Elicitor-induced formation of secondary products in plant cell cultures. Production of benzophenanthridine alkaloids in Eschscholtzia californica Cham. and hydroxycinnamic acid tyramides in Nicotiana glutinosa L

Abstract: Biotic and abiotic elicitors induce biosynthesis of secondary metabolites (phytoalexins) as a defense against pathogenic organisms in plants. The use of cell suspension cultures in combination with elicitors offer excellent experimental model systems for studies on various elicitor-induced responses of plant tissues. Various elicitors were added to cell suspension culture of Eschscholtzia californica and Nicotiana glutinosa to enhance secondary metabolite accumulation. Chitosan, a biotic elicitor, induced biosynthesis of hydroxycinnamoyltyramides in cell suspension cultures of N. glutinosa. These phenolic compounds were rapidly incorporated into insoluble cell wall material. Enzymes involved in the biosynthesis of the hydroxycinnamoyltyramides, i.e. phenylalanine ammonia lyase, 4-coumarate:CoA ligase, tyrosine decarboxylase (TDC) and hydroxycinnamoyl-CoA: tyramine hydroxycinnamoyl transferase, were all co-induced by elicitor treatment. The concentration of chitosan, resulting in elicitation but not in an irreversible permeabilization of the cells, was determined by a newly developed method based on measurements of conductivity of the cultivation medium. A carbohydrate fraction prepared from yeast extract, alginate or sodium orthovanadate induced the formation of benzophenthridine alkaloids (sanguinarine, chelerythrine and macarpine) in E. californica cell suspension cultures. The accumulation of benzophenanthridine alkaloids was accompanied by the stimulation of the activity of TDC, which is a key enzyme of isoquinoline alkaloid biosynthesis. A large portion of alkaloids produced (around 40 %) was excreted from elicitor-treated cells of E. califonica into the cultivation medium. An exception to this was cells treated with orthovanadate which did not excrete alkaloids to the same extent (less than 10 %). We ascribe this inhibition of alkaloid transport to be due to depolarization of the plasma membrane as a result of inhibition of plasma membrane H+ ATPase by orthovanadate. An active transport system, possibly specific for benzophenanthridine alkaloids, may be present in plasma membranes of E. californica. Cells of E. californica, immobilization by entrapment in Ca2+-alginate gel, showed an increased and extended (up to two weeks) biosynthesis of benzophenanthridine alkaloids. Prolonged induction of TDC was also observed in entrapped cells.