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

Abstract

A cell suspension of Vitis vinifera cv Gamay Fréaux 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), of cells grown in APM was observed. 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 has been purified approx. 35-fold with 3.4% enzyme activity recovery using anion exchange, chromatofocusing and gel filtration liquid chromatography. Mg 2+ 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 respect to the substrates Cy3G and S-adenosyl- l-methionine with K m values of 199 and 18 μM respectively. S-adenosyl- l-homocysteine showed a non-competitive inhibition with an approx. K i value of 50 μM. Relative to Cy3G, the activity of CGMT was 13% with cyanidin. No activity was observed with delphinidin or cyanidin 3- p-coumaroylglucoside.