A minimal serine/threonine protein kinase circadianly regulates phosphoenolpyruvate carboxylase activity in crassulacean acid metabolism-induced leaves of the common ice plant

Plant phosphoenolpyruvate carboxylase (PEPc) activity and allosteric proper ties are regulated by PEPc kinase (PPcK) through reversible phosphorylation of a specific serine (Ser) residue near the N terminus. We report the mole cular cloning of PPcK from the facultative Crassulacean acid metabolism (CA M) common ice plant (Mesembryanthemum crystallinum), using a protein-kinase -targeted differential display reverse transcriptase-polymerase chain react ion approach. M. crystallinum PPcK encodes a minimal, Ca2+-independent Ser/ threonine protein kinase that is most closely related to calcium-dependent Protein kinases, yet lacks both the calmodulin-like and auto-inhibitory dom ains typical of plant calcium-dependent protein kinase. In the common ice p lant PPcK belongs to a small gene family containing two members. McPPcK tra nscript accumulation is controlled by a circadian oscillator in a light-dep endent manner. McPPcK encodes a 31.8-kD polypeptide (279 amino acids), maki ng it among the smallest protein kinases characterized to date. Initial bio chemical analysis of the purified, recombinant McPPcK gene product document ed that this protein kinase specifically phosphorylates PEPc from CAM and C -4 species at a single, N-terminal Ser (threonine) residue but fails to pho sphorylate mutated forms of C-4 PEPc in which this specific site has been c hanged to tyrosine or aspartate. McPPcK activity was specific for PEPc, Ca2 +-insensitive, and displayed an alkaline pH optimum. Furthermore, recombina nt McPPcK was shown to reverse the sensitivity of PEPc activity to L-malate inhibition in CAM-leaf extracts prepared during the day, but not at night, documenting that PPcK contributes to the circadian regulation of photosynt hetic carbon flux in CAM plants.