Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant Kalancho� daigremontiana: testing the tonoplast-as-oscillator model

Abstract

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO2 uptake, stomatal conductance and leaf-internal CO2 pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phospho enolpyruvate carboxylase, controls the rate of CO2 uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO2 from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO2 removal: (i) starting at a trough of CO2 uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO2 increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO2 removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471-1479] that in extended continuous light, CO2 uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO2 uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.

Conservation and Divergence of Circadian Clock Operation in a Stress-Inducible Crassulacean Acid Metabolism Species Reveals Clock Compensation against Stress

“…However, Wyka et al (2004) found that preventing nocturnal malate accumulation in Kalanchoë daigremontiana did not cause the predicted phase delays generated by the aforementioned computer model. The authors conclude that their results rule out vacuolar malic acid accumulation as the central pacemaking process in Kalanchoë (Wyka et al, 2004). The current paradigm is that the central circadian oscillator responsible for all rhythmic outputs consists of a discrete suite of genes, which form an autoregulatory negative feedback loop.…”

“…Computer modeling of this biophysical oscillator has demonstrated that it could sustain robust rhythmicity of CAM over a range of temperatures (Lü ttge, 2000). However, Wyka et al (2004) found that preventing nocturnal malate accumulation in Kalanchoë daigremontiana did not cause the predicted phase delays generated by the aforementioned computer model. The authors conclude that their results rule out vacuolar malic acid accumulation as the central pacemaking process in Kalanchoë (Wyka et al, 2004).…”

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoë fedtschenkoi

“…Since the first discovery of PPCK activity and its circadian control in 1991 (Carter et al, 1991), through the subsequent cloning and characterization of the gene encoding this remarkable protein kinase in 1999 (Hartwell et al, 1999), there has been a long-held assumption that this circadian clock-controlled kinase is crucial for the temporal coordination and optimization of nocturnal atmospheric CO 2 fixation in CAM species, and the associated persistent circadian rhythm of CO 2 exchange observed under constant conditions in CAM species such as K. fedtschenkoi and K. daigremontiana (Wilkins, 1992;Nimmo, 2003;Wyka et al, 2004;Hartwell, 2006). However, it has not been possible previously to perturb genetically the level of PPCK activity and dark period PPC phosphorylation in a CAM species; thus, it has not been possible to test the validity of this long held assumption about the pivotal role of PPCK in the circadian control of CAM.…”

Evolution along the crassulacean acid metabolism continuum

“…CAM represents an noteworthy example of circadian clock specialisation and is one of the best-characterised physiological rhythms in plants (Wilkins 1992;Lüttge 2003;Wyka et al 2004). The presence of the CAM enzymatic machinery within a single cell requires strict temporal control of the competing carboxylation reactions by PEPC and RUBISCO.…”