Silencing PHOSPHOENOLPYRUVATE CARBOXYLASE1 in the Obligate Crassulacean Acid Metabolism Species Kalanchoë laxiflora causes Reversion to C3-like Metabolism and Amplifies Rhythmicity in a Subset of Core Circadian Clock Genes

Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines PEP with CO2 (as HCO3-), forming oxaloacetate that is rapidly converted to malate, leading to vacuolar malic acid accumulation that peaks phased to dawn. In the light period, malate decarboxylation concentrates CO2 around RuBisCO for secondary fixation. CAM mutants lacking PPC have not been described. Here, RNAi was employed to silence CAM isogene PPC1 in Kalanchoe 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 (LL) 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 were up-regulated and oscillated robustly. The regulation guard cell genes involved controlling stomatal movements was also altered in rPPC1-B. This provided direct evidence that altered regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.