Induction of 6a-hydroxymaackiain 3-O-methyltransferase and phenylalanine ammonia-lyase mRNA translational activities during the biosynthesis of pisatin

Abstract

The isoflavonoid phytoalexin pisatin is synthesized by pea (Pisum sativum L.) in response to microbial infection and certain other forms of stress. The terminal step in the biosynthesis of pisatin is catalyzation by the (+)-6a-hydroxymaackiain 3-O-methyltransferase (HMKMT). This enzyme, identified as a protein of Mr 43,000 by photoaffinity labeling (Preisig et al. (1989) Plant Physiol. 91, 559-566), was purified 280-fold from CuCl2-stressed pea seedlings and subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised in rabbit against this protein cut from the polyacrylamide gels. The antiserum against the purified enzyme inhibited HMKMT enzyme activity and showed high specificity for the Mr 43,000 protein on Western blots and in immunoprecipitations. This enzyme, present almost exclusively in the 95,000g supernatant after differential centrifugation, was induced in pea from a low constitutive level by treatment with CuCl2, suggesting that the HMKMT is newly synthesized in response to stress. HMKMT mRNA translational activity increased in peas with time after treatment with CuCl2. Peak translational activity occurred about 12 h after treatment, preceding peak enzyme activity by a few hours. Phenylalanine ammonia-lyase (PAL) mRNA abundance increased coordinately with that of HMKMT. The increase in PAL mRNA translational activity in response to stress is known to reflect transcriptional activation of PAL genes. Thus, the induction by stress of enzyme activity both at an early step and at the terminal step in the phenylpropanoid/isoflavonoid biosynthetic pathway appears to be at the transcriptional level.