Phosphoenolpyruvate carboxylase (EC 4.1.1.31, 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 M r 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 Ca 2+ -dependent protein kinase (CDPK).
: Regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in three CAM species, Kalanchoë pinnata, K. daigremontiana and Ananas comosus (pineapple) were examined. PEPC activity in the leaves of the three CAM species exhibited diurnal changes peaking during the first 2-h of darkness in Kalanchoë species and at midnight in pineapple, and then decreasing drastically until dawn. The oscillations of PEPC activity were far greater in Kalanchoë species than in pineapple. In the presence of 2 mM malate, the activity of PEPC decreased in all three CAM species, but the sensitivity of PEPC to malate was markedly different between pineapple and the Kalanchoë species. The malate sensitivity was 2-to 3-times higher in pineapple than in the Kalanchoë species during the dark period, but it was almost the same during the light period. PEPC in the three CAM species was phosphorylated only during the dark period. PEPC proteins were highly phosphorylated during the first 2-h of darkness in Kalanchoë species and at midnight in pineapple, and then they decreased drastically during the latter part of darkness. CAM-specific isoforms of PEPC in the leaves of the three CAM species contained a highly conserved phosphorylation site of Ser-11 at the N-terminus. These PEPC isoforms displayed diurnal changes in transcript abundance, with the peak of transcripts occurring during the dark period. The day/night changes in PEPC transcript abundance were mirrored by changes in the PEPC protein and corresponding enzyme activity over the diurnal cycle. These findings suggest that the diurnal regulation in PEPC activity is determined by the amount of PEPC protein as well as the posttranslational control in these CAM species.
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