Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin.
Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, approximately 20-50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.
Taguchi
G
G
Gene Research Center, Shinshu University, Ueda, Japan.
Yazawa
T
T
Hayashida
N
N
Okazaki
M
M
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D007210:Indoleacetic Acids / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D007210:Indoleacetic Acids; D011994:Recombinant Proteins; D005964:Glucosyltransferases; D020156:Salicylic Acid