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

In crassulacean acid metabolism (CAM) large amounts of malic acid art: redi stributed between vacuole and cytoplasm in the course of night-to-day trans itions. The corresponding changes of the cytoplasmic pH (pH(cyt)) were moni tored 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 agai n in the late-day-to-early-dark phase. In the physiological context the var iation in pH(cyt) may be a component of CAM regulation. Due to its pH sensi tivity, 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 val ue, an acid load of the cytoplasm by about 10 mM malic acid can be estimate d 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 dynam ics. 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.