THE EFFECTS OF HEAVY-METALS AND ROOT IMMERSION ON ISOFLAVONOID METABOLISM IN ALFALFA (MEDICAGO-SATIVA L.)

Modest increases in the concentration of medicarpin, C-fold in leaves and 4-fold in roots, were observed in alfalfa (Medicago sativa L.) see dlings treated with 1 mM metal salts for 72 h. However, medicarpin-3-O -glucoside-6''-O-malonate (MCM) and formononetin-7-O-glucoside-6''-O-m alonate (FCM) levels were up to 50-fold lower in metal-treated compare d to control roots. Approximately 1O% of the ''missing'' conjugates co uld be accounted for in the root treatment solution, where FCM and MCM transiently accumulated prior to their hydrolysis. Time-course studie s revealed that total isoflavonoid content (roots plus solution) incre ased slightly after CUCl2, treatment, whereas the levels of FCM and MC M increased rapidly in alfalfa roots immersed in water. This increase was reduced by aeration. The phenylalanine ammonia-lyase inhibitor L-a lpha-aminooxy-beta-phenylpropionic acid was used to show that immersio n of the roots reduced conjugate rates of degradation, which explains their accumulation. In contrast, conjugate rates of degradation were e levated in CuCl2-treated roots, with 50% of the increase being due to hydrolysis. Up to 90% of formononetin and medicarpin produced in respo nse to CuCl2 treatment arose via conjugate hydrolysis. Our results dem onstrate that both immersion/anaerobiosis and abiotic elicitation modi fy isoflavonoid metabolism in alfalfa, and that metal-stimulated accum ulation of phytoalexins may arise through the release from preformed s tores rather than de novo synthesis.