Date of this Version
Plant Physiol. (1995) 107: 775-782
Regulation of C3 phosphoenolpyruvate carboxylase (PEPC) and its protein-serine/threonine kinase (PEPC-PK) was studied in wheat (Trificum aesfivum) leaves that were excised from low-N-grown seedlings and subsequently illuminated and/or supplied with 40 mM KNO3. The apparent phosphorylation status of PEPC was assessed by its sensitivity to i-malate inhibition at suboptimal assay conditions, and the activity state of PEPC-PK was determined by the in vitro 32P labeling of purified maize dephospho-PEPC by [y32PIATP/ Mg. lllumination (±NO3-) for 1 h led to about a 4.5-fold increase in the 50% inhibition constant for i-malate, which was reversed by placing the illuminated detached leaves in darkness (minus NO3-). A 1 -h exposure of excised leaves to light, KNO3, or both resulted in relative PEPC-PK activities of 205, 11 9, and 659%, respectively, of the dark/O mM KNO3 control tissue. In contrast, almost no activity was observed when a recombinant sorghum phosphorylation-site mutant (S8D) form of PEPC was used as protein substrate in PEPC-PK assays of the light plus KNO3 leaf extracts. In vivo labeling of wheat-leaf PEPC by feeding 32P-labeled orthophosphate showed that PEPC from light plus KNO, tissue was substantially more phosphorylated than the enzyme in the dark minus-nitrate immunoprecipitates. Immunoblot analysis indicated that no changes in relative PEPC-protein amount occurred within 1 h for any of the treatments. Thus, C3 PEPC activity in these detached wheat leaves appears to be regulated by phosphorylation of a serine residue near the protein’s N terminus by a Ca2+-independent protein kinase in response to a complex interaction in vivo between light and N.