Characterization and activities of S-adenosyl-L-methionine:cyanidin 3-glucoside 3'-O-methyltransferase in relation to anthocyanin accumulation in Vitis vinifera cell suspension cultures.

摘要

A cell suspension of Vitis vinifera cv. Gamay Freaux was grown in a maintenance and an anthocyanin-promoting medium (APM). Time-course changes in anthocyanin accumulation and S-adenosyl-L-methionine:cyanidin 3-glucoside 3'-O-methyltransferase (CGMT)activity were examined throughout the growth cycle. An increase in anthocyanin accumulation mainly due to peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside, i.e. 3'-methylated derivatives of cyanidin 3-glucoside (Cy3G), was observed in cells grown in APM. The higher activity of CGMT in cells grown in the APM might be responsible for the enhanced formation of 3'-methylated anthocyanins. CGMT isolated from cell suspension cultures was purified ?5-fold with 3.4% enzyme activity recovery using anion exchange, chromatofocusing and gel filtration liquid chromatography. Mg2+ ions strongly enhanced the reaction rate at a concentration of 5 mM. Optimal pH of CGMT ranged from 7.75 to 9.75. Michaelis-Menten kinetics were observed at pH 8.75 with respectto the substrates Cy3G and S-adenosyl-L-methionine with Km values of 199 and 18 鍹, respectively. S-adenosyl-L-homocysteine showed a non-competitive inhibition with an approximate Ki value of 50 鍹. Relative to Cy3G, the activity of CGMT was 13% with cyanidin. No activity was observed with delphinidin or cyanidin 3-p-coumaroylglucoside.