On the Mechanism of Reinitiation of Endogenous Crassulacean Acid Metabolism Rhythm by Temperature Changes

Abstract

Under continuous light the endogenous Crassulacean acid metabolism (CAM) rhythm of Kalanchoe daigremontiana Hamet et Perrier de Ia Bâthie disappears at high (>29.0"C) or low (<8.0"C) temperatures. We investigated the reinitiation of rhythmicity when temperature was reduced from above the upper and increased from below the lower threshold leve1 via measurements of (a) short-term changes in carbon-isotope discrimination to illustrate shifts between C, and C, carboxylation in vivo, and (b) the malate sensitivity of phosphoenolpyruvate carboxylase (PEPC) in vitro. When the net C0,-exchange rhythm disappears at both temperatures, the instantaneous discrimination indicates low PEPC activity. Leaf malate concentration and osmolarity attain high and low values at low and high temperatures, respectively. After small temperature increases or reductions from the iow and high temperatures, respectively, the rhythm is reinitiated, with phases shifted by 180" relative to each other. This can be related to the contrasting low and high leaf malate concentrations due t o direct inhibition of PEPC and possibly also of the phosphorylation of PEPC by malate. The experimental results were satisfactorily simulated by a mathematical CAM-cycle model, with temperature acting only on the passive efflux of malate from the vacuole. We stress the important role of the tonoplast i n malate compartmentation and of malate itself for the reinitiation and generation of endogenous CAM rhythmicity.CAM is an ecophysiological adaptation of plants to limited availability of water or CO, (Kluge and Ting, 1978). At night CO, is fixed via the C, carboxylation enzyme PEPC, with the product, malate, stored in the vacuole. During the day the decarboxylation of malate releases CO,, which is refixed by the C, carboxylation enzyme Rubisco when the stomata are closed.

Metabolite Control Overrides Circadian Regulation of Phosphoenolpyruvate Carboxylase Kinase and CO2 Fixation in Crassulacean Acid Metabolism

“…It has been suggested that the disruption of the circadian oscillations of CO 2 exchange in CAM plants by high temperature may be a consequence of increased efflux of malate from the vacuole to the cytosol, the site of PEPc activity (Wilkins, 1983;Grams et al, 1997). Figure 5 illustrates the physiological consequences of exposing either control leaves or leaves prevented from accumulating malate over the first half of the dark period (half N 2 ) to an 8°C increase in temperature in the middle of the night (from 2:30-3 am).…”

“…Thus, in order to completely inhibit PEP carboxylation at night, individual leaves of intact plants were enclosed in an atmosphere of N 2 overnight, as described by Borland and Griffiths (1997), thereby preventing access to external CO 2 and inhibiting the release of internal (respiratory) sources of CO 2 (full N 2 ). Some leaves were enclosed in an atmosphere of N 2 for the first half of the dark period (until 2 pm) and then exposed to ambient air for the remainder (half N 2 ).…”

“…Physiological manipulations of dark CO 2 uptake and malate accumulation have indicated that the storage capacity of the vacuole for malate plays a key role in determining the timing of the inactivation of PEPc (Winter and Tenhunen, 1982;Fischer and Kluge, 1984;Borland and Griffiths, 1997). Thus, in plants prevented from accumulating malate overnight in an atmosphere of N 2 , flux through PEPc increases substantially at the start of the day in ambient air, and the inactivation of PEPc is delayed by 2 to 3 h (Borland and Griffiths, 1997).…”

“…Thus, in plants prevented from accumulating malate overnight in an atmosphere of N 2 , flux through PEPc increases substantially at the start of the day in ambient air, and the inactivation of PEPc is delayed by 2 to 3 h (Borland and Griffiths, 1997). Observations that the circadian rhythms of phosphorylation of PEPc and CO 2 exchange can be disrupted and re-initiated by temperature changes have also pointed to a key role for the tonoplast in malate compartmentation, and for malate itself in the generation of the endogenous rhythm of PEPc activity (Wilkins, 1983;Carter et al, 1991;Grams et al, 1997). Malate inhibits PEPc kinase by binding to PEPc (Carter et al, 1991;Chollet 1993, 1994), although it is not clear whether this effect is physiologically significant.…”

“…This results in a circadian rhythm in the phosphorylation state of PEPc (Nimmo et al, 1987), which plays an important role in generating the endogenous rhythms of CO 2 exchange in CAM plants first described by Wilkins (1959). To date, the exact nature of the circadian oscillator in CAM is unknown, but recent observations indicate that the timing of PEPc activation/deactivation varies between different CAM species grown under identical environmental conditions (Borland and Griffiths, 1997).…”