Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant

In crassulacean acid metabolism (CAM) large amounts of malic acid art: redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pH(cyt)) were monitored in mesophyll protoplasts from the CAM plant Kalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2',7'-bis-(2-carboxye thyl)-5-(and-6-)carboxyfluoresce in as a pH(cyt) indicator. At the beginning of the light phase, pH(cyt) was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pH(cyt) may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring Delta pH(cyt) in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H+-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pH(cyt) of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pH(cyt). A diurnal oscillation in pH(cyt) and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.