pH regulation in apoplastic and cytoplasmic cell compartments of leaves

The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect.. Apoplastic responses to CO2, HCl or NH3 differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO2 either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO2 shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO2 was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO2-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO2 under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO2 stress increased in the presence of oxygen with the length of previous exposure to high CO2. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO2 was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO2, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO2 stress was similar to the time require. for establishing steady-state pH values in the cytosol under add stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m(2) leaf area)(-1) s(-1) or less. Proton export under acid stress into the vacuole was about 1200 nmol m(-2) s(-1). The kinetics of pH responses to the addition or withdrawal of CO2 indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast.