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.
Highlights
2 3 Crassulacean acid metabolism (CAM) is a pathway of photosynthetic CO2 fixation 4 found in species adapted to low rainfall and/or periodic drought, such as the Madagascan-endemic succulent, Kalanchoë laxiflora Baker (Family: Crassulaceae; Order: Saxifragales) (Hartwell et al, 2016)
(Figure 2E), and was likely to be inactive. Consistent with this prediction, loss of the light period dephosphorylation of pyruvate orthophosphate dikinase (PPDK) in rPPC1-B correlated with a significant decrease in PPDK activity in the light, whereas the wild type and line rPPC1-A showed strongly light-induced levels of PPDK activity that correlated with the detected level of PPDK dephosphorylation in the light (Figure 2F and Supplemental Table 2)
By comparing the impacts of the different levels of PPC1 679 silencing in these transgenic lines of Kalanchoë, it is possible to conclude that the wild type level of PPC1 transcript enables the plant to adapt to drought more rapidly by increasing nocturnal CO2 fixation and malate accumulation and by decreasing the magnitude and duration of phases II and IV of CAM in the light
Summary
As K. laxiflora is relatively slow-growing, with flowering and seed set taking approximately 9-months seed-to-seed (Hartwell et al, 2016), data are presented for independent primary transformants that were propagated clonally via leaf margin plantlets and/or stem cuttings. In line rPPC1-B, PPCK1, encoding the protein kinase that phosphorylates the CAM-associated protein PPC1, was down-regulated during its dark period phased peak and appeared to be slightly up-regulated at 2 h after dawn (Figure 1E). PPDK was phosphorylated throughout the 24 h cycle in rPPC1-B (Figure 2E), and was likely to be inactive Consistent with this prediction, loss of the light period dephosphorylation of PPDK in rPPC1-B correlated with a significant decrease in PPDK activity in the light, whereas the wild type and line rPPC1-A showed strongly light-induced levels of PPDK activity that correlated with the detected level of PPDK dephosphorylation in the light
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