REGULATION OF ISOFLAVONOID METABOLISM IN ALFALFA

Isoflavonoids are believed to play important roles in plant-microbe in teractions. During infection of alfalfa (Medicago sativa) leaves with the fungal pathogen Phoma medicaginis, rapid increases in mRNA levels and enzyme activities of isoflavone reductase, phenylalanine ammonia-l yase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite s ativan increase beginning at 4 and 8 hours, respectively, along with o ther isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of t he general phenylpropanoid and flavonoid genes phenylalanine ammonia-l yase and chalcone synthase increases while mRNA levels for the phytoal exin-specific isoflavone reductase decrease. The total isoflavonoid co ntent of colonized roots increases with time and is higher than that o f uninoculated roots, but the accumulation of the antifungal medicarpi n is somehow suppressed. An isoflavone reductase genomic clone has bee n isolated, promoter regions have been fused to the reporter gene P-gl ucuronidase, and the promoter-reporter fusions have been transformed i nto tobacco and alfalfa, Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-sp ecific gene in whole plants at a more detailed level than other method s allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgeni c alfalfa plants by Phoma causes an increase in P-glucuronidase staini ng, as does elicitation of transgenic alfalfa cell cultures, indicatin g that this promoter fusion is a good indicator of phytoalexin biosynt hesis in alfalfa.