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

Abstract

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, < 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.

Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavonol and 3-hydroxyl group of coumarin from tobacco cells

“…NtGT2 was obtained by the screening of the tobacco cDNA library [22] using a fragment of the Perilla glucosyltransferase gene (3R4, [12]) containing the PSPG box.…”

“…Affinity-purified enzymes were used to determine substrate specificity and enzymatic parameters. The enzyme reaction for the determination of the substrate specificity of rNTGT2 using UDP-[ 14 C] glucose was performed as described previously [22]. For the reaction with the anthocyanin substrate, the enzyme was extracted using 50 mM Tris-HCl (pH 8.0) containing 10 mM 2-mercaptoethanol, according to Yamazaki et al [12].…”

“…Moreover, several GTase genes have been reported, namely, genes of IEGT, TOGT, jasmonate-inducible GTase gene (JIGT) [19], and salicylic acid GTase gene [20]. In our previous studies, the glucosylation activity in cultured tobacco cells was investigated [21][22][23]. We isolated GTase genes (NtGT1a, NtGT1b and NtGT3) whose encoding enzymes have glucosylation activity against many kinds of phenolics, such as flavonoids, coumarins and naphthols [22,23].…”

“…In our previous studies, the glucosylation activity in cultured tobacco cells was investigated [21][22][23]. We isolated GTase genes (NtGT1a, NtGT1b and NtGT3) whose encoding enzymes have glucosylation activity against many kinds of phenolics, such as flavonoids, coumarins and naphthols [22,23]. These enzymes glucosylate mainly on 3-OH of flavonoids.…”

A single amino acid in the PSPG‐box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

“…In the present investigation we have used site-directed mutagenesis, and domain swapping experiments between CaUGT2 and NtGT1b, to try to identify specific amino acids within the PSPG-box of CaUGT2 that control its catalytic function and substrate recognition properties. NtGT1b is a tobacco group E UGT which displays very little ability to glucosylate curcumin [24]. These experiments revealed that a single relatively non-conserved amino acid residue plays an important role in defining the catalytic activity of CaUGT2.…”