The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1,200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%.Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, and affinity chromatography on UDP-glucuronic acid agarose. The purified enzyme had a pH optimum of 7.5, an isoelectric point (pI) of 5.0, and a molecular mass of 49 kDa. The enzyme did not require metal cofactors for activity. Its activity was inhibited by Zn 2+ , Co 2+ and Cu 2+ ions, as well as by SH-blocking reagents. The Km values for UDP-glucose, scopoletin and esculetin were 43 µM, 150 µM and 25 µM, respectively. A study of the initial rate of the reaction suggested that the reaction proceeded via a sequential mechanism. The purified enzyme preferred hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequence of CGTase revealed strong homology to the amino acid sequences of other glucosyltransferases in plants.

The plasmid DNA containing a NPTII as a selectable marker gene and a tobacco PAL cDNA combined with the CaMV 35S promoter and NOS terminator was transformed into tobacco cells by a particle bombardment device based on flowing helium. The most efficient transformation was achieved when the amount of DNAcoated tungsten particle was 0.2mg per projectile, the amount of plasmid DNA was 41g/mg of tungsten particles, the helium pressure accelerated was 3 kg/cm2, and the distance between the sample and the projectile was 15 cm. Genetic analysis of kanamycin-resistant transformants obtained showed that the PAL cDNA construct integrated into the genome of tobacco cells. PAL activity of the transformant increased almost 4-fold and scopoletin content increased more than 2-fold as compared to nontransformed cells.