A role for phosphorylation in the down-regulation of phenylalanine ammonia-lyase in suspension-cultured cells of french bean

Abstract

A role for the phosphorylation of phenylalanine ammonia-lyase (PAL) which catalyses a key metabolic step in the biosynthesis of plant phenolics has been identified. Using immunoprecipitation of proteins radiolabelled with [ 32P]orthophosphate in cells in vivo, the M r 70 000 subunit degradation product was found to be phosphorylated. This phosphorylation was amplified by treatment of the cells with high concentrations of forskolin, but not elicitor alone, relative to the level of [ 35S]methionine labelled M r 77 000 and 70 000 subunit forms newly synthesised and turned-over as a consequence of elicitor action. Forskolin treatments bring about a correspondingly decreased specific activity of the extracted enzyme. Although a high level of phosphorylation was observed for a large number of species of polypeptides in cell-free extracts in vitro, including one of M r 69 000, this was not immunoprecipitated. The results are consistent with phosphorylation of the M r 70 000 PAL degradation product being specific and completed in vivo. Since forskolin brings about the down-regulation of elicitor-induced PAL, it is probable that the transient appearance of the enzyme activity and its turnover is in part regulated by phosphorylation, which is possibly cAMP dependent, in addition to other inactivating processes.