CDNA cloning and characterization of a C4 PEPC kinase from Flaveria trinervia

Abstract

PEPC activity in C4 plants is regulated by reversible phosphorylation mainly in response to light. The cDNA for this specific protein kinase (PEPC-PK) has not yet been cloned, though several cDNAs for PEPC-PKs from C3 and CAM plants have been isolated recently. A complete cDNA (~1.4 kb) encoding PEPC-PK was isolated from Flaveria trinervia, a dicot C4 plant, by screening cDNA library with a F. trinervia PEPC-PK fragment prepared by PCR with degenerate primers. The cloned PEPC-PK contained an ORF for a protein of 281 amino acid residues, and it was composed of the kinase domain only. The recombinant PEPC-PK expressed in E. coli phosphorylated a specific serine residue of maize C4-form PEPC near the N-terminus, whereas it did not phosphorylate a mutant PEPC in which the serine residue of the phosphorylation-site have been substituted for aspartate (S15D). The activity of the PEPC-PK was Ca2+-independent. The interesting enzymatic properties will also be presented. Northern blot analysis showed that the transcripts accumulated abundantly in leaves in daytime, but not at night. The transcripts were not detected in roots and stems. Genomic Southern blot analysis under the high-stringency conditions indicated that the F. trinervia genome contained more than two copies. These results suggested that this PEPC-PK plays a major role in the regulatory phosphorylation of PEPC for C4 photosynthesis.