REGULATION OF ISOFLAVONOID METABOLISM IN ALFALFA

Isoflavonoids are believed to play important roles in plant-microbe interactions. 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-lyase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite sativan increase beginning at 4 and 8 hours, respectively, along with other isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of the general phenylpropanoid and flavonoid genes phenylalanine ammonia-lyase and chalcone synthase increases while mRNA levels for the phytoalexin-specific isoflavone reductase decrease. The total isoflavonoid content of colonized roots increases with time and is higher than that of uninoculated roots, but the accumulation of the antifungal medicarpin is somehow suppressed. An isoflavone reductase genomic clone has been isolated, promoter regions have been fused to the reporter gene P-glucuronidase, and the promoter-reporter fusions have been transformed into tobacco and alfalfa, Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-specific gene in whole plants at a more detailed level than other methods allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgenic alfalfa plants by Phoma causes an increase in P-glucuronidase staining, as does elicitation of transgenic alfalfa cell cultures, indicating that this promoter fusion is a good indicator of phytoalexin biosynthesis in alfalfa.