INDUCTION OF ROSMARINIC ACID ACCUMULATION IN CELL-SUSPENSION CULTURES OF ORTHOSIPHON-ARISTATUS AFTER TREATMENT WITH YEAST EXTRACT

Cell suspension cultures of Orthosiphon aristatus were shown to accumulate rosmarinic acid (RA) at concentrations of 1.0-2.0-mu-mol g-1 fr. wt. Addition of yeast extract (4-6 g 1(-1)) to the liquid growth media resulted in a large increase of RA accumulation in treated cells independent of the growth stage. The highest concentration of RA observed in treated cells (ca 10-mu-mol g-1 fr.wt) was usually reached 72-96 hr after addition of yeast extract. When cells present in the stationary phase were treated with yeast extract a second phenolic was shown to accumulate which presumably originated by oxidative decarboxylation of RA. The induction of RA accumulation by yeast extract was due to de novo synthesis as shown by feeding experiments with C-14-tracers and by analysis of the activities of phenylalanine ammonia lyase (PAL) and tyrosine aminotransferase (TAT) which are the key enzymes of RA biosynthesis. Following addition of yeast extract both enzyme activities showed a strong transient increase which preceded the peak of RA accumulation. Fractionation of yeast extract by acetone precipitation, ion exchange and gel permeation chromatography yielded two active fractions (elicitor A and B) capable of inducing RA accumulation. Both elicitors were shown to be carbohydrate polymers (M(r) of elicitor A ca 22 000, of elicitor B ca 7 000) containing mainly mannose, glucose and to a lesser degree galactose. The elicitors are, thus, not identical to a glucan elicitor previously reported from yeast extract.