Use of Immunotitration to Demonstrate Phytochrome-Mediated Synthesis de novo of Chalcone Synthase and Phenylalanine Ammonia Lyase in Mustard Seedling Cotyledons

The suggestion [H. J. Newbury and H. Smith, Eur. J. Biochem . 117, 575 — 580 (1981)] of a posttranslational activation of accumulated enzyme precursor by phytochrome was tested in the case of phytochrome-mediated induction of chalcone synthase (EC 2.3.1.74) in mustard seedlings. The approach of immunotitration of enzyme activity was adopted since it allows to relate the amount of antigenic material present in a particular extract to the extract’s enzymatic activity. The data obtained show that under all experimental conditions enzyme activity of an extract is propor­ tional to the amount of immunoresponsive material. No indication was found either o f lightdependent modification of the enzyme or of enzymatically inactive immunoresponsive material in extracts prepared from dark-grown mustard seedlings or from light-treated seedlings placed again in darkness. In model experiments with purified ribulose-bisphosphate carboxylase (EC 4 .1.1.39) heat-inactivated enzyme was detected by the antibodies. Experiments on induction of phenyl­ alanine ammonia lyase (EC 4.3.1.5) by phytochrome in mustard cotyledons support the result — synthesis de novo o f enzyme protein — obtained previously [W. Tong and P. Schöpfer, Proc. Nat. Acad. Sei. U .S .A . 43, 4017—4021 (1976)] with the technique of density labelling. It is concluded that the phytochrome-mediated induction of enzymes involved in anthocyanin formation in mus­ tard cotyledons is due to synthesis de novo of enzyme protein.