Erhard E. Pfündel

The pH Dependence of Violaxanthin Deepoxidation in Isolated Pea Chloroplasts.

The absorbance change at 505 nm was used to monitor the kinetics of violaxanthin deepoxidation in isolated pea (Pisum sativum) chloroplasts under dark conditions at various pH values. In long-term measurements (65 min) a fast and a slow exponential component of the 505-nm absorbance change could be resolved. The fast rate constant was up to 10 times higher than the slow rate constant. The asymptote value of the fast kinetic component was twice that of the slow component. The pH dependency of the parameters of the fast kinetic component was analyzed from pH 5.2 to pH 7.0. It was found that the asymptote value dropped slightly with increasing pH. The rate constant was zero at pH values greater than 6.3 and showed maximum values at pH values less than 5.8. Hill plot analysis revealed a strong positive cooperativity for the pH dependency of the fast rate constant (Hill coefficient nH = 5.3). The results are discussed with respect to published activity curves of violaxanthin deepoxidation.

UV screening by phenolics in berries of grapevine (Vitis vinifera)

The role of phenolics in UV-screening was investigated in berries of a white grape cultivar (Vitis vinifera L. cv. Bacchus). Fluorescence microscopy revealed accumulation of phenolics in the skin of berries and, by high performance liquid chromatography and mass spectrometry, flavonols and hydroxycinnamic acids were identified as the main groups of UV-absorbing phenolics. Relationships between natural radiation and the synthesis of phenolics were studied in plants that were cultivated in the absence of UV radiation in a greenhouse before outdoor exposure to three different light regimes: the entire solar spectrum, the solar spectrum minus UV-B radiation and only visible radiation. During six days of exposure, flavonol synthesis was significantly stimulated by natural UV, in particular UV-B, but concentrations of hydroxycinnamic acids decreased under all conditions. Direct comparison of fluorimetrically-determined skin absorbance with absorbance of extracted flavonols or hydroxycinnamic acids suggested that acclimation of UV screening depends almost exclusively on flavonol synthesis. While increased flavonol levels resulted in efficient UV-A shielding, UV-B shielding was incomplete, probably due to decreased levels of the UV-B-absorbing hydroxycinnamic acids during exposure.

Intrathylakoid pH in Isolated Pea Chloroplasts as Probed by Violaxanthin Deepoxidation

Light-driven violaxanthin deepoxidation was measured in isolated pea (Pisum sativum) chloroplasts without ATP synthesis (basal conditions) and with ATP synthesis (coupled conditions). Thylakoids stored in high salt (HS) or low salt (LS) storage medium were tested. In previous experiments, HS thylakoids and LS thylakoids were related to delocalized and localized proton coupling, respectively.Light-driven deepoxidase activity was compared to the pH dependence of deepoxidase activity established in dark reactions. At an external pH of 8, light-driven deepoxidation indicated effective pH values close to pH 6 for all reaction conditions. Parallel to deepoxidation, the thylakoid lumen pH was estimated by the fluorescent dye pyranine.In LS thylakoids under coupled conditions the lumen pH did not drop below pH 6.7. At pH 6.7, no deepoxidase activity is expected based on the pH dependence of enzyme activity. The results suggest that deepoxidation activity is controlled by the pH in sequestered membrane domains, which, under localized proton coupling, can be maintained at pH 6.0 when the lumen pH is far above pH 6.0. The extent of violaxanthin conversion (availability), however, appeared to be regulated by lumenal pH. Dithiothreitol-sensitive nonphotochemical quenching of chlorophyll fluorescence was dependent on zeaxanthin and not related to lumenal pH. Thus, zeaxanthin-dependent quenching[mdash]known to be pH dependent[mdash]appeared to be triggered by the pH of localized membrane domains.

Analyzing the Light Energy Distribution in the Photosynthetic Apparatus of C4 Plants Using Highly Purified Mesophyll and Bundle-Sheath Thylakoids.

The chlorophyll fluorescence characteristics of mesophyll and bundle-sheath thylakoids from plant species with the C4 dicarboxylic acid pathway of photosynthesis were investigated using flow cytometry. Ten species with the NADP-malic enzyme (NADP-ME) biochemical type of C4 photosynthesis were tested: Digitaria sanguinalis (L.) Scop., Euphorbia maculata L., Portulaca grandiflora Hooker, Saccharum officinarum L., Setaria viridis (L.) Beauv., Zea mays L., and four species of the genus Flaveria. This study also included three species with NAD-ME biochemistry (Atriplex rosea L., Atriplex spongiosa F. Muell., and Portulaca oleracea L.). Two C4 species of unknown biochemical type were investigated: Cyperus papyrus L. and Atriplex tatarica L. Pure mesophyll and bundle-sheath thylakoids were prepared by flow cytometry and characterized by low-temperature fluorescence spectroscopy. In pure bundle-sheath thylakoids from many species with C4 photosynthesis of the NADP-ME type, significant amounts of photosystem II (PSII) emission can be detected by fluorescence spectroscopy. Simulation of fluorescence excitation spectra of these thylakoids showed that PSII light absorption contributes significantly to the apparent excitation spectrum of photosystem I. Model calculations indicated that the excitation energy of PSII is efficiently transferred to photosystem I in bundle-sheath thylakoids of many NADP-ME species.

Thylakoid lumenal pH determination using a fluorescent dye: Correlation of lumen pH and gating between localized and delocalized energy coupling

Abstract Thylakoid lumen pH measurements were made using the fluorescent dye 8-hydroxypyrene 1,3,6-trisulfonic acid (HPTS; pyranine). The purpose was twofold: (1) to develop the method for use in the fluorescence ratio mode (permitting assays independent of lumenal dye concentration and lumen volume); (2) to use the technique to measure transmembrane ΔpH values in thylakoids predicted by previously used criteria to be energizing ATP formation with a delocalized or localized protonmotive force. The dye was readily loaded into thylakoids at pH 6 and the loading, washing and resuspension steps did not disrupt the localized or delocalized energy coupling patterns, tested by the effect of a permeable amine on the flash number required to initiate ATP formation as described in earlier work (Beard, W.A. and Dilley, R.A. (1988) J. Bioenerg. Biomembr. 20, 129). Light-dependent dye fluorescence changes were recorded in continuous illumination over 10–20 s with low- or high-salt-stored thylakoids under either coupled or basal conditions to assess the extent of the ΔpH developed across the membranes. Low-salt stored membranes - which were predicted by the criteria developed earlier to show localized Δ \ gm H + energy coupling - gave a ΔpH below the thermodynamically predicted energetic requirement for ATP formation at external pH of 7.8 to 8.9. At an external pH of 7.8 or 8.0 the lumen pH under coupled conditions in the low-salt-stored thylakoids was about 6.8 to 7.0, respectively, (ΔpH ≈ 1.0 unit), and at pH 8.2 the ΔpH was about 1.4 units (lumen mpH = 6.8). At pH 8.9 the lumen pH reached near 7.3 (ΔpH 1.6) under the coupled conditions for low-salt-stored thylakoids. Under basal conditions (no ADP) low-salt-stored membranes gave a ΔpH of 2.4 to 2.8 units, at moderate and high intensities, respectively, when the external pH was 8.9. The pH calibration is not sensitive enough at pH values less than about 6.0 to 6.3 to obtain accurate internal pH data in that range. For that reason, when the external pH was 7.8 to 8.0 all that can be said is that the basal ΔpH could be observed to be >1.7 units. At an external pH of 8.9, high-salt-stored thylakoids - predicated to have a delocalized Δ \ gm H + coupling mode - showed a μpH >2.3 in all cases, coupled or basal, but in the coupled mode the ΔpH was near 2.3 at low light and near 2.7 at high light intensity. That is the expected result of the faster electron-proton transport at the high intensity. These lumen pH measurements support the concept developed with other techniques that thylakoids can maintain either localized or delocalized energy coupling Δ \ gm H + gradients, and that in the localized energy coupling mode, the H+ ions involved in driving ATP formation do not equilibrate with the lumen.

FLOW CYTOMETRY OF MESOPHYLL AND BUNDLE SHEATH CHLOROPLAST THYLAKOIDS OF MAIZE (ZEA MAYS L.)

Chlorophyll fluorescence at short and long wavelengths was used to sort thylakoid membranes of maize, a plant with the C4 dicarboxylic acid pathway of photosynthesis, in a flow cytometer. The method yielded two distinct particle populations that were identified as mesophyll and bundle sheath thylakoids by low-temperature fluorescence spectroscopy and by the pigment ratio of chlorophyll a/b. Mesophyll and bundle sheath thylakoids were essentially pure after sorting by flow cytometry. Fluorescence data and chlorophyll a/b pigment ratios of thylakoids separated by flow cytometry were compared with earlier data of preparations obtained by conventional isolation procedures. Our results indicate that impure mesophyll and bundle sheath membranes were used in most previous investigations. We were unable to detect the major light-harvesting complex of PS II (LHC II) in our pure bundle sheath thylakoids using fluorescence excitation spectroscopy. Therefore, we believe that the previously reported presence of LHC II in bundle sheath chloroplasts of maize can be attributed to mesophyll contamination.

IS ZEAXANTHIN CAPABLE OF ENERGY TRANSFER TO CHLOROPHYLL a IN PARTIALLY GREENED LEAVES? A STUDY OF FLUORESCENCE EXCITATION SPECTRA DURING VIOLAXANTHIN DEEPOXIDATION

Absorbance spectra and excitation spectra of chlorophyll a fluoresence were recorded during the light‐induced deepoxidation of violaxauthin to zeaxanthin in bean leaves (Phaseolus coccineus) greened under intermittent light. Light minus dark fluorescence excitation difference spectra showed distinct minima at 440, 465, and 500 nm corresponding to maxima in the absorbance difference spectra. Both difference spectra were prevented by the deepoxidase inhibitor dithiothreitol and were inverted when zeaxanthin was epoxidized. The fluorescence excitation difference spectra were successfully modeled by considering the absorbance differences between violaxanthin and zeaxanthin, assuming no energy transfer from the two pigments to chlorophyll a, and accounting for light‐induced scattering changes. The pigment stoichiometry and the scattering changes of the simulation were in accordance with experimental data. The results indicate that, in the early stage of leaf development, light absorbed by the cycle pigments violaxanthin and zeaxanthin is not transferred to chlorophyll.