Light-induced pH changes in leaves of C4plants

摘要

Light-induced changes in the fluorescence of the pH-indicating dyes pyranine or 5-(and 6-)carboxy-2′, 7′-dichlorofluorescein (CDCF) which had been fed to leaves were examined to monitor cellular pH changes. After short-term feeding of pyranine (pK7.3) to leaves ofAmaranthus caudatusL., a NAD-malic-enzyme-type C4plant, vascular bundles and surrounding cells became fluorescent. Fluorescence emission from mesophyll cells required longer feeding times. In CO2-free air, pyranine fluorescence increased much more on illumination after mesophyll cells had become fluorescent than when only the vascular bundles and the bundle sheath ofAmaranthusleaves had been stained. After short feeding times and in the absence of actinic illumination, CO2decreased pyranine fluorescence very slowly inAmaranthusand rapidly in C3leaves. After prolonged feeding times, the extent of the light-dependent increase in pyranine fluorescence was several times greater in different C4plants than in C3species. The kinetics of the fluorescence changes were also remarkably different in C3and C4plants. Carbon dioxide (500 μl · l−1) suppressed the light-induced increase in pyranine fluorescence more in C4than in C3leaves. Light-dependent changes in light scattering, which are indicative of chloroplast energization, and in 410-nm transmission, which indicate chloroplast movement, differed kinetically from those of the changes in pyranine fluorescence. Available evidence indicated that light-dependent changes in pyranine fluorescence did not originate from the apoplast of leaf cells. Microscopic observation led to the conclusion that, after prolonged feeding times or prolonged incubation, changes in pyranine fluorescence emitted from C4leaves reflect pH changes mainly in the cytosol of mesophyll cells. A transient acidification reaction indicated by quenching of pyranine fluorescence in the dark-light transient and not observed in C3species is attributed to the carboxylation of phosphoenolpyruvate. After short feeding times and in the absence of actinic illumination, CO2(250 μl μ l−1) decreased pyranine fluorescence very slowly inAmaranthusand more rapidly in C3leaves. After prolonged feeding times, both the rate and the extent of CO2-dependent quenching of pyranine fluorescence increased, but the increase was insufficient to indicate the presence of highly active carbonic anhydrase in the compartment from which pyranine fluorescence was emitted. In contrast to pyranine, CDCF (pK4.8) did not increase but rather decreased its fluorescence on illumination of anAmaranthusleaf, indicating acidification of an acidic compartment, most probably the vacuole of green leaf cells. The pattern of the acidification reaction was similar in C4and C3leaves. The remarkably large extent of the light-dependent increase in pyranine fluorescence from leaves of C4species and its slow kinetics are proposed to be caused by an alkalization of the cytosol which in the absence of CO2is larger in the mesophyll than in the bundle sheath. It gives rise to deprotonation of dye originally located in the mesophyll and, in addition, of dye which diffuses from the bundle sheath into the mesophyll following a pH gradient. Implications of slow diffusional transport of pyranine and CO2between mesophyll and bundle-sheath cells and the fast metabolite transport required in C4photosynthesis ar