The effect of phenylalanine on biosynthesis of protoberberine alkaloids in the cell culture of low meadow-rue

Abstract

The addition of 7 mM phenylalanine to the nutrient medium for low meadow-rue (Thalictrum minus L.) cell culturing on the 7th or 8th day doubled berberine secretion into medium. Simultaneously, the content of phenolic compounds increased in the cells and medium. Investigation of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activities showed that exogenous Phe activated PAL by 35% and inactivated TAL by 20%. When the crude extract was separated on DEAE-Sephacel column, two proteins were isolated. One of them displayed both PAL and TAL activities, whereas another protein displayed only PAL activity. This activity disappeared after cell culturing longer than 20 days and also under the effect of Phe at a concentration reducing alkaloid biosynthesis. Phe addition to medium also increased the content of protein in both the cells and culture medium. The proportion of low-molecular proteins in the medium increased. Testing antimicrobial activity of the medium showed that it was determined by berberine and to a lesser degree by palmatine. Protein fraction also demonstrated antimicrobial activity. An improved antimicrobial activity after Phe adding to medium resulted from alkaloid and protein accumulation. The conclusion was made that one of the mechanisms of Phe action was the control of alkaloid biosynthesis with the involvement of the enzyme system of the early steps of the phenylpropanoid pathway, which, in its turn, is one of the stages in stress-induced plant response to pathogen action.