Kalanchoë PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling 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 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.

Boxall SF ，Kadu N ，Dever LV ，Kneřová J ，Waller JL ，Gould PJD ，Hartwell J ... -  《-》

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO(2) Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoë fedtschenkoi.

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.

Boxall SF ，Dever LV ，Kneřová J ，Gould PD ，Hartwell J ... -  《-》

Transgenic perturbation of the decarboxylation phase of Crassulacean acid metabolism alters physiology and metabolism but has only a small effect on growth.

Mitochondrial NAD-malic enzyme (ME) and/or cytosolic/plastidic NADP-ME combined with the cytosolic/plastidic pyruvate orthophosphate dikinase (PPDK) catalyze two key steps during light-period malate decarboxylation that underpin secondary CO(2) fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced activities of NAD-ME or PPDK. Transgenic line rNAD-ME1 had 8%, and rPPDK1 had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CAM CO(2) fixation. In well-watered conditions, these lines fixed all of their CO(2) in the light; they thus performed C(3) photosynthesis. The alternative malate decarboxylase, NADP-ME, did not appear to compensate for the reduction in NAD-ME, suggesting that NAD-ME was the key decarboxylase for CAM. The activity of other CAM enzymes was reduced as a consequence of knocking out either NAD-ME or PPDK activity, particularly phosphoenolpyruvate carboxylase (PPC) and PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPC kinase transcript levels and activity, and the classic circadian rhythm of CAM CO(2) fixation were lost, or dampened toward arrhythmia, under constant light and temperature conditions. Surprisingly, oscillations in the transcript abundance of core circadian clock genes also became arrhythmic in the rNAD-ME1 line, suggesting that perturbing CAM in K. fedtschenkoi feeds back to perturb the central circadian clock.

Dever LV ，Boxall SF ，Kneřová J ，Hartwell J ... -  《-》

The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolism (CAM) species Kalanchoë fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness.

Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis characterized by a diel pattern of stomatal opening at night and closure during the day, which increases water-use efficiency. Starch degradation is a key regulator of CAM, providing phosphoenolpyruvate as a substrate in the mesophyll for nocturnal assimilation of CO2. Growing recognition of a key role for starch degradation in C3 photosynthesis guard cells for mediating daytime stomatal opening presents the possibility that starch degradation might also impact CAM by regulating the provision of energy and osmolytes to increase guard cell turgor and drive stomatal opening at night. In this study, we tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been reprogrammed during evolution to enable nocturnal stomatal opening and daytime closure. Biochemical and genetic characterization of wild-type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi with reduced activity of plastidic phosphoglucomutase (PGM) constituted a preliminary approach for the understanding of starch metabolism and its implications for stomatal regulation in CAM plants. Starch deficiency reduced nocturnal net CO2 uptake but had negligible impact on nocturnal stomatal opening. In contrast, daytime stomatal closure was reduced in magnitude and duration in the starch-deficient rPGM RNAi lines, and their stomata were unable to remain closed in response to elevated concentrations of atmospheric CO2 administered during the day. Curtailed daytime stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll. Nocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring daytime stomatal closure in this CAM species.

Hurtado-Castano N ，Atkins E ，Barnes J ，Boxall SF ，Dever LV ，Kneřová J ，Hartwell J ，Cushman JC ，Borland AM ... -  《-》

Crassulacean acid metabolism guard cell anion channel activity follows transcript abundance and is suppressed by apoplastic malate.

Plants utilising crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reducing transpirational water loss associated with photosynthesis. Unlike stomata of C3 and C4 species, CAM stomata open at night for the mesophyll to fix CO2 into malate (Mal) and store it in the vacuole. CAM plants decarboxylate Mal in the light, generating high CO2 concentrations within the leaf behind closed stomata for refixation by RuBisCO. CO2 may contribute to stomatal closure but additional mechanisms, plausibly including Mal activation of anion channels, ensure closure in the light. In the CAM species Kalanchoë fedtschenkoi, we found that guard cell anion channel activity, recorded under voltage clamp, follows KfSLAC1 and KfALMT12 transcript abundance, declining to near zero by the end of the light period. Unexpectedly, however, we found that extracellular Mal inhibited the anion current of Kalanchoë guard cells, both in wild-type and RNAi mutants with impaired Mal metabolism. We conclude that the diurnal cycle of anion channel gene transcription, rather than the physiological signal of Mal release, is a key factor in the inverted CAM stomatal cycle.

Lefoulon C ，Boxall SF ，Hartwell J ，Blatt MR ... -  《-》