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

Theng, V., Agarie, S., Nose, A.
Biologia plantarum 2008 v.52 no.2 pp. 281-290
Ananas comosus, Crassulacean acid metabolism, active sites, amino acids, antagonists, calcium, calmodulin, cycloheximide, genes, leaves, phosphoenolpyruvate carboxylase, phosphorylation, pineapples, protein synthesis inhibitors, puromycin, reverse transcriptase polymerase chain reaction, scotophase
Phosphoenolpyruvate carboxylase (EC, PEPC) in the three crassulacean acid metabolism (CAM) plants: Kalanchoë pinnata, K. daigremontiana and Ananas comosus (pineapple) undergoes regulatory phosphorylation during the dark period. We cloned PEPC kinase gene from two CAM Kalanchoë 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 Kalanchoë species was abundant at the beginning of dark phase, but that in pineapple cross-hybridized with Kalanchoë 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 Kalanchoë species. Furthermore, the transcript abundance of PEPC kinase matched the apparent in vivo phosphorylation state of PEPC in the Kalanchoë species, but unmatched that in the pineapple. These results implicated that the phosphorylation state of PEPC in Kalanchoë 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).