IN-VIVO REGULATION OF WHEAT-LEAF PHOSPHOENOLPYRUVATE CARBOXYLASE BY REVERSIBLE PHOSPHORYLATION

Regulation of C-3 phosphoenolpyruvate carboxylase (PEPC) and its prote in-serine/threonine kinase (PEPC-PK) was studied in wheat (Triticum ae stivum) leaves that were excised from low-N-grown seedlings and subseq uently illuminated and/or supplied with 40 mM KNO3. The apparent phosp horylation status of PEPC was assessed by its sensitivity to L-malate inhibition at suboptimal assay conditions, and the activity state of P EPC-PK was determined by the in vitro P-32 labeling of purified maize dephospho-PEPC by [gamma-P-32]ATP/Mg. Illumination (+/-NO3-) for 1 h l ed to about a 4.5-fold increase in the 50% inhibition constant for L-m alate, which was reversed by placing the illuminated detached leaves i n darkness (minus NO3-). A 1-h exposure of excised leaves to light, KN O3, or both resulted in relative PEPC-PK activities of 205, 119, and 6 59%, respectively, of the dark/0 mM KNO3 control tissue. In contrast, almost no activity was observed when a recombinant sorghum phosphoryla tion-site mutant (S8D) form of PEPC was used as protein substrate in P EPC-PK assays of the light plus KNO3 leaf extracts. In vivo labeling o f wheat-leaf PEPC by feeding P-32-labeled orthophosphate showed that P EPC from light plus KNO3 tissue was substantially more phosphorylated than the enzyme in the dark minus-nitrate immunoprecipitates. Immunobl ot analysis indicated that no changes in relative PEPC-protein amount occurred within 1 h for any of the treatments. Thus, C-3 PEPC activity in these detached wheat leaves appears to be regulated by phosphoryla tion of a serine residue near the protein's N terminus by a Ca2+-indep endent protein kinase in response to a complex interaction in vivo bet ween light and N.