Changes in the accumulation of flavonoid and isoflavonoid conjugates associated with plant age and nodulation in alfalfa (Medicago sativa)

Medicarpin 3-0-glucoside-6"-0-malonate (MGM), formononetin 7-0-glucoside-6"-0-malonate (FGM) and two glycosides of coumestrol (CG), one of which was characterised as a malonylated glucoside, have been identified as major isotlavonoid metabolites in the roots of healthy alfalfa (Medicago sativa) plants. Foliage contained a conjugate of the flavonoid apigenin, very low levels of medicarpin and MGM, and depending on the cultivar studied, formononetin and FGM. In the foliage the isoflavonoids were restricted to the stems. Seeds contained conjugates of quercetin, luteolin and 7,4'dihydroxyflavone but no isoflavonoid conjugates. When alfalfa plants were grown under controlled conditions, isoflavonoid conjugates were first observed in the roots 24 h after emerging, and continued to accumulate as the plants matured over a 57 day period. Inoculating the plants with a commercial formulation of Rhizobium meliloti (Nodulaid) reduced FGM accumulation in the roots but did not affect MGM or CG content. Under field conditions plants accumulated higher concentrations of the isoflavonoid conjugates in both leaves and roots than plants grown under controlled conditions. Field-grown plants which were not inoculated with Nodulaid contained higher levels of both MGM and FGM in the roots than nodulated plants. The conjugation of isoflavonoids in altalfa cell cultures resembled that of the roots in that they accumulated FGM and MGM as major metabolites. However, cultures accumulated a higher proportion of their conjugated isoflavonoids as MGM and tormononetin glucoside than did the roots and did not synthesise coumestrol. Comparisons with clover species revealed that FGM and MGM were common metabolites in forage legumes but alfalfa was unusual in having negligible levels of isoflavonoid conjugates in the foliage. Our results clarify the nature and distribution of isoflavonoid conjugates in altalfa and demonstrate that their accumulation is regulaled in response to both plant age and nodulation