CHANGES IN ACTIVITIES OF ENZYMES INVOLVED IN FLAVONOID METABOLISM DURING THE INITIATION AND SUPPRESSION OF ANTHOCYANIN SYNTHESIS IN CARROT SUSPENSION-CULTURES REGULATED BY 2,4-DICHLOROPHENOXYACETIC ACID

When anthocyanin synthesis was induced in cell suspension cultures of carrot (Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6-7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized. PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3-6 h of 2,4-D addition, CHS activity was completely repressed 24-36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.