Mark P. Robbins

Comparative biochemistry of chalcone isomerases

Abstract Fungal elicitor-inducible chalcone isomerase activity was present in suspension cultured cells of Phaseolus vulgaris , Glycine max and Medicago sativa , and a non-inducible activity was found in the purple sections of callus cultures of Petunia hybrida strain AK-5000. The enzyme from the legume sources catalysed the isomerisation of both 2′,4,4′- trihydroxy-and 2′,4,4′,6′-tetrahydroxychalcones whereas the Petunia enzyme was specific for the tetrahydroxychalcone. Apparent differences in the properties of the enzyme from different sources were observed in relation to kinetic parameters, M r values for holoenzyme and subunits newly synthesised in vitro from mRNA, antigenic cross-reactivity and cDNA cross-hybridisation. Our data confirm and extend previous observations suggesting differences and anomalies between the properties of chalcone isomerases from different sources.

Elicitor-mediated induction of chalcone isomerase in Phaseolus vulgaris cell suspension cultures

Approximately fourfold increases in the extractable activity of the enzyme chalcone isomerase (CHI, EC 5.5.1.6) were observed within 24 h of treatment of cell suspension cultures of Phaseolus vulgaris with a crude elicitor preparation heatreleased from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The induction of CHI activity was highly dependent upon elicitor concentration, with maximum induction occurring in two discrete concentration ranges. A basal half-life for CHI>32 h in control cultures was determined by labelling with 2H from 2H2O followed by analysis of the equilibrium distribution of enzyme activity in CsCl density gradients. Comparative density labelling indicated that at both the lower and higher effective elicitor concentrations, the induced appearance of CHI activity was the result of an apparent initial activation of pre-existing enzyme followed by an increase in the rate of de-novo synthesis of the enzyme as compared with non-elicited controls. The increased appearance of the enzyme over the first 8 h in elicitor-treated cultures was inhibited by cycloheximide, cordycepin and actinomycin D. The results are discussed in relation to the mechanisms of co-ordinate enzyme induction operating in French-bean cell cultures exposed to fungal elicitors.

Trans-cinnamic acid as a modulator of chalcone isomerase in bean cell suspension cultures

Abstract Trans-cinnamic acid, but not the biosynthetically related 4-coumaric and ferulic acids, induced an increase in the extractable activity of chalcone isomerase (CHI, EC 5.5.1.6) in cell suspension cultures of Phaseolus vulgaris. The kinetics of isomerase induction in response to 10−3 M cinnamic acid were similar to those observed following exposure of cell cultures to a fungal elicitor preparation; however, extractable activity of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) was markedly decreased by cinnamic acid in contrast to its induction by elicitor. Treatment of elicited cell cultures with L-α-aminooxy-β-phenylpropionic acid (AOPP), a potent inhibitor of phenylanine ammonia-lyase, and therefore cinnamic acid production in vivo, resulted in superinduction of extractable lyase activity but reduced extractable isomerase activity. Aminooxyphenylprypionic acid does not inhibit CHI activity in vitro. The results suggest that cinnamic acid plays a role in the induction of CHI in vivo; these findings are discussed in the content of recent evidence for the induced synthesis of both active and inactive forms of the isomerase in elicitor-treated cell cultures.

Molecular Targets for Elicitor Modulation in Bean (Phaseolus Vulgaris) Cells

This chapter reviews recent work on the biochemistry of disease resistance expression in relation to the French bean (Phaseolus vulgaris)/Colletotrichum lindemuthianum interaction. The basic biological and genetical factors which make this system highly amenable to studies at the biochemical and molecular genetic levels have been reviewed previously [1], and the advantages and disadvantages of using elicitor-treated cell suspension cultures as a model system in initial biochemical studies have also been discussed [2].