Regulatory phosphorylation of phosphoenolpyruvate carboxylase in the leaves of Kalanchoë pinnata, K. daigremontiana and Ananas comosus

Phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) in the three crassulacean acid metabolism (CAM) plants: Kalanchoe pinnata, K. daigremontiana and Ananas comosus (pineapple) undergoes regulatory phosphorylation during the dark period. We cloned PEPC kinase gene from two CAM Kalanchoe species using conventional RT-PCR approach. The PEPC kinase transcripts comprise only a protein kinase catalytic domain, encoding 272–276 amino acids with predicted Mr of 30.6–31.0 kDa. The expression of PEPC kinase gene in the Kalanchoe species was abundant at the beginning of dark phase, but that in pineapple cross-hybridized with Kalanchoe PEPC kinase probes was abundant at the end of dark phase. The PEPC kinase was encoded by a small gene family containing at least two members in each species. Treatment of detached leaves with the protein synthesis inhibitors cycloheximide and puromycin blocked the nocturnal appearance of PEPC kinase activity and maintained PEPC in the dephosphorylated state in the three CAM species. The calcium/calmodulin antagonist W7 blocked the apparent phosphorylation state of PEPC in pineapple, but not in Kalanchoe species. Furthermore, the transcript abundance of PEPC kinase matched the apparent in vivo phosphorylation state of PEPC in the Kalanchoe species, but unmatched that in the pineapple. These results implicated that the phosphorylation state of PEPC in Kalanchoe species is largely controlled by PEPC kinase transcript abundance, while that in pineapple may be controlled by both PEPC kinase transcript abundance and Ca2+-dependent protein kinase (CDPK).