Enzymological aspects of phytoalexin accumulation in Phaseolus vulgaris

Abstract

In wounded cotyledons of Phaseolus vulgaris the accumulation of the 5-hydroxy isoflavonoids 21-hydroxygenistein and kievitone preceded the major increases in the levels of the 5-deoxy compounds coumestrol and phaseollin. Increased phytoalexin (kievitone and phaseollin) levels werepreceded by transient increases in the extractable activities of L-phenyl-alanine ammonia-lyase (EC 4.3.1.5), chalcone synthase and chalcone isomerase (EC 5.5.1.6). 13C-NMR analysis of kievitone and phaseollin produced after feeding [l,2-13C2] acetate to wounded cotyledons demonstrated the incorporation of intact acetate units into the aromatic A-rings. Phaseollin showed a specific folding of the polyketide chain whereas kievitone exhibited a randomisation of the label in keeping with the intermediacy of a 2',4',6'-trihydroxychalcone during its formation. The biosynthetic routes to these 5-hydroxy and 5-deoxy isoflavonoids thus diverge prior to chalcone formation. The results suggest the involvement of a 6'-deoxychalcone synthase in the biosynthesis of 5-deoxy isoflavonoid compounds.However, efforts to locate the activity of such an enzyme were unsuccessful 6'-Hydroxy chalcone synthase and chalcone isomerase were purified from cell suspension cultures of P. vulgaris. Chalcone synthase showedmany similarities to the enzyme purified from Petroselinum hortense butwas much less stable, had significantly different aoparent K values formthe two substrates and produced different in vitro release products following purification. With malonyl CoA as variable substrate, double reciprocal plots for naringenin formation were sigmoidal suggesting positive cooperativity which may favour the formation of chalcones in vivo. Only a single form of chalcone isomerase was found; however, the enzyme catalysed the isomerisation of both 6-hydroxy and 6'-deoxy chalcones to the corresponding flavanones. Kievitone strongly inhibited the activity of both enzymes whilst coumestrol was a potent inhibitor of chalcone isomerase A range of other 5-hydroxy and 5-deoxy isoflavonoid compounds tested had little or no effect on the activity of the two enzymes. The possible relevance of the inhibitory effects of such compounds is discussed in relation to the role of both enzymes in the induced accumulation of isoflavonoid phytoalexins in P. vulgaris.