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

ABSTRACT

Unlike C ₃ plants, Crassulacean acid metabolism (CAM) plants fix CO ₂ in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines PEP with CO ₂ (as HCO ₃ ⁻), 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 CO ₂ around RuBisCO for secondary fixation. CAM mutants lacking PPC have not been described. Here, RNAi was employed to silence CAM isogene PPC1 in Kalanchoë laxiflora. Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO ₂ 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 CO ₂ 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.