Unlike Cplants, Crassulacean acid metabolism (CAM) plants fix COin the dark using phosphopyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphopyruvate with CO(as HCO), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates COaround Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogeneinLinelackedtranscripts, PPC activity, dark period COfixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM COfixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lackingFurthermore, a subset of gene transcripts within the central circadian oscillator was upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed inThese findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM. 3 2 2 3 2 2 2enol enol PPC1Kalanchoë laxiflora rPPC1-B PPC1PPC1rPPC1-B -
