Adaptation of Leguminous Plants to Environmental Microbes through Expression of Genes Encoding Enzymes of Flavonoid Biosynthetic Pathway

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Legume flavonoids possessing characteristic structures (5-deoxy, isoflavonoid, etc) function as defense compounds against phytopathogens and chemical signals during the symbiotic nitrogen fixation with Rhizobia. In this study, the enzymes responsible for the construction of theses structures were characterized through molecular cloning of cDNAs, and the relationship between the gene expression and the response of the plant against the environmental microorganisms was studied.From a leguminous plant licorics (Glycyrrhiza echinata), cDNAs encoding O-methyltransferases and cytochrome P450s of flavonoid pathway were cloned. By expressing in heterologous eucaryotic cells, the functions of P450s were determined : i.e., flavanone 2-hydroxylase involved in the construction of the skeleton of flavones acting as symbiotic signals ; 2-hydroxyisoflavanone synthase and isoflavone 2'-hydroxylase both of which are essential in phytoalexin biosynthesis. Also, a cDNA encoding an OMT similar to that producing the alfalfa symbiotic signal was cloned from licorice, and was shown to be involved in retrochalcone biosynthesis. The instability of the chalcone product in licorice cells was demonstrated. Furthermore, chalcone isomerase isozymes were characterized, and a new scheme of the biosynthesis of formononetin, a precursor of phytoalexins, was proposed.A phytoalexin of the model legume Lotus japanicus was identified, and cDNAs of some of the enzymes in its biosynthetic pathway were cloned. A new culture line of licorice was found to produce isoflavonoid phytoalexins. In these plant materials, coordinated expression of flavonoid biosynthetic genes were demonstrated, confirming the essential defensive roles of flavonoids in legumes. Finally, transformation methods of L. japonicus and other non-legume plants were established, and experimental systems for the flavonoid response to environmental organisms using transgenes are being constructed.