Phosphopyruvate carboxylase (PPC; EC 4.1.1.31) catalyzes primary nocturnal COfixation in Crassulacean acid metabolism (CAM) species. CAM PPC is regulated posttranslationally by a circadian clock-controlled protein kinase called phosphopyruvate carboxylase kinase (PPCK). PPCK phosphorylates PPC during the dark period, reducing its sensitivity to feedback inhibition by malate and thus enhancing nocturnal COfixation to stored malate. Here, we report the generation and characterization of transgenic RNAi lines of the obligate CAM specieswith reduced levels oftranscripts. Plants with reduced or no detectable dark phosphorylation of PPC displayed up to a 66% reduction in total dark period COfixation. These perturbations paralleled reduced malate accumulation at dawn and decreased nocturnal starch turnover. Loss of oscillations in the transcript abundance ofwas accompanied by a loss of oscillations in the transcript abundance of many core circadian clock genes, suggesting that perturbing the only known link between CAM and the circadian clock feeds back to perturb the central circadian clock itself. This work shows that clock control ofprolongs the activity of PPC throughout the dark period in, optimizing CAM-associated dark COfixation, malate accumulation, CAM productivity, and core circadian clock robustness. enol enol Kalanchoë fedtschenkoi KfPPCK1KfPPCK1KfPPCK1K. fedtschenkoi2 2 2 2
