Appearance and Accumulation of C 4 Carbon Pathway Enzymes in Developing Maize Leaves and Differentiating Maize A 188

Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C4 photosynthesis, for cell morphology, and for '4C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5bisphosphate carboxylase (RuBPC, EC 4.1.139) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later. Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C3 leaves and from etiolated C4 leaf tissues, but differing from the corresponding enzymes in the mature leaf. Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology. Analysis of the labeled products of 4C02 fixation by nonchlorophylIous calli indicated ,-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in B-carboxylation products was even greater. Analysis of enzyme levels in young leaf sections by protein blot and of '4C-labeling patterms in the present study are in general agreement with enzyme activity determinations of previous studies, providing additional information about PPDK levels, and supporting the model proposed for developing young leaves. These results suggest that maize leaves begin to express C4 enzymes during ontogeny through several stages from greening and cell differentiation as seen in the callus and then shoot formation, and finally acquire capacity for full C4 photosynthesis during leaf development concomitant with the development of Kranz anatomy and accumulation of large amounts of enzymes involved in carbon metabolism.