Studies on the System Regulating Proton Movement across the Chloroplast Envelope : Effects of ATPase Inhibitors, Mg, and an Amine Anesthetic on Stromal pH and Photosynthesis

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H⁺ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg²⁺ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg²⁺ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg²⁺ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H⁺ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg²⁺ modulated) H⁺ flux linked to monovalent cation antiport, and ATPase-dependent H⁺ efflux.     

Stromal pH and Photosynthesis Are Affected by Electroneutral K and H Exchange through Chloroplast Envelope Ion Channels

Potassium movement across the limiting membrane of the chloroplast inner envelope is known to be linked to counterex-change of protons. For this reason, K⁺ efflux is known to facilitate stromal acidification and the resultant photosynthetic inhibition. However, the specific nature of the chloroplast envelope proteins that facilitate K⁺ fluxes, and the biophysical mechanism which links these cation currents to H⁺ counterflux, is not characterized. It was the objective of this work to elucidate the nature of the system regulating K⁺ flux linked to H⁺ counterflux across the chloroplast envelope. In the absence of external K⁺, exposure of spinach (Spinacia oleracea) chloroplasts to the K⁺ ionophore valinomycin was found to increase the rate of K⁺ efflux and H⁺ influx. These data were interpreted as suggesting that H⁺ counterexchange must be indirectly linked to movement of K⁺ across the envelope. Studies using the K⁺ channel blocker tetraethylammonium indicated that K⁺ likely moves, in a uniport fashion, into or out of the stroma through a monovalent cation channel in the envelope. Blockage of K⁺ efflux from the stroma by exposure to tetraethylammonium was found to restrict H⁺ influx, further substantiating an indirect linkage of these cation currents. Further studies comparing the effect of exogenous H⁺ ionophores and K⁺/H⁺ exchangers suggested that K⁺ uniport through this ion channel likely is the main endogenous pathway for K⁺ currents across the envelope. These experiments were also consistent with the presence of a proton channel in the envelope. Movement of H⁺ through this channel was speculated to be regulated and rate limited by an electroneutral requirement for K⁺ countercurrents through the separate K⁺ uniport pathway. K⁺ and H⁺ fluxes across the chloroplast envelope were envisioned to be interrelated via this mechanism. The significant effect of cation currents across the envelope, as mediated by these channels, on photosynthetic capacity of the isolated chloroplast was also demonstrated.     

Surface Charge-Mediated Effects of Mg on K Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis

Studies of Spinacia oleracea L. were undertaken to characterize further how Mg²⁺ external to the isolated intact chloroplast interacts with stromal K⁺, pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed hypothesis that millimolar levels of external, unchelated Mg²⁺ result in lower stromal K⁺, which somehow is linked to stromal acidification. Stromal acidification directly results in photosynthetic inhibition. These effects were attributed to Mg²⁺ interaction (binding) to negative surface charges on the chloroplast envelope. Chloroplast envelope-bound Mg²⁺ was found to decrease the envelope membrane potential (inside negative) of the illuminated chloroplast by 10 millivolts. It was concluded that Mg²⁺ effects on photosynthesis were likely not mediated by this effect on membrane potential. Further experiments indicated that envelope-bound Mg²⁺ caused lower stromal K⁺ by restricting the rate of K⁺ influx; Mg²⁺ did not affect K⁺ efflux from the stroma. Mg²⁺ restriction of K⁺ influx appeared consistent with the typical effects imposed on monovalent cation channels by polyvalent cations that bind to negatively charged sites on a membrane surface near the outer pore of the channel. It was hypothesized that this interaction of Mg²⁺ with the chloroplast envelope likely mediated external Mg²⁺ effects on chloroplast metabolism.     

Effects of Magnesium on Intact Chloroplasts : II. CATION SPECIFICITY AND INVOLVEMENT OF THE ENVELOPE ATPase IN (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE ENVELOPE

Addition of exogenous Mg²⁺ (2 millimolar) to illuminated intact spinach (Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K⁺. Addition of K⁺ (10-50 millimolar) reversed both stromal acidification and K⁺ efflux from the chloroplast caused by Mg²⁺. These data suggested that Mg²⁺ induced reversible H⁺/K⁺ fluxes across the chloroplast envelope. Ca²⁺ and Mn²⁺ (2 millimolar) were as effective as 4 millimolar Mg²⁺ in causing K⁺ efflux from chloroplasts and inhibition of O₂ evolution. In contrast, 10 millimolar Ba²⁺ induced only a small amount of inhibition. The lack of strong inhibition by Ba²⁺ indicated that the effects of divalent cations such as Mg²⁺ cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg²⁺ was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O₂ evolution (43%) induced by Mg²⁺.     

Leaf k interaction with water stress inhibition of nonstomatal-controlled photosynthesis

The relationship between leaf K⁺ concentration, in vitro dehydration, and nonstomatal-controlled photosynthesis was investigated using leaf slices that were vacuum infiltrated with media containing varying sorbitol concentrations. The leaf slices were from plants either supplied with complete or K⁺-deficient medium throughout a 35-day growth period. During this time, leaf K⁺ concentration, water potential, osmotic potential, and turgor pressure were monitored. Leaf K⁺ concentration averaged 239 micomoles per gram (fresh weight) in control plants, and dropped to 74.3 micromoles per gram (fresh weight) in K⁺-deficient plants. Less negative osmotic potentials and resultant turgor loss in K⁺-deficient plants indicated that the osmotically active pool of cellular K⁺ was lower in those plants.

The decrease in leaf K⁺ concentration enhanced the dehydration inhibition of photosynthesis. For example, increasing sorbitol from 0.33 to 0.5 molar during incubation inhibited photosynthesis in the controls by 14% or less. This same protocol resulted in an inhibition of photosynthesis by as much as 41% in K⁺-deficient tissue. In contrast to the data obtained with leaf slices, dehydration inhibition of isolated chloroplast photosynthesis was not affected by K⁺ status of parent plant material. These data are consistent with the hypothesis that one effect of leaf K⁺ deficiencies on photosynthetic response to dehydration may be mediated by extra-choloroplastic factors.

Ammonium ions, which facilitate stromal alkalinization, reversed the increased sensitivity of K⁺-deficient leaf slice photosynthesis to cell dehydration. However, NH₄⁺ had no effect on photosynthesis of K⁺-deficient leaf slices under nonhypertonic conditions. These data suggest that endogenous extra-chloroplastic K⁺ may modulate dehydration inhibition of photosynthesis, possibly by facilitating stromal alkalinization.

     

Effects of Alkalinization and ATPase Inhibition on Stromal Free Mg2+ Concentration in Spinach Chloroplasts

Our earlier studies indicate that stromal alkalinization is essential for light-induced increase in free Mg²⁺ concentration ([Mg²⁺]) in chloroplast. Stromal [Mg²⁺] was increased by dark incubation of chloroplasts in the K⁺-gluconate medium (pH 8.0), or by NH₄Cl. These results indicate that stromal alkalinization can induce an increase in stromal [Mg²⁺] without illumination. Some inhibitors of envelope proton-translocating ATPase activity involved in H⁺ efflux inhibited the alkalinization-induced increase in [Mg²⁺].     

Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+release

Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K⁺-permeable channel was coined fast-activating chloroplast cation channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K⁺ conductance domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K⁺ uptake. Nuclear POLLUX ion channels cofunction with Ca²⁺ channels to generate Ca²⁺ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca²⁺ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca²⁺ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant’s stress-related Ca²⁺ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.     

Chloroplast osmotic adjustment and water stress effects on photosynthesis

Previous studies have suggested that chloroplast stromal volume reduction may mediate the inhibition of photosynthesis under water stress. In this study, the effects of spinach (Spinacia oleracea, var `Winter Bloomsdale') plant water deficits on chloroplast photosynthetic capacity, solute concentrations in chloroplasts, and chloroplast volume were studied. In situ (gas exchange) and in vitro measurements indicated that chloroplast photosynthetic capacity was maintained during initial leaf water potential (Ψ_w) and relative water content (RWC) decline. During the latter part of the stress period, photosynthesis dropped precipitously. Chloroplast stromal volume apparently remained constant during the initial period of decline in RWC, but as leaf Ψ_w reached −1.2 megapascals, stromal volume began to decline. The apparent maintenance of stromal volume over the initial RWC decline during a stress cycle suggested that chloroplasts are capable of osmotic adjustment in response to leaf water deficits. This hypothesis was confirmed by measuring chloroplast solute levels, which increased during stress. The results of these experiments suggest that stromal volume reduction in situ may be associated with loss of photosynthetic capacity and that one mechanism of photosynthetic acclimation to low Ψ_w may involve stromal volume maintenance.     

Maintenance of photosynthesis at low leaf water potential in wheat : role of potassium status and irrigation history

The interaction of low water potential effects on photosynthesis, and leaf K⁺ levels in wheat (Triticum aestivum L.) plants was studied. Plants were grown at three K⁺ fertilization levels; 0.2, 2, and 6 millimolar. With well watered plants, 2 millimolar K⁺ supported maximal photosynthetic rates; 0.2 millimolar K⁺ was inhibitory, and 6 millimolar K⁺ was superoptimal (i.e. rates were no greater than at 2 millimolar K⁺). Photosynthesis was monitored at high (930 parts per million) and low (330 parts per million) external CO₂ throughout a series of water stress cycles. Plants subjected to one stress cycle were considered nonacclimated; plants subjected to two successive cycles were considered acclimated during the second cycle. Sensitivity of photosynthesis to declining leaf water potential was affected by K⁺ status; 6 millimolar K⁺ plants were less sensitive, and 0.2 millimolar K⁺ plants were more sensitive than 2 millimolar K⁺ plants to declining water potential. This occurred with nonacclimated and acclimated plants at both high and low assay CO₂. It was concluded that the K⁺ effect on photosynthesis under stress was not mediated by treatment effects on stomatal resistance. Differences between the K⁺ treatments were much less pronounced, however, when photosynthesis of nonacclimated and acclimated plants was plotted at a function of declining relative water content during the stress cycles. These results suggest that K⁺ effects on the relationship between relative water content and water potential in stressed plants was primarily responsible for the bulk of the K⁺-protective effect on photosynthesis in stressed plants. In vitro experiments with chloroplasts and protoplasts isolated from 2 millimolar K⁺ and 6 millimolar K⁺ plants indicated that upon dehydration, K⁺ efflux from the chloroplast stroma into the cytoplasm is less pronounced in 6 millimolar K⁺ protoplasts.     

The role of monovalent cations for photosynthesis of isolated intact chloroplasts

The role of monovalent cations in the photosynthesis of isolated intact spinach chloroplasts was investigated. When intact chloroplasts were assayed in a medium containing only low concentrations of mono- and divalent cations (about 3 mval l^-1), CO₂-fixation was strongly inhibited although the intactness of chloroplasts remained unchanged. Addition of K⁺, Rb⁺, or Na⁺ (50–100 mM) fully restored photosynthesis. Both the degree of inhibition and restoration varied with the plant material and the storage time of the chloroplasts in low-salt medium. In most experiments the various monovalent cations showed a different effectiveness in restoring photosynthesis of low-salt chloroplasts (K⁺>Rb⁺>Na⁺). Of the divalent cations tested, Mg²⁺ also restored photosynthesis, but to a lesser extent than the monovalent cations.In contrast to CO₂-fixation, reduction of 3-phosphoglycerate was not ihibited under low-salt conditions. In the dark, CO₂-fixation of lysed chloroplasts supplied with ATP, NADPH, and 3-phosphoglycerate strictly required the presence of Mg²⁺ but was independent of monovalent cations. This finding excludes a direct inactivation of Calvin cycle enzymes as a possible basis for the inhibition of photosynthesis under low-salt conditions.Light-induced alkalization of the stroma and an increase in the concentration of freely exchangeable Mg²⁺ in the stroma, which can be observed in normal chloroplasts, did not occur under low-salt conditions but were strongly enhanced after addition of monovalent cations (50–100 mM) or Mg²⁺ (20–50 mM).The relevance of a light-triggered K⁺/H⁺ exchange at the chloroplast envelope is discussed with regard to the light-induced increase in the pH and the Mg²⁺ concentration in the stroma, which are thought to be obligatory for light activation of Calvincycle enzymes.     

Effects of Magnesium on Intact Chloroplasts: I. EVIDENCE FOR ACTIVATION OF (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE CHLOROPLAST ENVELOPE

Exogenous Mg²⁺ (2 millimolar) altered the stromal pH of intact spinach chloroplasts. Without added KCl in the medium, Mg²⁺ decreased the stromal pH in the light by approximately 0.3 pH unit. External KCl (25 millimolar) largely prevented the acidification caused by Mg²⁺. Effects on the stromal pH were not caused by changes in H⁺ pumping across the thylakoid membrane because Mg²⁺ had no effect on the light-induced quenching of atebrin fluorescence by intact chloroplasts. However, Mg²⁺ affected H⁺ fluxes across the envelope. Addition of Mg²⁺ to intact chloroplasts in the dark caused a significant acidification of the medium that was dependent on the presence of K⁺.     

Modulation of water stress effects on photosynthesis by altered leaf k

Wheat irrigated with nutrient solutions containing 0, 0.2, 0.5, 1, 2, or 6 millimolar K⁺ had maximum photosynthetic rates at 1 to 2 millimolar K⁺ concentrations. Rates in the 6 millimolar K⁺-grown plants were not higher than the 2 millimolar K⁺-grown wheat, and rates were inhibited below 0.5 millimolar K⁺. Photosynthesis was measured by both attached whole leaf CO₂ uptake and by ¹⁴CO₂ fixation of leaf slices in solution. Exposure of leaf slices from 0.2, 2, and 6 millimolar K⁺-grown wheat to various assay media water potentials showed that photosynthesis of the 0.2 millimolar K⁺-grown wheat decreased from control (high water potential) rates by 35%, that of the 2 millimolar K⁺-grown wheat by 20.4%, and that of the 6 millimolar K⁺-grown wheat by only 8.3% at −3.11 megapascals. Also, photosynthesis of the 6 millimolar K⁺-grown wheat was enhanced by 28% over that of the 2 millimolar K⁺ wheat at the most severe water stress (−3.11 megapascals), indicating that the excess leaf K⁺ in the 6 millimolar K⁺-grown wheat partially reversed dehydration effects on photosynthesis. Oligomycin eliminated the protective effects of high K⁺ on photosynthesis in dehydrated leaf slices. These results suggest that the protective effect of high K⁺ under water stress may involve the exchange of K⁺ in the cytoplasm for stroma H⁺, thus altering stromal pH and restoring photosynthesis. The protective effect of high K⁺ was also observed in attached whole leaf photosynthesis of in situ water-stressed wheat grown on 0.2, 2, and 6 millimolar K⁺. Under water stress, rates of the 6 millimolar K⁺-grown wheat were enhanced by 66.2% and 113.9% over that of 2 millimolar K⁺-grown wheat in two separate experiments. Internal CO₂ concentration of the 6 millimolar K⁺-grown wheat was lower than that of the 0.2 and 2 millimolar K⁺-grown wheat. These results suggest that the high K⁺ effects on chloroplast photosynthesis seen in leaf slices also occur at the whole plant level.     

Photosynthesis in chloroplasts rapidly isolated from primary leaves of oat (Avena sativa L.): effects of orthophosphate, metal ion and chelators

Abstract A simple mechanical method for the rapid isolation of chloroplasts with high rates of photosynthesis from young leaves of oat (Avena sativa L.) was described. The photosynthetic activity of these chloroplasts was stable for at least 2 h with rates of CO₂-dependent O₂ evolution of 30–40 μmol g ¹ Chl s ¹. The photosynthetic properties of these chloroplasts were similar to those reported for spinach and pea chloroplasts isolated by mechanical disruption. The pH optimum for photosynthetic O₂ evolution was pH 7.6. The induction time was 0.5–2 min. Maximal rates of photosynthetic O₂ evolution in these chloroplast preparations were obtained in the absence of both divalent cations and EDTA. Addition of divilent cations strongly inhibited photosynthesis which could be partially restored by the subsequent addition of EDTA. But when these cations were not present in the assay medium the addition of EDTA greater than 1 mol m ³ decreased photosynthetic activity. The optimal orthophosphate concentration required for photosynthesis in these chloroplast preparations was 0.2–0.3 mol m ³. In contrast, the addition of pyrophosphate either in the light or dark inhibited photosynthesis. In a comparative study, chloroplasts were also isolated from oat and wheat (Triticum aestivum L., cultivar Hybrid C306) protoplasts. These chloroplast preparations were found to have properties similar to those determined for oat chloroplasts isolated by the mechanical method reported above.     

Development and use of chlorotetracycline fluorescence as a measurement assay of chloroplast envelope-bound mg

Experiments were conducted to develop chlorotetracycline (CTC) fluorescence as an assay of Mg²⁺ bound to the envelope of the intact chloroplast. This assay technique has been widely used to measure envelope associated divalent cations in animal cell and subcellular systems, but has not been used with chloroplasts. Chloroplast envelope-associated Mg²⁺ was altered by pretreatment with Mg²⁺ and divalent cation chelating agents and by additions of Mg²⁺ to the CTC assay medium. Results indicated that for a given chloroplast preparation, relative changes in envelope-associated Mg²⁺ can be effectively monitored with CTC fluorescence. It was concluded that the limitations of this assay system are: (a) chlorophyll strongly quenches CTC fluorescence signal, so a constant chlorophyll concentration must be maintained, (b) measurements must be made quickly, and (c) use of the technique to compare different chloroplast preparations may not be valid. Studies with ²⁸Mg²⁺ confirmed our interpretation of the fluorescence results, and also suggested that the chloroplast envelope is fairly impermeable to Mg²⁺. It was concluded that changes in Mg²⁺ associated with the chloroplast due to incubation of plastids in solutions containing up to 5 millimolar Mg²⁺ may be exclusively due to increased envelope-associated Mg²⁺. The CTC assay was used in experiments to demonstrate that increases in chloroplast envelope-associated Mg²⁺ inhibit photosynthetic capacity. This inhibition can be partially overcome by the presence of K⁺ in the photosynthetic reaction media.     

Transport of mono- and divalent cations across chloroplast membranes mediated by the ionophore A23187

Effects of the ionophore A23187 on isolated broken and intact chloroplasts in the pH range of 6.2 to 7.6 have been studied. In both types of chloroplasts, uncoupling of photosynthetic electron transport by A23187 (6–10 μm) was mediated either by Mg²⁺ or—in the absence of divalent cations (i.e., when EDTA was added to the medium)—by high concentrations of Na⁺, but not of K⁺ ions. At increased concentrations of the ionophore (above about 10 μm) and high pH (7.2 to 7.6), uncoupling in broken chloroplasts was also mediated by K⁺ ions. The inhibition of the energy-dependent slow decline of chlorophyll fluorescence in intact chloroplasts by the ionophore (which denotes uncoupling) is reversed by EDTA in the presence of K⁺, but not of Na⁺ ions. In 3-(3′,4′-dichlorophenyl)1,1-dimethylurea-poisoned intact chloroplasts, the yield of variable chlorophyll fluorescence is lowered by A23187 + EDTA and increased again by addition of NaCl or KCl. Chlorophyll fluorescence spectra at 77 °K of intact chloroplasts incubated with A23187 + EDTA indicated that the distribution of excitation energy had changed in favor of photosystem I, as expected from a depletion of Mg²⁺. This change was reversed by MgCl²⁺, KCl, or NaCl. From a comparison of low-temperature fluorescence spectra of broken and intact chloroplasts at different levels of Mg²⁺ in the medium, the concentration of free Mg²⁺ in the stroma of the intact chloroplasts at pH 7.6 in the dark was estimated at 1 to 4 mm. The results show that in chloroplasts the specificity of A23187 for divalent cations is limited. In the presence of EDTA, the ionophore mediates fast $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange across thylakoid membranes, whereas K⁺ is transferred much less efficiently. Both Na⁺ and K⁺ ions seem to be transported readily across the chloroplast envelope by the action of the ionophore, leading to an exchange of Mg²⁺ for monovalent cations at the thylakoid membrane surfaces in intact chloroplasts.     

Properties of inwardly rectifying K+ channels in ventricular myocytes

Summary The voltage- and time-dependent properties of whole-cell, multi-channel (outside-out), and single channel inwardly-rectifying K⁺ currents were studied using adult and neonatal rat, and embryonic chick ventricular myocytes. Inward rectification of the current-voltage relationship was found in the whole-cell and single channel measurements. The steady-state single channel probability of opening decreased with hyperpolarization from E_K, as did the mean open time, thereby explaining the time-dependent inactivation of the macroscopic current. Myocytes dialysed with a Mg⁺⁺-free K⁺ solution (to remove the property of inward rectification) displayed a quasi-linear current-voltage relationship. The outward K⁺ currents flowing through the modified inward rectifier channels were able to be blocked by the local anesthetic and anti-arrhythmic agent, lidocaine.     

Photosynthesis under osmotic stress

The reversibility of the inhibition of photosynthetic reactions by water stress was examined with four systems of increasing complexity—stromal enzymes, intact chloroplasts, mesophyll protoplasts, and leaf slices. The inhibition of soluble chloroplast enzymes by high solute concentrations was instantly relieved when solutes were properly diluted. In contrast, photosynthesis was not restored but actually more inhibited when isolated chloroplasts exposed to hypertonic stress were transferred to conditions optimal for photosynthesis of unstressed chloroplasts. Upon transfer, chloroplast volumes increased beyond the volumes of unstressed chloroplasts, and partial envelope rupture occurred. In protoplasts and leaf slices, considerable and rapid, but incomplete restoration of photosynthesis was observed during transfer from hypertonic to isotonic conditions. Chloroplast envelopes did not rupture in situ during water uptake. It is concluded that inhibition of photosynthesis by severe water stress is at the biochemical level brought about in part by reversible inhibition of chloroplast enzymes and in part by membrane damage which requires repair mechanisms for reversibility. Both soluble enzymes and membranes appear to be affected by the increased concentration of internal solutes, which is caused by dehydration.     

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.     

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.

     

Uptake of bicarbonate ion in darkness by isolated chloroplast envelope membranes and intact chloroplasts of spinach

Bicarbonate uptake by isolated chloroplast envelope membranes and intact chloroplasts of spinach (Spinacia oleracea L. var. Viroflay) in darkness exhibited a similar dependency upon temperature, pH, time, and concentrations of isolated or attached envelope membranes. This similarity in uptake properties demonstrates the usefulness of the envelope membranes for the study of chloroplast permeability. Maximal rates for dark HCO₃^- uptake by isolated envelope membranes and intact chloroplasts were more than sufficient to account for the maximal rates of photosynthetic CO₂ fixation observed with intact chloroplasts. The active species involved in the uptake process was found to be HCO₃^- and not CO₂. The significance of HCO₃^- uptake and its relationship to carbonic anhydrase and ribulose diphosphate carboxylase is discussed. Conditions for maximal HCO₃^- uptake in darkness by intact chloroplasts were found to be similar to those required for maximal photosynthetic CO₂ fixation, suggesting that HCO₃^- uptake by the envelope membrane may regulate photosynthetic CO₂ fixation.