Light-induced pH changes in leaves of C4 plants

Abstract

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 (pK 7.3) to leaves of Amaranthus caudatus L., a NAD-malic-enzyme-type C4 plant, 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 of Amaranthus leaves had been stained. After short feeding times and in the absence of actinic illumination, CO2 decreased pyranine fluorescence very slowly in Amaranthus and rapidly in C3 leaves. After prolonged feeding times, the extent of the light-dependent increase in pyranine fluorescence was several times greater in different C4 plants than in C3 species. The kinetics of the fluorescence changes were also remarkably different in C3 and C4 plants. Carbon dioxide (500 μl · l−1) suppressed the light-induced increase in pyranine fluorescence more in C4 than in C3 leaves. 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 C4 leaves 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 C3 species 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 in Amaranthus and more rapidly in C3 leaves. 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 (pK 4.8) did not increase but rather decreased its fluorescence on illumination of an Amaranthus leaf, indicating acidification of an acidic compartment, most probably the vacuole of green leaf cells. The pattern of the acidification reaction was similar in C4 and C3 leaves. The remarkably large extent of the light-dependent increase in pyranine fluorescence from leaves of C4 species and its slow kinetics are proposed to be caused by an alkalization of the cytosol which in the absence of CO2 is 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 CO2 between mesophyll and bundle-sheath cells and the fast metabolite transport required in C4 photosynthesis are discussed.