Electron paramagnetic resonance-spectroscopic studies on spin-labeled purified tonoplast membranes showed that in the obligate crassulacean-acid-metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. the fluidity of the tonoplast decreased during acclimation to higher temperatures. This phenotypic change in tonoplast fluidity was paralleled by a decrease in the mobilization of malic acid from the vacuoles during CAM in the light. The shift from the C3 to the CAM mode of photosynthesis in the facultative CAM plant Mesembryanthemum crystallinum L. also led to a decrease in the fluidity of the tonoplast membrane. The results are consistent with the hypothesis that the ability to store malic acid during CAM in the vacuoles depends largely on the actual fluidity of the tonoplast membrane.

The thermotropic phase behaviour of tonoplast material isolated from leaf mesophyll protoplasts of the obligatory CAM plant Kalanchoe daigremontiana was investigated by electron power magnetic resonance (EPR) spectroscopy using a spin label technique. The data clearly show that at temperatures below 9°C the tonoplast membrane is in a rigid state. Above 9 "C, an increasing fluidization of the tonoplast occurs. Two distinct temperature ranges were observed: a cooperative melting process between 9 and 14 "C being followed by a second broad melting process starting at 18 "C, with continuously increasing membrane fluidity up to 51 "C, which was the highest temperature tested. These results are important for a better understanding of the mechanism of the temperature modulation of CAM. The data support the hypothesis that temperature affects CAM via the permeability of the tonoplast membrane, which determines the rates of the passive malic acid efflux from the vacuole and thus the capability of the plant to accumulate malic acid in the vacuoles overnight at a given temperature.

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