Ion Relations of Symplastic and Apoplastic Space in Leaves from Spinacia oleracea L. and Pisum sativum L. under Salinity

Salt tolerant spinach (Spinacia oleracea) and salt sensitive pea (Pisum sativum) plants were exposed to mild salinity under identical growth conditions. In order to compare the ability of the two species for extra- and intracellular solute compartmentation in leaves, various solutes were determined in intercellular washing fluids and in aqueously isolated intact chloroplasts. In pea plants exposed to 100 millimolar NaCl for 14 days, apoplastic salt concentrations in leaflets increased continuously with time up to 204 (Cl⁻) and 87 millimolar (Na⁺), whereas the two ions reached a steady concentration of only 13 and 7 millimolar, respectively, in spinach leaves. In isolated intact chloroplasts from both species, sodium concentrations were not much different, but chloride concentrations were significantly higher in pea than in spinach. Together with data from whole leaf extracts, these measurements permitted an estimation of apoplastic, cytoplasmic, and vacuolar solute concentrations. Sodium and chloride concentration gradients across the tonoplast were rather similar in both species, but spinach was able to maintain much steeper sodium gradients across the plasmamembrane compared with peas. Between day 12 and day 17, concentrations of other inorganic ions in the pea leaf apoplast increased abruptly, indicating the onset of cell disintegration. It is concluded that the differential salt sensitivity of pea and spinach cannot be traced back to a single plant performance. Major differences appear to be the inability of pea to control salt accumulation in the shoot, to maintain steep ion gradients across the leaf cell plasmalemma, and to synthesize compatible solutes. Perhaps less important is a lower selectivity of pea for K⁺/Na⁺ and NO₃⁻/Cl⁻ uptake by roots.     

Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO₃ ⁻–N, NH₄ ⁺–N, NH₄NO₃). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO₃ ⁻–N) or mixed (NH₄NO₃) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO₃ ⁻–N or NH₄NO₃. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO₃ ⁻-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH₄ ⁺–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH₄ ⁺–N the enzyme activity in roots was even higher than in those supplied with NO₃ ⁻–N.     

Boron induces hyperpolarization of sunflower root cell membranes and increases membrane permeability to k

Although many studies have alluded to a role for boron (B) in membrane function, there is little evidence for a direct effect of B on the plasmalemma of higher plant cells. These studies were conducted to demonstrate, by electrophysiological techniques, a direct effect of B on the membrane potential (E_m) of sunflower (Helianthus annuus [L.], cv Mammoth Grey Stripe) root tip cells and to determine if the response to B occurs rapidly enough to account for the previously observed effects of B on ion uptake. By inserting a glass microelectrode into an individual cell in the root tip, the E_m of the cell was determined in basal salt medium (BSM), pH 6.0. The perfusion solution surrounding the root tissue was then changed to BSM + 50 micromolar H₃BO₃, pH 6.0. The exposure to B induced a significant plasmalemma hyperpolarization in sunflower root cells within 20 minutes. After just 3 minutes of exposure to B, the change in E_m was already significantly different from the negligible change in E_m observed over time in root cells never exposed to B. Membrane hyperpolarization could be caused by a stimulation of the proton pump or by a change in the conductance of one or more permeable ions. Since B has been shown to affect K⁺ uptake by plants, the electrophysiological techniques described above were used to determine if B has an effect on membrane permeability to K⁺, and could thereby lead to an increased diffusion potential. When sunflower root tips were pretreated in 50 micromolar B for 2 hours, cell membranes exhibited a significantly greater depolarization with each 10-fold increase in external [K⁺] than minus-B cells. Subsequent studies demonstrated that the depolarization due to increased external [K⁺] was also significantly greater when tissue was exposed to B at the same time as the 10-fold increase in [K⁺], indicating that the effect of B on K⁺ permeability was immediate. Analysis of sunflower root tips demonstrated that treatment in 50 micromolar B caused a significantly greater accumulation of K⁺ after 48 hours. The B-induced increase in K⁺ uptake may cause a subsequent stimulation of the H⁺-ATPase (proton pump) and lead to the observed hyperpolarization of root cell membranes. Alternatively, B may stimulate the proton pump, with the subsequent hyperpolarization resulting in an increased driving force for K⁺ influx.     

Photosynthesis and ion content of leaves and isolated chloroplasts of salt-stressed spinach

Spinach (Spinacia oleracea) plants were subjected to salt stress by adding NaCl to the nutrient solution in increments of 25 millimolar per day to a final concentration of 200 millimolar. Plants were harvested 3 weeks after starting NaCl treatment. Fresh and dry weight of both shoots and roots was decreased more than 50% compared to control plants but the salt-stressed plants appeared healthy and were still actively growing. The salt-stressed plants had much thicker leaves. The salt-treated plants osmotically adjusted to maintain leaf turgor. Leaf K⁺ was decreased but Na⁺ and Cl⁻ were greatly increased.

The potential photosynthetic capacity of the leaves was measured at saturating CO₂ to overcome any stomatal limitation. Photosynthesis of salt-stressed plants varied only by about 10% from the controls when expressed on a leaf area or chlorophyll basis. The yield of variable chlorophyll a fluorescence from leaves was not affected by salt stress. Stomatal conductance decreased 70% in response to salt treatment.

Uncoupled rates of electron transport by isolated intact chloroplasts and by thylakoids were only 10 to 20% below those for control plants. CO₂-dependent O₂ evolution was decreased by 20% in chloroplasts isolated from salt-stressed plants. The concentration of K⁺ in the chloroplast decreased by 50% in the salt-stressed plants, Na⁺ increased by 70%, and Cl⁻ increased by less than 20% despite large increases in leaf Na⁺ and Cl⁻.

It is concluded that, for spinach, salt stress does not result in any major decrease in the photosynthetic potential of the leaf. Actual photosynthesis by the plant may be reduced by other factors such as decreased stomatal conductance and decreased leaf area. Effective compartmentation of ions within the cell may prevent the accumulation of inhibitory levels of Na⁺ and Cl⁻ in the chloroplast.

     

In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots

The plasmalemma vesicles isolated from cucumber and maize roots were used to study the effect of Cu²⁺ and Cd²⁺ on the hydrolytic and proton pumping activities of ATPase. In vivo application of metal ions to the plant growth solutions resulted in stimulation of the proton transport in maize. In cucumber roots the action of metals was not the same: cadmium stimulated the H⁺ transport through plasmalemma whereas Cu²⁺ almost completely inhibited it. Copper ions decreased the hydrolytic activity of H⁺-ATPase in cucumber, without any effect on this activity in membranes isolated from maize roots. The effect of cadmium on the hydrolytic activities was opposite: ATP-hydrolysis activity in plasmalemma was not altered in cucumber, whereas in maize its stimulation was observed. The amount of accumulated metals was not the main reason of different influence of metals on H⁺-ATPase activity in tested plants. In in vitro experiments Cu²⁺ inhibited H⁺ transport in the cucumber, to a higher degree than Cd²⁺ and both metals did not change this H⁺-ATPase activity of plasmalemma isolated from corn roots. Cu²⁺ added into the incubation medium reduced the hydrolytic activity of ATPase in the plasma membrane isolated from cucumber as well as from corn roots. Cd²⁺ diminished the hydrolytic activity of ATPase in cucumber, and no effect of Cd²⁺ in the plasmalemma isolated from corn roots was found. Our results indicated different in vitro and in vivo action of both metals on H⁺-ATPase and different response of this enzyme to Cu²⁺ and Cd²⁺ in maize and cucumber.     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Isolation and bicarbonate transport of chloroplast envelope membranes from species of differing net photosynthetic efficiency

A three-phase discontinuous sucrose gradient yielded two fractions of chloroplast envelope membranes from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L., mesophyll and undifferentiated chloroplasts). These species were selected to represent plants with fast photorespiration and slow net photosynthesis, fast photorespiration yet fast net photosynthesis, and slow photorespiration and fast net photosynthesis, respectively. Buoyant densities were 1.08 and 1.11 g cm^-3. The light fraction contained primarily single (incomplete) membrane vesicles and the heavy fraction double (complete) ones. Enzymic, chemical, and electron microscopic examination of the complete envelope membranes showed a lack of microbial, microsomal, mitochondrial, and lamellar membrane contamination as well as stromal contamination. Envelope membranes for all species examined were found to contain 2 to 4% of the total chloroplast protein and yields of about 0.2 to 0.4 mg of protein were obtained from 40 g leaves. An Mg²⁺-dependent nonlatent ATPase, a marker enzyme for chloroplast envelope membranes, had the following activities (μmoles of phosphate released/hr^-1 mg protein^-1): spinach, 77; sunflower, 163; old maize, 126; and young maize, 87. Bicarbonate transport was directly correlated with levels of ATPase activity in spinach and sunflower envelope membranes. Transport of HCO₃⁻ with sunflower envelope membranes approached that of young maize.     

Lipid and Fatty Acid composition of chloroplast envelope membranes from species with differing net photosynthesis

Lipid and fatty acid compositions were determined for chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) leaves. The lipid composition was similar in sunflower, spinach, and undifferentiated maize chloroplast envelope membranes and different in maize mesophyll chloroplast envelope membranes. The predominant lipid constituents in all envelope membranes were monogalactosyldiglyceride (27 to 46%), digalactosyldiglyceride (18 to 33%), and phosphatidylcholine (7 to 30%). The fatty acid composition was also similar in sunflower and spinach chloroplast envelope membranes in comparison to those from maize. The major acyl fatty acids of the chloroplast envelope membrane were palmitic (C_16:0, 41 and 36%) and linolenic (C_18:3, 29 and 40%) acids for spinach and sunflower; palmitic (77%) and stearic (C_18:0, 12%) acids for young maize; and palmitic (61%), stearic (14%), and linolenic (13%) acids for mature maize. The differences in lipid and acyl fatty acid compositions among these plants which vary in their rates of net photosynthesis were largely quantitative rather than qualitative.     

The strategy of Na+ compartmentation and growth of Atriplex centralasiatica in adaptation to saline environments

In this study, we investigated the adaptation strategy employed by Atriplex centralasiatica Iljin in response to high salinity. When grown in high saline environments (100–200 mM NaCl), A. centralasiatica plants were larger and more succulent. This increased growth and water uptake was correlated with a large and specific cellular accumulation of sodium, demonstrating that in A. centralasiatica Na⁺ is beneficial rather than toxic. More than 95% of Na⁺ absorbed by salt-treated A. centralasiatica plants accumulated in shoots, especially in leaves; approximately 98% of Na⁺ that accumulated in leaves was localized in leaf protoplasts, a situation that was responsible for the decreased photosynthetic rate observed with increasing salt concentration. Because of the greater leaf area per plant found under saline conditions, no reduction in biomass of individual plants was observed. Measurements on isolated tonoplast-enriched membrane vesicles derived from the leaves of A. centralasiatica revealed increased V-H⁺-ATPase hydrolytic activity and V-H⁺-ATPase proton pump activity in salt-treated leaves compared with controls. These results suggest that, as an adaptation to saline environments, A. centralasiatica can efficiently sequester Na⁺ into vacuoles, thereby increasing leaf area to maintain its CO₂ assimilation capabilities.     

Protein kinase activities in tonoplast and plasmalemma membranes from corn roots

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H⁺-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K_m value for MgATP²⁻ of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg²⁺ and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca²⁺, but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H⁺-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.     

Aquaporin Content in Cell Membranes of Mesembryanthemum crystallinum as Affected by Plant Transition from C3 to CAM Type of Photosynthesis

Western-blot analysis was used to determine the contents of aquaporin isoforms MIP A, MIP B, and MIP C in cell membranes isolated from roots and leaves of Mesembryanthemum crystallinum plants with C₃ and Crassulacean acid metabolism (CAM) types of photosynthesis. These membrane preparations were also used to assess osmotic water permeability; to this end, the rate of osmotic vesicle shrinking was registered as the light scattering intensity by the method of stopped flow. The cell membranes represented by the plasmalemma and the tonoplast-enriched fraction were obtained by separating the microsomes in a two-phase polymer system. Plant transition from C₃ to CAM-photosynthesis occurred in the course of plant development or was induced by salinization. All three isoforms under study were found in the plasma membranes of roots and leaves of the C₃ plants, whereas in the CAM plants, independent of the transition-inducing factor, the aquaporin contents notably decreased in the leaf membranes and remained unchanged in the roots. In the membranes isolated from roots and leaves of the C₃ plants, the values of osmotic water permeability exceeded two–threefold the corresponding indices characteristic of the CAM plants. The authors believe that aquaporin isoforms in M. crystallinum are under the organ- and tissue-specific control.     

Characterisation of a plasma membrane-associated phospholipase A2 activity increased in response to cold acclimation

We here demonstrate the presence of a plasma membrane-associated phospholipase A₂ (EC 3.1.1.4; PLA₂) activity in spinach (Spinacia oleracea) leaves. The pH profile of the spinach plasma membrane PLA₂ activity revealed two peaks, one at pH 4.4 and one at pH 5.5. The activity at pH 5.5 had an absolute requirement of Ca²⁺, with full enzyme activity at 10 μmol/L Ca²⁺. The Ca²⁺-dependent PLA₂ activity was both heat sensitive and stimulated by diacylglycerol, whereas ATP completely inhibited the activity. Thus, the spinach plasma membrane contains a Ca²⁺-dependent PLA₂ activity, which has not previously been characterised in plants. Cold acclimation of spinach resulted in a 2.2-fold higher plasma membrane PLA₂ activity whereas the plasma membrane phospholipase D activity remained unaffected. Taken together, our data suggest a role of PLA₂ in cold acclimation in plants.     

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: comparison of reconstituted vesicles with native membrane vesicles

A Na⁺-specific and Na⁺-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na⁺ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K⁺, Li⁺, and choline⁺. The electrochemical effect of the lipophilic anion, SCN^?, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl^? or SO₄^2?. The Na⁺-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K_m of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na⁺-specific and Na⁺-stimulated concentrative α-aminoisobutyric acid uptake.     

K stimulation of ATPase activity associated with the chloroplast inner envelope

Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg²⁺ and Mg·ATP complex effects on ATPase activity revealed that any Mg²⁺ stimulation of activity was likely a function of the use of the Mg·ATP complex as a substrate by the enzyme; free Mg²⁺ may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K⁺. This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K⁺. K⁺ stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K⁺ effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the K_m increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K⁺-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K⁺ effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H⁺-ATPase). Such ATPases are thought to be indirectly involved in active K⁺ uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K⁺ effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo.     

Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes

The association of K⁺-stimulated, Mg²⁺-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg²⁺-ATPase activity to stimulation by K⁺ and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg²⁺-ATPase activity was stimulated up to 700% by addition of K⁺. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg²⁺. Ca²⁺ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO₃ and about 40% by diethylstilbestrol.     

Electron donation to photosystem I

Electron donation to photosystem I was studied in highly resolved particles from spinach. Divalent cations increased the efficiency of electron donation from spinach plastocyanin to P700⁺ through a decrease in the apparent K_m for plastocyanin. Cytochrome f was not an efficient electron donor for P700⁺ in the presence or absence of divalent cations. Cytochrome f photooxidation could be observed in the presence of both plastocyanin and divalent cations.     

Buffer capacities of leaves, leaf cells, and leaf cell organelles in relation to fluxes of potentially acidic gases

Since environmental pollution by potentially acidic gases such as SO₂ causes proton release inside leaf tissues, homogenates of needles of spruce (Picea abies) and fir (Abies alba) and of leaves of spinach (Spinacia oleracea) and barley (Hordeum vulgare) were titrated and buffer capacities were determined as a function of pH. Titration curves of barley leaves were compared with titration curves of barley mesophyll protoplasts. From the protoplasts, chloroplasts and vacuoles were isolated and subjected to titration experiments. From the titration curves, the intracellular distribution of buffering capacities could be deduced. Buffering was strongly pH-dependent. It was high at the extremes of pH but still significant close to neutrality. Owing to its large size, the vacuole was mainly responsible for cellular buffering. However, on a unit volume basis, the cytoplasm was much more strongly buffered than the vacuole. Potentially acidic gases are trapped in the anionic form. They release protons when trapped. The magnitude of diffusion gradients from the atmosphere into the cells, which determines flux, depends on intracellular pH. In the light, the chloroplast stroma, as the most alkaline leaf compartment, has the highest trapping potential. Acidification of the chloroplast stroma inhibits photosynthesis. The trapping potential of the chloroplast is followed by that of the cytosol. Compared with the cytoplasm, the vacuole possesses little trapping potential in spite of its large size. It is particularly small in the acidic vacuoles of conifer needles. In the physiological pH range (slightly above neutrality), chloroplast buffering was about 1 microequivalents H⁺ per milligram chlorophyll per pH unit or 35 microequivalents H⁺ per milliliter per pH unit in barley or spinach chloroplasts. This compares with SO₂-generated H⁺ production of somewhat more than 1 microequivalent H⁺ per milligram chlorophyll per hour, which results from observed SO₂ uptake of leaves when stomata were open and the atmospheric SO₂ concentration was 0.4 microliters per liter (GE Taylor Jr, DT Tingey 1983 Plant Physiol 72: 237-244). At lower SO₂ concentrations, similar H⁺ generation inside the cells requires correspondingly longer exposure times.