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.