The pH of the medium during CO2 uptake into the intracellular inorganic carbon (Ci) pool of a high CO2-requiring mutant (E1) and wild type of Anacystis nidulans R2 was measured. Experiments were performed under conditions where photosynthetic CO2 fixation is inhibited. There was an acidification of the medium during CO2 uptake in the light and an alkalization during CO2 efflux after darkening. A one to one stoichiometry existed between the amounts of H+ appearing in the medium and CO2 taken up into the intracellular Ci pool, regardless of the carbon species transported. The results indicate that (a) CO2 is taken up simultaneously with an efflux of equimolar H+, probably produced as a result of CO2 hydration during transport and (b) HCO3− produced by hydration of CO2 in the medium was transported into the cells without accompanying net flux of H+ or OH−. The influx and efflux of Ci during Ci transport produced nonequilibrium between CO2 and HCO3− in the medium, with the concentration of HCO3− being higher than that expected under equilibrium conditions. The nonequilibrium was present even under the conditions where the influx of Ci is compensated by its efflux. The direction of this nonequilibrium suggested that efflux of HCO3− occurs during uptake of Ci.
Potassium ferricyanide (K3Fe[CN]6) was added to aerated and stirred nonbuffered suspensions of mechanically isolated photosynthetically competent Asparagus sprengeri Regel mesophyll cells. Rates of Fe(CN)63− reduction and H+ efflux were measured with or without illumination. On the addition of 1 millimolar Fe(CN)63− to nonilluminated cell suspensions acidification of the medium indicated an H+ efflux of 1.54 nanomoles H+/106 cells per minute. Simultaneous Fe(CN)63− reduction occurred at a rate of 1.55 nanomoles Fe(CN)63−/106 cells per minute. Illumination stimulated these rates 14 to 17 times and corresponding values were 26.1 nanomoles H+/106 cells per minute and 22.9 nanomoles Fe(CN)63−/106 cells per minute. These two processes appeared to be tightly coupled and were rapidly inhibited when illuminated suspensions were transferred to darkness or treated with 1 micromolar 3-(3,4-dichlorophenyl)-1,1 dimethylurea. Addition of 0.1 millimolar diethylstilbestrol eliminated ATP dependent H+ efflux in illuminated or nonilluminated cells but had no influence on Fe(CN)63− dependent H+ efflux. Recent reports indicate that a transmembrane redox system spans the plasma membrane of root cells and is coupled to the efflux of H+. The present report extends these observations to photosynthetically competent mesophyll cells. The results indicate a transport process independent of ATP driven H+ efflux which operates with a H+/e− stoichiometry of one.
A carotenoid-associated membrane protein was isolated from Anacystis nidulans R2 thylakoids. Sodium pyrophosphate and sodium bromide washed thylakoids were solubilized with the nonionic detergents dodecyl-β-D-maltoside and octyl-β-D-glucopyranoside, and these detergent extracts were fractionated on a sucrose density gradient. A yellow fraction from the sucrose gradient was further purified by anion-exchange and organomercuric-affinity column chromatography to yield a fraction virtually free of chlorophyll and highly enriched in both carotenoids and a 42 kilodalton polypeptide. Evidence presented in this paper suggests that the carotenoid-containing 42 kilodalton protein is thylakoid associated rather than cytoplasmic membrane associated. The purified 42 kilodalton polypeptide was used to raise polyclonal antibodies in rabbits. Immuno-chemical detection of the 42 kilodalton polypeptide on Western blots demonstrated an increased accumulation of this polypeptide in cells grown under high-light conditions relative to cells grown under low light.
The irradiance dependence of the efficiencies of photosystems I and II were measured for two pea (Pisum sativum [L.]) varieties grown under cold conditions and one pea variety grown under warm conditions. The efficiencies of both photosystems declined with increasing irradiance for all plants, and the quantum efficiency of photosystem I electron transport was closely correlated with the quantum efficiency of photosystem II electron transport. In contrast to the consistent pattern shown by efficiency of the photosystems, the redox state of photosystem II (as estimated from the photochemical quenching coefficient of chlorophyll fluorescence) exhibited relationships with both irradiance and the reduction of P-700 that varied with growth environment and genotype. This variability is considered in the context of the modulation of photosystem II quantum efficiency by both photochemical and nonphotochemical quenching of excitation energy.
Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H+/Ca2+ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H+/Ca2+ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H+/Ca2+ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH4Cl gradient (NH4+in » NH4+out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the Km for calcium (∼15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H+/Ca2+ antiporter has been reconstituted in active form.
Acclimation of the photosynthetic apparatus to light absorbed primarily by photosystem I (PSI) or by photosystem II (PSII) was studied in the unicellular red alga Porphyridium cruentum (ATCC 50161). Cultures grown under green light of 15 microeinsteins per square meter per second (PSII light; absorbed predominantly by the phycobilisomes) exhibited a PSII/PSI ratio of 0.26 ± 0.05. Under red light (PSI light; absorbed primarily by chlorophyll) of comparable quantum flux, cells contained nearly five times as many PSII per PSI (1.21 ± 0.10), and three times as many PSII per cell. About 12% of the chlorophyll was attributed to PSII in green light, 22% in white light, and 39% in red light-grown cultures. Chlorophyll antenna sizes appeared to remain constant at about 75 chlorophyll per PSII and 140 per PSI. Spectral quality had little effect on cell content or composition of the phycobilisomes, thus the number of PSII per phycobilisome was substantially greater in red light-grown cultures (4.2 ± 0.6) than in those grown under green (1.6 ± 0.3) or white light (2.9 ± 0.1). Total photosystems (PSI + PSII) per phycobilisome remained at about eight in each case. Carotenoid content and composition was little affected by the spectral composition of the growth light. Zeaxanthin comprised more than 50% (mole/mole)...
To clarify the kinetic characteristics and ionic requirements of the tonoplast H+-translocating inorganic pyrophosphatase (H+-PPiase), PPi hydrolysis and PPi-dependent H+ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H+-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H+-PPiase activity was maximal in the presence of K+ (K50 approximately 3 millimolar), with PPi-dependent H+ transport being more selective for K+ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H+-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg2+ for activation. At saturating concentrations of free Mg2+, H+-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi2− but not Mg2PPi, demonstrating that MgPPi2− was the true substrate of the enzyme. The apparent Km (MgPPi2−) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H+ transport (7 micromolar). Free Mg2+ was shown to be an allosteric activator of the H+-PPiase...
A pressure chamber technique was used to study the root uptake and xylem translocation of nonradiolabeled cinmethylin and its analogs in detopped soybean (Glycine max) roots. Quantifications of compounds were achieved by gas chromatography analysis using a mass spectrometry detector under selected ion monitoring. The compounds tested, with octanol-water partition coefficients (log Kow values) ranging from 0.96 to 5.3, were all nonionizable under the experimental conditions. Root efflux curves of all compounds exhibited a steady-state kinetic profile. The time required to achieve the steady state efflux concentration in the xylem sap correlated with log Kow values in a manner very similar to the root binding profile reported previously by GG Briggs et al. ( Pestic Sci 13: 495-504). After reaching the steady state efflux, the concentration ratio of each compound in the xylem sap to the final concentration in the pressure chamber was taken as the transpiration stream concentration factor (TSCF). A nonlinear relationship was observed between TSCF and log Kow values. The highest TSCF value was between 0.6 to 0.8 for compounds with log Kow between 2.5 to 3.5. The range of optimal log Kow values was slightly higher than that reported earlier by Briggs et al. ( Pestic Sci 13: 495-504). After taking into account the binding of the compound to soil...
Effects of NO2−, ClO3−, and ClO2− on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO3− influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m−3 NO2− fully induced NO3− transport but failed to induce NRA. Similar pretreatments with ClO3− and ClO2− induced neither NO3− transport nor NRA. Net ClO3− uptake was induced by NO3− but not by ClO3− itself, indicating that NO3− and ClO3− transport occur via the NO3− carrier. At the uptake step, NO2− and ClO2− strongly inhibited NO3− influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO3− proved to be a weak inhibitor of NO3− influx (Ki = 16 mol m−3) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO3− analogs as screening agents for the isolation of mutants defective in NO3− transport.
The absorption of K+ by excised roots of barley (Hordeum vulgare L. cv California Mariout) has been systematically compared with that of entire, undisturbed seedlings. Some experiments have also been done with wheat (Triticum aestivum L.) and an amphiploid obtained from a cross between it and salt-tolerant tall wheatgrass (Lophopyrum elongatum Host Löve [syn. Agropyron elongatum Host]). For all three genotypes, the rate of K+ absorption measured in a 20-min period was identical for entire 8-d-old seedlings and their excised roots within the experimental error. Manipulation gentler than root excision, viz. careful transfer of seedlings from one experimental solution to another, was also without effect on the rate of K+ absorption. Absorption of K+ measured by assay of its 86Rb label in the tissue was identical with that measured by K+ depletion of the experimental solutions assayed chemically. For the plant materials and conditions of these experiments, the excised root technique for studying ion transport into roots is validated. The advantages of the technique, and findings differing from the present ones, are discussed.
Triazine-resistant and -susceptible Brassica napus L. plants grown under low photon flux density (PFD) have previously been shown to exhibit a similar photon yield. In contrast, high PFD-grown resistant plants have a lower photon yield than high PFD-grown susceptible plants (JJ Hart, A Stemler  Plant Physiol 94: 1295-1300). In this work we tested the hypothesis that high PFD can induce a differential decrease in photon yield in low PFD-grown plants. We measured photon yield, variable fluorescence/maximum fluorescence, and O2 flash yield in low PFD-grown resistant and susceptible leaf discs before and after exposure to high PFD exposure. The results demonstrated that high PFD exposure results in a greater decrease in photosystem II (PSII) activity in resistant plants. Characteristics of recovery and other evidence suggest that the differential decrease in PSII efficiency in resistant leaf discs is caused by photoinhibitory damage. We propose that the differential reduction in photon yield and photosynthesis often observed in resistant plants is the result of increased sensitivity to photoinhibition.
In maize (Zea mays L., cv Contessa), nitrogen (NO3−) limitation resulted in a reduction in shoot growth and photosynthetic capacity and in an increase in the leaf zeaxanthin contents. Nitrogen deficiency had only a small effect on the quantum yield of CO2 assimilation but a large effect on the light-saturated rate of photosynthesis. Linear relationships persisted between the quantum yield of CO2 assimilation and that of photosystem II photochemistry in all circumstances. At high irradiances, large differences in photochemical quenching and nonphotochemical quenching of Chl a fluorescence as well as the ratio of variable to maximal fluorescence (Fv/Fm) were apparent between nitrogen-deficient plants and nitrogen-replete controls, whereas at low irradiances these parameters were comparable in all plants. Light intensity-dependent increases in nonphotochemical quenching were greatest in nitrogen-deficient plants as were the decreases in Fv/Fm ratio. In nitrogen-deficient plants, photochemical quenching decreased with increasing irradiance but remained higher than in controls at high irradiances. Thermal dissipative processes were enhanced as a result of nitrogen deficiency (nonphotochemical quenching was elevated and Fv/Fm was lowered) allowing PSII to remain relatively oxidised even when carbon metabolism was limited via nitrogen limitation.
A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S  Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.
Low concentrations of oligomycin, which strongly inhibit mitochondrial oxidative phosphorylation but do not affect chloroplast photophosphorylation, caused an inhibition of photosynthesis by 30 to 40% in barley (Hordeum vulgare L.) leaf protoplasts. This inhibition is reversed and the full rate of photosynthesis is regained when the protoplasts are ruptured so as to leave the chloroplasts intact. Oligomycin fed into barley leaves by the transpiration stream inhibited photosynthesis in these leaves by up to 60%. The measurement of metabolites in protoplast and leaf extracts showed that oligomycin caused a decrease in the ATP/ADP ratio and an increase in the content of glucose- and fructose 6-phosphate. Subcellular analysis of protoplasts revealed that the decrease in ATP/ADP ratio in the cytosol was larger than in the stroma and that the increase in hexose monophosphates was restricted to the cytosol, whereas the stromal hexosemonophosphates decreased upon the addition of oligomycin. Moreover, oligomycin caused an increase in the triosephosphate-3-phosphoglycerate ratio. It is concluded from these results that during photosynthesis of a plant leaf cell mitochondrial oxidative phosphorylation contributes to the ATP supply of the cell and prevents overreduction of the chloroplast redox carriers by oxidizing reductive equivalents generated by photosynthetic electron transport.
A NADH dehydrogenase was isolated from an inner membrane-enriched fraction of beetroot mitochondria (Beta vulgaris L.) by solubilization with sodium deoxycholate and purified using gel filtration and affinity chromatography. The NADH dehydrogenase preparation contained a minor ATPase contamination. Beetroot mitochondria were chosen as the isolation material for purifying the enzymes responsible for oxidizing matrix NADH due to the absence of the externally facing NADH dehydrogenase in the variety we have used. The purified NADH dehydrogenase complex catalyzed the reduction of various electron acceptors with NADH as the electron donor, was not sensitive to rotenone inhibition, and had a slow NADPH-ubiquinone 5 reductase activity. The isolated complex contained 14 major polypeptides. It was concluded that the dehydrogenase represented a form of the plant mitochondrial complex I and not the internally facing rotenone-insensitive NADH dehydrogenase found in plant mitochondria because of its complex structure, its cross-reactivity with antisera raised against bovine heart mitochondrial complex I, and the similarity of its kinetics and inhibitor responses to rotenone-sensitive NADH oxidation by beetroot submitochondrial particles.
Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K+ channel blocker, tetraethyl-ammonium+ (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (−60 millivolts) at which K+ channels are inactivated. These results are indicative of FC activation of a H+ pump. FC effects on the magnitude of inward and outward K+ currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H+ ATPase of guard cells is a primary locus for the FC effect on stomatal apertures.
Net fluxes of NH4+ and NO3− into roots of 7-day-old barley (Hordeum vulgare L. cv Prato) seedlings varied both with position along the root axis and with time. These variations were not consistent between replicate plants; different roots showed unique temporal and spatial patterns of uptake. Axial scans of NH4+ and NO3− net fluxes were conducted along the apical 7 centimeters of seminal roots of intact barley seedlings in solution culture using ion-selective microelectrodes in the unstirred layer immediately external to the root surface. Theoretically derived relationships between uptake and concentration gradients, combined with experimental observations of the conditions existing in our experimental system, permitted evaluation of the contribution of bulk water flow to ion movement in the unstirred layer, as well as a measure of the spatial resolution of the microelectrode flux estimation technique. Finally, a method was adopted to assess the accuracy of this technique.
Cucumber plants (Cucumis sativus L.) with incipient Fe deficiency showed increased root capacity to reduce chelated Fe3+ compared to Fe-sufficient plants. When Fe-ethylenediaminete-traacetate was added to the root medium of the Fe-deficient plants, the reductase activity was associated with acidification of the medium and an increase in the net apparent K+ efflux. In the presence of the H+-ATPase inhibitor N,N′-dicyclohexylcarbodiimide the net apparent H+ efflux was completely suppressed, though some reductase activity was preserved, and the net apparent K+ efflux was significantly increased. The inhibition of the reductase activity by N,N′-dicyclohexylcarbodiimide was similar whether the pH of the medium was buffered or not. Anoxia and the protonophore carbonyl cyanide m-chlorophenyl hydrazone also caused a similar inhibition of the reductase activity. It is proposed that this redox system transports electrons only and that its activity is inhibited by plasmamembrane depolarization and anoxia. The H+ and K+ efflux associated with the reductase activity may be a result of the plasmamembrane depolarization it causes.
Using photoacoustic spectroscopy, state 1-state 2 transitions were demonstrated in vivo in intact sugar maple leaves (Acer saccharum Marsh.) by following the changes in energy storage of photosystems (PS) I and II. Energy storage measured with 650 nm modulated light (light 2) in the presence of background white light indicated the total energy stored by both photosystems (ESt), and in the presence of background far-red light showed the energy stored by PSI (ESpsi). The difference between ESt and ESpsi gave the energy stored by PSII (ESpsii). While ESt remained nearly constant during state transitions, both ESpsi and ESpsii changed considerably. The ratio of ESpsii to ESpsi, an indicator of the energy distribution between the two photosystems, decreased or increased during transition to state 2 or state 1, respectively. State transitions were completed in about 20 min and were fully reversible. During transition from state 1 to state 2, the fraction of excitation energy gained by PSI was nearly equal to that lost by PSII. This fraction of excitation energy transferred from PSII to PSI accounted for about 5% of the absorbed light (fluorescence is not considered), 19% of ESt, 34% of ESpsii, and 43% of ESpsi in state 2. NaF treatment inhibited the transition to state 1. Data in the present study confirm the concept of changes in absorption cross-section of photosystems during state transitions.
At low water potential (ψw), dehydration reduces the symplast volume of leaf tissue. The effect of this reduction on photosynthetic capacity was investigated. The influence of osmotic adjustment on this relationship was also examined. To examine these relationships, comparative studies were undertaken on two wheat cultivars, one that osmotically adjusts in response to water deficits (`Condor'), and one that lacks this capacity (`Capelle Desprez'). During a 9-day stress cycle, when water was withheld from plants grown in a growth chamber, the relative water content of leaves declined by 30% in both cultivars. Leaf osmotic potential (ψs) declined to a greater degree in Condor plants. Measuring ψs at full turgor indicated that osmotic adjustment occurred in stressed Condor, but not in Capelle plants. Two methods were used to examine the degree of symplast (i.e. protoplast) volume reduction in tissue rapidly equilibrated to increasingly low ψw. Both techniques gave similar results. With well-watered plants, symplast volume reduction from the maximum (found at high ψw for each cultivar) was the same for Condor and Capelle. After a stress cycle, volume was maintained to a greater degree at low ψw in Condor leaf tissue than in Capelle. Nonstomatally controlled photosynthesis was inhibited to the same degree at low ψw in leaf tissue prepared from well-watered Condor and Capelle plants. However...