The isoflavonoid conjugates medicarpin-3-O-glucoside-6''-O-malonate (MGM), 
afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6''-O-malonate (AGM) 
were isolated and characterized from cell suspension cultures of alfalfa 
(Medicago sativa L.), where they were the major constitutive secondary 
metabolites. They were also found in alfalfa roots but not in other parts of the 
plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured 
cells treated with elicitor from Colletotrichum lindemuthianum, and this was 
subsequently accompanied by an increase in the levels of MGM. In contrast, net 
accumulation of afrormosin conjugates was not affected by elicitor treatment. 
Labeling studies with [(14)C]phenylalanine indicated that afrormosin conjugates 
were the major de novo synthesized isoflavonoid products in unelicited cells. 
During elicitation, [(14)C]phenylalanine was incorporated predominantly into 
medicarpin, although a significant proportion of the newly synthesized 
medicarpin was also conjugated. Treatment of (14)C-labeled, elicited cells with 
l-alpha-aminooxy-beta-phenylpropionic acid, a potent inhibitor of PAL activity 
in vivo, resulted in the initial appearance of labeled medicarpin of very low 
specific activity, suggesting that the phytoalexin could be released from a 
preformed conjugate under these conditions. Our data draw attention to the 
involvement of isoflavone hydroxylases during the constitutive and 
elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa 
cell cultures.