Characterization of a flavonoid 3-O-glucosyltransferase and its activity during cotyledon growth in buckwheat (Fagopyrum esculentum)

The flavonoid 3-O-glucosyltransferase (3GT) was isolated and purified from buckwheat (Fagopyrum esculentum) cotyledons. It was purified about 171-fold with final specific activities of 88.8 pmol of isoquercitrin (quercetin-3-glucoside) generation from quercetin per min per mg protein. The enzyme was determined to be a monomer by gel filtration (56.0 kDa) and electrophoresis (58.6 kDa). The Km values were calculated to be 27 μM for quercetin (as a sugar acceptor) and 1.04 mM for UDP-Glc (as a sugar donor). In addition, changes in both flavonoid concentration and 3GT activity during cotyledon growth were investigated to ascertain the physiological roles of 3GT in cotyledons. The 3GT activity increased linearly together with rutin accumulation and then rapidly decreased when the rutin concentration reached a maximum value. On the other hand, quercetin (a sugar acceptor of 3GT) and isoquercitrin (a product of 3GT) concentrations were 2 or 3 orders of magnitude less than the rutin concentration, which indicates that the isoquercitrin generated by 3GT is an intermediate for rutin biosynthesis in the plant tissues. The 3GT activity and rutin concentration were high even when buckwheat cotyledons were grown under darkness. This result suggests that rutin in the buckwheat cotyledon has roles other than as a UV screen (e.g., enhancement of the defense system against stressful conditions).