Light-dependent proton transport into mesophyll vacuoles of leaves of C3 plants as revealed by pH-indicating fluorescent dyes: a reappraisal

Esculin, a pH-sensitive fluorescent dye, was used to indicate light-dependent pH changes in leaves of Spinacia oleracea L. and Pelargonium zonale L. Shortly after its introduction into the leaves via the transpiration stream, esculin was localized mainly in the symplasm. An increase in its blue fluorescence on illumination with red actinic light indicated that the cytosolic pH had increased. A similar light-dependent alkalinization was seen when the green fluorescence of pyranine was used to monitor changes in the cytosolic pH. After esculin had been transferred into the vacuoles, a light-dependent vacuolar acidification was indicated by a decrease in its blue fluorescence. Since the pK of esculin is close to neutrality, it is suitable as an indicator of proton transport into vacuoles provided the vacuolar sap is only moderately acidic. In leaf cells with very acidic vacuoles, esculin therefore responds only to cytosolic pH changes as long as it remains in the cytosol. The observations made with esculin after it had entered the vacuoles confirmed earlier conclusions on light-dependent proton transport into the vacuoles of mesophyll cells. Previous measurements had been made with 5-carboxy-2,7-dichlorofluoresceine (CDCF), which has a pK of 4.8. In contrast to esculin, CDCF can, in principle, record pH changes in very acidic vacuoles. However, earlier conclusions made on the basis of observed CDCF fluorescence are now recognized to have no unambiguous basis because new measurements, reported here, show that CDCF fluorescence is influenced not only by pH changes but also by changes in light scattering. The latter are, like pH changes, light-dependent and originate from the thylakoid system of chloroplasts. They indicate both the formation of a large transthylakoid proton gradient and the dissipation of excess light energy as heat. Decreased green fluorescence of leaves which had been fed CDCF may therefore, depending on conditions, indicate vacuolar acidification or the dissipation of excess light energy absorbed by the pigment system of chloroplasts, or both. Pyranine fluorescence was found to be much less influenced by light scattering than CDCF fluorescence.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluoresceine - P₇₀₀ primary donor of PS I - PFD photon flux density - Q_A primary quinone acceptor of PS II - Q_P, Q_N photochemical, non-photochemical quenching of chlorophyll fluorescence, respectivelyThis work was supported by the Deutsche Forschungsgemeinschaft within the framework of the research of the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful to Drs. U. Schreiber and K.-J. Dietz and to Mrs. B. Hollenbach (all from our Institute) for discussions.