Regulation of PEP-carboxylase by biological clock in a CAM plant

The endogenous circadian rhythm in a crassulacean acid metabolism (CAM) plant Graptopetalum paraguayense was investigated. Phosphoenolpyruvate carboxylase (PEP-C) takes two forms: the malate-sensitive day form and the malate-insensitive night form. We monitored the state of PEP-C by measuring the sensitivity to malate as a parameter of the circadian rhythm. We also measured vacuolar pH and malate concentration, and contents of oxaloacetate, pyruvate and phosphoenolpyruvate (PEP).A free-running circadian oscillation was observed under continuous dim light (5 klux) after 12 h/12 h light/dark cycles at 20°C. The period of the rhythm was about 20 h. Under continuous light (18 klux), the rhythm was less clear but the length of the period was not affected. On the other hand, the rhythms of the vacuolar pH and the malate concentration were evident under the continuous light, but were not clear under the continuous dim light. The rhythm disappeared in continuous darkness. The content of PEP changed simultaneously with the transformation of PEP-C during the normal day-night cycles and under the continuous light, but stayed at a low level under the continuous dim light. This indicated that the transformation of PEP-C was not sufficient to maintain the rhythm in the carbon metabolism.Shift of the timing of the start or end of the dark period prior to the continuous illumination shifted the phase of the PEP-C rhythm without changing the period length significantly. At 30°C, the rhythm of PEP-C was less clear, but the period length was not affected.These results suggest that the biological clock controls CO₂ uptake and day-night CAM cycle through regulation of PEP-C transformation.