Abstract
Phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31) catalyzes primary nocturnal CO2 fixation in Crassulacean acid metabolism (CAM) species. CAM PPC is regulated posttranslationally by a circadian clock-controlled protein kinase called phosphoenolpyruvate carboxylase kinase (PPCK). PPCK phosphorylates PPC during the dark period, reducing its sensitivity to feedback inhibition by malate and thus enhancing nocturnal CO2 fixation to stored malate. Here, we report the generation and characterization of transgenic RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced levels of KfPPCK1 transcripts. Plants with reduced or no detectable dark phosphorylation of PPC displayed up to a 66% reduction in total dark period CO2 fixation. These perturbations paralleled reduced malate accumulation at dawn and decreased nocturnal starch turnover. Loss of oscillations in the transcript abundance of KfPPCK1 was 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 of KfPPCK1 prolongs the activity of PPC throughout the dark period in K. fedtschenkoi, optimizing CAM-associated dark CO2 fixation, malate accumulation, CAM productivity, and core circadian clock robustness.
Highlights
Crassulacean acid metabolism (CAM) is one of several higher plant photosynthetic CO2-concentrating mechanisms
Comparison of KfPPCK1 transcript levels over a 12-h-light/12-h-dark cycle revealed that, relative to the wild type, the two best RNA interference (RNAi) lines had a large reduction in the transcript abundance of the target transcript (Figure 1A). While both lines displayed reduced levels of KfPPCK1 transcripts relative to the wild type, line rPPCK1-1 had a higher level of KfPPCK1 transcripts in the middle of the dark period than line rPPCK1-3, and neither line displayed a complete loss of KfPPCK1 transcript accumulation in the dark (Figure 1A)
This work on a C4 species surprised the wider community because decades of prior work had argued that phosphoenolpyruvate carboxylase (PPC) phosphorylation by phosphoenolpyruvate carboxylase kinase (PPCK) was vital for alleviating malate/aspartate inhibition of PPC in planta and for optimizing photosynthetic CO2 fixation in C4 and CAM species (Vidal and Chollet, 1997; Nimmo, 2003)
Summary
Crassulacean acid metabolism (CAM) is one of several higher plant photosynthetic CO2-concentrating mechanisms. It is noteworthy for its high water-use efficiency (WUE) relative to C3 and C4 photosynthesis (Borland et al, 2009; Cushman et al, 2015). The functional genomics and evolutionary biology of CAM species has recently received a dramatic upsurge in interest due to the potential of CAM crops, such as agaves and opuntias, as water-wise sources of biomass for biofuels, platform chemicals, and food (Cushman et al, 2015; Yang et al, 2015). Efforts are underway to engineer CAM into C3 species using synthetic biology approaches (Borland et al, 2014, 2015). The further exploitation of CAM in these ways will require detailed characterization of CAM gene function, especially with respect to their daily regulation by the circadian clock (Yang et al, 2015; Hartwell et al, 2016)
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