Kalanchoë PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes

Abstract

Unlike C₃ plants, Crassulacean acid metabolism (CAM) plants fix CO₂ in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphoenolpyruvate with CO₂ (as HCO₃⁻), 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 CO₂ around Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogene PPC1 in Kalanchoë laxiflora. Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO₂ fixation, 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 CO₂ fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1. Furthermore, 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 in rPPC1-B. These 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.