Light-dependent Proton Uptake Research Articles

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 μM, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO2-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 μM CO2) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO2-dependent O2evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO2-dependent O2evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C3-plants are discussed.

Chilling effects on leaf photosynthesis and seed yields of Phaseolus vulgaris

Three common bean cultivars (climbing ‘Negro Mecentral’, bushlike ‘Negro Mecentral’, and ‘Tendergreen’) were studied during seed maturity. A single dark period of a chilling treatment (4 °C, 12 h) resulted in a sizeable reduction of the photochemical and photosynthetic activities of the leaves during the seed maturity stage. Leaf net photosynthesis, transpiration rate, and stomatal conductance for water vapor were drastically reduced in ‘Tendergreen’ at 28 days after anthesis, but in bushlike ‘Negro Mecentral’ these values decreased until 30 days after anthesis. Chilling drastically reduced electron transport of freshly lysed chloroplasts isolated from bushlike ‘Negro Mecentral’ and ‘Tendergreen’ at 32 days after anthesis. This contrasts with climbing ‘Negro Mecentral’ plants, in which electron transport decreased progressively between 28 and 32 days after anthesis. Photophosphorylation rates at 32 days after anthesis were reduced by 31, 58, and 45% in climbing ‘Negro Mecentral’, bushlike ‘Negro Mecentral’, and ‘Tendergreen’ plants, respectively. Light-dependent proton uptake was more damaged in ‘Tendergreen’ (88%) at 32 days after anthesis than in bushlike ‘Negro Mecentral’ (31%) and climbing ‘Negro Mecentral’ (38%). Seed yield per plant tended to decrease with treatment in climbing ‘Negro Mecentral’ (45% at 32 days after anthesis) and in bushlike ‘Negro Mecentral’ (34% at 32 days after anthesis), whereas in ‘Tendergreen’ the yield apparently was not affected. However, seed weight and size of climbing ‘Negro Mecentral’ decreased following the treatment, while they increased in bushlike ‘Negro Mecentral’ and ‘Tendergreen’. Seed protein content increased dramatically at 28, 30, and 32 days after anthesis in treated bushlike ‘Negro Mecentral’ (52, 78, and 42%, respectively), but it did not change in climbing ‘Negro Mecentral’ and ‘Tendergreen’. In conclusion, the chilling effects on photochemical activities, seed yield, and seed composition of common bean plants at seed maturity vary according to cultivar and plant developmental stage. Key words: Phaseolus vulgaris, photosynthesis, chilling effect.

A conserved carboxylic acid group mediates light-dependent proton uptake and signaling by rhodopsin.

A carboxylic acid residue is conserved at the cytoplasmic border of the third transmembrane segment among nearly all G protein-coupled receptors. In the visual receptor rhodopsin, replacement of the conserved Glu134 by a neutral glutamine results in enhanced transducin activation. Here we show that a key event in forming the active state of rhodopsin is proton uptake by Glu134 in the metarhodopsin II (MII) photoproduct. Site-directed mutants E134D and E134Q were studied by flash photolysis, where formation rates of their photoproducts and rates of pH change could be monitored simultaneously. Both mutants showed normal MII formation rates. However, E134D displayed a slowed rate of proton uptake and E134Q displayed a loss of light-induced uptake of two protons from the aqueous phase. Thus, Glu134 mediates light-dependent proton uptake by MII. We propose that receptor activation requires a light-induced conformational change that allows protonation of Glu134 and subsequent protonation of a second group. The strong conservation of Glu134 in G protein-coupled receptors implies a general requirement for a proton acceptor group at this position to allow light- or ligand-dependent receptor activation.

Effect of Frost Hardening on Lipid and Fatty Acid Composition of Chloroplast Thylakoid Membranes in Two Wheat Varieties of Contrasting Hardiness

Lipid and fatty acid composition of chloroplast thylakoid membranes was determined in two varieties of wheat (Triticum aestivum L.), the hardy Miranovskaja and the sensitive Penjamo. Plants were grown at room temperature or under frost hardening conditions (1.5 degrees C). Changes in lipid and fatty acid composition of the isolated thylakoids could be related to the temperature dependence of light-stimulated proton uptake. Changes in the thylakoid phospholipids upon hardening of the two varieties did not show any direct relation with low temperature tolerance of light-dependent H(+) uptake; neither did changes in phospholipid fatty acid chain lengthening to 20 and 22 C-atoms in combination with increased desaturation up to 6 double bonds. Increased low temperature tolerance of light-induced H(+) uptake by hardening was correlated with the following glycolipid changes: maintained glycolipid level, a proportionally increased digalactosyl diglyceride fraction, a decrease in thylakoid monogalactosyl diglyceride, increased sulfolipid fatty acid chain lengthening (20 and 22 C-atoms), and increased sulfolipid desaturation (4-6 double bonds). We suggest that the above mentioned changes in glycolipids have adaptive value for low temperature tolerance of light-dependent proton uptake.

Light-induced generation of a protonmotive force and Ca 2+-transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes

The light-driven primary proton pump bacteriorhodopsin has been incorporated in the cytoplasmic membrane of Streptococcus cremoris, in order to generate a protonmotive force across these membranes. This has been achieved by fusion of S. cremoris membrane vesicles with bacteriorhodopsin proteoliposomes. This fusion occurred when both preparations were mixed at low pH (less than 6.0), as shown by sucrose density gradient centrifugation and by dilution of fluorescent phospholipids incorporated into the bacteriorhodopsin proteoliposomes. Fusion was strongly enhanced by the presence of negatively charged phospholipids in the liposomal bilayer. When proteoliposomes were used that showed light-dependent proton uptake, the orientation of bacteriorhodopsin in the fused membranes was inside-out with respect to the in vivo orientation in Halobacterium halobium. Consequently, in the light a ΔΨ, interior positive and a ΔpH, interior acid were generated. This protonmotive force could drive calcium uptake in the fused membranes. The uptake increased hyperbolically with increasing light intensity and was abolished by bleaching of bacteriorhodopsin. Addition of the ionophore valinomycin stimulated calcium uptake and led to an increase of the ΔpH. Calcium uptake was strongly decreased in the dark and in the light in the presence of uncouplers, nigericin or both valinomycin and nigericin.

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg 2+-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca 2+-ATPase activity of the soluble coupling factor from chromatophores ( R. rubrum F 1) was inactivated by NBD-Cl with kinetics resembling those described for Mg 2+-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD- R. rubrum F 1 fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca 2+-ATPase activity of R. rubrum F 1 were fully protected by 5 mM P i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca 2+-ATPase activity by P i indicated that NBD-Cl and P i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F 1 underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.

Reconstitution of delipidated bacteriorhodopsin with endogenous polar lipids.

Delipidated bacteriorhodopsin has been reconstituted with endogenous polar lipids from Halobacterium halobium. The vesicle (diameter, 250-500 A) formed are very stable, relatively homogeneous in bacteriorhodopsin and lipid content, and almost optically clear; a minor turbid fraction can be separated by gel filtration. Bacteriorhodopsin in the reconstituted vesicles has an inside out orientation and, on illumination, translocates protons efficiently from the medium to the interior of the vesicles in the presence of the ionophore valinomycin. In the absence of the latter, both the rate and the extent of light-dependent proton uptake by the vesicles are decreased 3-6- and 5-15-fold, respectively, depending on the salt in the assay medium. Both the stimulation by valinomycin and the proton-translocating activity are higher in NaCl than in KCl. Bacteriorhodopsin in these vesicles as in purple membrane, undergoes light adaptation as indicated by a red shift (7-8 nm) of the absorption maximum. At low pH, the absorption maximum of reconstituted protein shows a 50-nm red shift, possibly due to protonation of an ionizable group which interacts with the chromophore. The latter group appears to be accessible only from the external medium.

Quercetin interaction with the chloroplast ATPase complex

1. Quercetin, a flavonoid which acts as an energy transfer inhibitor in photophosphorylation is shown to inhibit the P-ATP exchange activity of membrane-bound CF 1 and the ATPase activity of isolated CF 1. Quercetin, affects also the proton uptake in chloroplasts in a manner similar to that of dicyclohexyl-carbodiimide. 2. The light-dependent proton uptake in EDTA-treated chloroplasts is stimulated by quercetin. In untreated chloroplasts quercetin has a dual effect: it enhances at pH above 7.5 while at lower pH values it decreases the extent of H + uptake. Similar effects were obtained with dicyclohexylcarbodiimide. 3. Like quercetin, dicyclohexylcarbodiimide was also found to inhibit the ATPase activity of isolated CF 1. 4. Quercetin inhibits uncoupled electron transport induced by either EDTA-treatment of chloroplasts or by addition of uncouplers. Quercetin restores H + uptake in both types of uncoupled chloroplasts. 5. The mode of action of quercetin and dicyclohexylcarbodiimide in photo-phosphorylation is discussed, and interaction with both CF 1 and F 0 is suggested.

ATP synthesis linked to light-dependent proton uptake in a red mutant strain of Halobacterium lacking bacteriorhodopsin

Intact cells of Halobacterium halobium R 1mR, a red strain deficient in bacteriorhodopsin, pumped protons only in an inward direction when illuminated, in contrast to R 1 cells which showed proton transfer in both directions. The cellular ATP level of R 1mR, as well as R 1 cells, under nitrogen, was increased upon illumination to the aerobic level. The proton uptake and ATP synthesis observed with both R 1 and R 1mR occurred even after the majority of bacteriorhodopsin (in R 1) had been bleached with NH 2OH, but they were abolished by brief heat treatment of the cells. These results suggest a mechanism common to both strains which is responsible for the observed proton uptake and ATP synthesis. When R 1mR cells were grown in the presence of nicotine, an inhibitor of carotenoid biosynthesis, both the proton uptake and ATP synthesis were completely depressed, but recovered after all-trans retinal was added externally. The action spectrum by R 1mR, NH 2OH-treated R 1, or nicotinegrown R 1mR reconstituted with retinal consistently exhibited a maximum between 580 and 600 nm. Hence the mechanism is independent of bacteriorhodopsin of the purple membrane. Instead, our results indicate the possible presence of a new chromoprotein which involves retinal as the chromophore and participates in the mechanism of cellular ATP synthesis.

Alterations in Chloroplast Thylakoids during an in Vitro Freeze-Thaw Cycle

Plastocyanin and chloroplast coupling factor 1 (CF(1)) are released from spinach (Spinacia oleracea L.) thylakoids during a slow freezethaw cycle. CF(1) addition increases the proton uptake of thylakoids previously frozen in sucrose concentrations of 15 mm to 100 mm. Addition of CF(1) and plastocyanin restores the proton uptake of thylakoids frozen in 100 mm sucrose. Plastocyanin and CF(1) release is a manifestation, not the cause, of freeze-thaw damage.Frozen-thawed thylakoids appear to exhibit two levels of response to sucrose as measured by light-dependent proton uptake. Different levels of protection afforded by sucrose may be due, in part, to quantitative differences in CF(1) release. The results suggest at least three freeze-induced lesions in light-dependent proton uptake by thylakoids: plastocyanin release, CF(1) release, and disruption of the semi-permeability of thylakoids.

Alterations in Chloroplast Thylakoids during Cold Acclimation

Freeze-fracture electron microscopy reveals a decreased particle concentration on the inner fracture face of acclimated thylakoids, suggestive of some alteration(s) in the hydrophobic region. Sonic oscillation causes a reversal of the altered particle concentration in acclimated thylakoids and suggests that increases in unsaturation of fatty acids can, at most, account for only part of the altered particle concentration. The particles on the inner fracture face of acclimated thylakoids are of one size group (+/- 140 A) as compared to two size groups (+/- 100 A and +/- 165 A) for nonacclimated thylakoids. The paracrystalline array might be associated with the acclimated state of thylakoids. Nonacclimated thylakoids require 50 mm sucrose for maximum protection of light-dependent proton uptake, while acclimated thylakoids require 25 mm sucrose, and the protection afforded acclimated thylakoids during a freeze-thaw cycle is greater. Sucrose is required for alterations in acclimated thylakoids to be manifested. Apparently increased hardiness is not only associated with changes in cellular environment but also alterations in membranes.

Changes in chlorophyll fluorescence in relation to light-dependent cation transfer across thylakoid membranes

Based on cation effects on chlorophyll a fluorescence and the light scattering behaviour of chloroplasts, a new interpretation of energy-dependent fluorescence quenching in intact leaves and isolated spinach chloroplasts is given. This type of fluorescence quenching is suggested to reflect movement of Mg 2+ and other cations from the thylakoids to the stroma compartment. Cation efflux processes are associated with light-dependent proton uptake by thylakoids. Since cations strongly increase the fluorescence yield, their efflux leads to fluorescence lowering, apparently by means of structural changes of the membrane system. Similarly, the light-induced increase of apparent absorbance at 535 nm (caused by increased light scattering), which parallels fluorescence quenching, may reflect structural changes due to cation efflux from the thylakoids. In the dark these processes are reversed. The following results support this view: 1. 1. After the envelopes of intact chloroplasts had been ruptured by osmotic shock in a medium of low cation content, the fluorescence yield was drastically lowered, and the long-term fluorescence quenching, as well as the light-dependent absorbance increase were missing. This is understood as being caused by loss of cations, which had been retained within the envelope. 2. 2. Addition of certain cations to these thylakoid preparations largely restored the fluorescence signal characteristic of intact chloroplasts. 3. 3. A dark period of 2–3 min in the presence of cations was required to produce the maximum fluorescence response. 4. 4. When chloroplasts were ruptured in the presence of 5 mM MgCl 2, both the signals of fluorescence and apparent absorbance at 535 nm remained very similar to those of the intact chloroplasts. 5. 5. The described cation-dependent phenomena are sensitive to FCCP and closely correlated with light-induced proton uptake into the thylakoids, thus showing a relation to the energy conserving mechanism of photosynthesis.

Biogenesis of chloroplast membranes IX. Development of photophosphorylation and proton pump activities in greening Chlamydomonas reinhardi y-1 as measured with an open-cell preparation

Chlamydomonas reinhardi y-1 cells are rendered permeable to substrates and cofactors for photosynthetic electron transfer and photophosphorylation by applying low shearing forces in a hypertonic viscous medium. The chloroplast integrity is well preserved in such open-cell preparations which exhibit high and stable photophosphorylation rates for both photosystems and proton pump activity. These activities were measured during greening of dark-grown y-1 cells using open-cell preparations. It was found that photophosphorylation and light-dependent proton uptake are absent in preparations obtained from cells grown in the dark for 5–6 generations which still contained 1–2 μg chlorophyll per 10 7 cells. The activities became measurable following exposure of the cells to the light and increased rapidly reaching a maximal rate per chlorophyll unit 3–4 times higher than that exhibited by light-grown cells after 2–3 h of illumination when the chlorophyll content of the cells has increased only slightly. As chlorophyll continues to accumulate the activity per chlorophyll unit declined and became equal to that usually found in light grown cells. Photosynthetic ATP formation and pH rise do not develop in cells greening in the presence of chloramphenicol which specifically inhibits the synthesis of chloroplast made proteins. Chloroplast membranes formed in the presence of chloramphenicol which are enriched in proteins of cytoplasmic origin resume the light-dependent proton pump activity and photophosphorylation by both photosystems if the cells are further incubated in absence of chloramphenicol. The repair of the inactive membranes requires synthesis of proteins within the chloroplast and does not require light and additional synthesis of chlorophyll or proteins of cytoplasmic origin.

Inactivation of energy-linked functions of chloroplasts by polyanions at low pH

Various anions when present at low concentrations in the acid stage of an acid-base phosphorylation experiment cause severe inhibition of subsequent ATP formation. Polyvalent anions, such as heparinate, polygalacturonate, polyvinylsulfate, and ferri- or ferrocyanide are most effective; sulfate is much less effective and chloride ions the least so. Brief pretreatment with polyanions at low pH also inhibits Mg-dependent ATPase of chloroplasts activated by acid-base transition, and light-dependent proton uptake and photophosphorylation supported either by cyclic or noncyclic electron flow. A similar sensitivity of the Ca-dependent ATPase activity of solubilized chloroplast coupling factor, and preliminary evidence for its aggregation under these conditions, suggests denaturation of this enzyme as one basis for the action of polyanions. Further evidence indicates that the coupling factor, before or after denaturation, is detached from the chloroplast thylakoid membranes.

Freezing Injury and Uncoupling of Phosphorylation from Electron Transport in Chloroplasts

Freezing of chloroplast membranes uncouples photophosphorylation from electron transport and inactivates the light-dependent and thiol-requiring ATPase, conformational changes and the light-dependent proton uptake. All of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to photophosphorylation is demonstrated by the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere with the protection afforded by sucrose.In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloroplast coupling factor, which is released from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by freezing.The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a structure involved in the formation of a non-phosphorylated high energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the permeability properties of chloroplast membranes-frozen membrane vesicles no longer function as osmometers-it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH gradient across the vesicle membranes owing to proton leakage through the membranes. From the results, the basic injury caused by freezing appears to consist in the alteration of permeability properties of biological membranes due to the dehydration which accompanies freezing.