobacterium rhizogenes produced and released the phytoalexin of umbelliferone into the culture medium by treatment with copper sulfate. However, this umbelliferone was immediately recovered in the hairy roots as its glucoside, skimmin, after a few hours. To analyze the details of these phenomena, we searched for glucosyltransferase (GTase) genes related to skimmin production and isolated two full-length cDNAs, named PNgt1 and PNgt2. They contain the plant secondary product glucosyltransferase (PSPG) box that is a common signature of plant GTases. Phylogenetic analyses of PNgt1 and PNgt2 indicated that they encode a broad substrate-specific GTase and an unclassified GTase, respectively. An enzyme assay of the recombinant PNGT1 protein showed a high glucosylation rate against coumarin and benzaldehyde derivatives, especially umbelliferone. The expression level of PNgt1 after treatment with umbelliferone showed a correlation with skimmin production. Moreover, we found the expression levels of PNgt1 were altered by the plant hormones salicylic acid and methyl jasmonate. These results suggest the importance of plant GTases in phytoalexin production and plant defense mechanisms.