ANALYSIS OF STRESS RESPONCES IN SOME MEDICINAL PLANT CELL CULTURES

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1. Among the enzymes involved in isoflavonoid biosynthesis in Pueraria lobata, complementary as well as genomic DNAs encoding chalcone synthase (CHS) and reductase (CHR) co-acting with CHS have been cloned and sequenced. In the promoter regions of both genomic clones, elicitor responding elements reported in Phaseolus vulgaris were identified. Plasmid containing this 500 bp region connected to beta-glucuronidase (GUS) gene as a reporter was constructed. Hairy roots of tobacco plant transformed with this plasmid expressed GUS protein responding to the elicitation stress. This type of promoter is useful for the time and tissue specific expressions of foreign genes in plants.2. Elicitor inducible isoflavonoid metabolism in Pueraria lobata consisted of two distinct phases. In the first phase (0-4 hr after elicitation), constitutive isoflavone malonylglucosides (IMG) were bound to cell walls by lignification. Reaccumulation of IMG in the second phase (6-24 hr after elicitation) was brought about by de novo biosynthesis. The rapid lignification of isoflavonoid found in the first phase as a consequence of disruption of subcellular compartmentation of IMG and hydrolytic enzymes. This type of responce has never been reported in Legume plants, however, it can possibly function as a rapid defense mechanism prior to others which require de novo biosynthesis of enzymes.3. Elicitor active components of yeast extract which induce p-coumarolyamino acids in Ephedra distachya cultures were separated, purified and identified as mannanglycoprotein. This is the first demonstration of active elicitor having mannan structure as the main sugar component D-Alanine was also found to be an elicitor active component of yeast extract. A new D-aminoacid specific p-coumaroyltransferase was detected in E.distachya cultures which serves as a key enzyme in the biosynthesis of p-coumarolyamino acids.