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

Abstract

Abstract. 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 * Present address: Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, UK Abbreviations: CDCF=5-(and 6-)carboxy-2',7'-dichlorofluoresceine; PT00 = primary donor of PS I; PFD = photon flux density; QA = primary quinone acceptor of PS II; Qp, QN = photochemical, non-photochemical quenching of chlorophyll fluorescence, respectively Correspondence to: K. Hiive; FAX: 49 (931) 888 6158 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.

Intercellular Diffusion of a Fluorescent Sucrose Analog via the Septal Junctions in a Filamentous Cyanobacterium

“…Esculin fluorescence is pH dependent ( 51 ), and fluorescence emission spectra at pH 4.5 to 10 show that the fluorescence yield increases with the pH (see Fig. S7A in the supplemental material).…”

Light-Induced Changes in Hydrogen, Calcium, Potassium, and Chloride Ion Fluxes and Concentrations from the Mesophyll and Epidermal Tissues of Bean Leaves. Understanding the Ionic Basis of Light-Induced Bioelectrogenesis1

“…Linnemeyer et al (1990) discussed at least four different mechanisms for this process, and there is clearly a need for more experiments in this direction. It has been argued that light activation of H ϩ uptake from the stroma to the thylakoid lumen should result in alkalinization of the cytosol (Hansen et al, 1987;Linnemeyer et al, 1990;Heber et al, 1994;Okazaki et al, 1994;Yin et al, 1996). According to the findings of Linnemeyer et al (1990), this shift in cytosolic pH from neutral to more alkaline should result in a significant decrease in PM ATPase activity (the optimal pH for which is close to 6.5 for bean mesophyll cells).…”

Effects of magnesium availability on the activity of plasma membrane ion transporters and light-induced responses from broad bean leaf mesophyll

“…Upon illumination, the cytosolic CO 2 pool is quickly depleted, leading to a significant influx of CO 2 from external media into the cell (Hansen et al 1993). That shifts the equilibrium between HCO 3 À and CO 2 in the apoplastic solution towards CO 2 formation and results in significant alkalinization of the measuring solution (Yin et al, 1996), which is interpreted by the MIFE technique as an apparent influx (Shabala and Newman 1999). As a result, activation of plasma membrane H + pump, known to occur in response to light (Elzenga 1997), is masked by this phenomenon.…”