Regulation of Cytoplasmic and Vacuolar pH in Maize Root Tips under Different Experimental Conditions

³¹P-Nuclear magnetic resonance spectra of perfused maize (Zea mays L., hybrid WW x Br 38) root tips, obtained at 10-minute intervals over 12 hours or longer, indicate that no cytoplasmic or vacuolar pH changes occur in these cells in the presence of 25 millimolar K₂SO₄, which induces extrusion of 4 to 5 microequivalents H⁺ per gram per hour. In contrast, hypoxia causes cytoplasmic acidification (0.3-0.6 pH unit) without a detectable change in vacuolar pH. The cytoplasm quickly returns to its original pH on reoxygenation. Dilute NH₄OH increases the vacuolar pH more than it does the cytoplasmic pH; after NH₄OH is removed, the vacuole recovers its original pH more slowly than does the cytoplasm. The results indicate that regulation of cytoplasmic pH and that of vacuolar pH in plant cells are separate processes.     

Active, Irreversible Accumulation of Extreme Levels of H(2)SO(4) in the Brown Alga, Desmarestia

The brown algae Desmarestia ligulata var. ligulata (Lightf.) Lamour., and D. viridis (Mull.) Lamour., accumulate H₂SO₄ until their average internal pH is 0.5 to 0.8. A related species, D. aculeata (L.) Lamour., does not accumulate acid. The H₂SO₄ accumulation is accompanied by a reduction in the K⁺ and Cl⁻ content, presumedly to maintain osmotic balance. Measurements of the membrane potential and H⁺ and SO₄²⁻ concentrations indicate that both ions are accumulated in the vacuole against their electrochemical potential gradients.

The internal pH remains constant in all three species over the growing season, despite striking changes in the algal morphology. The pH is not affected by periods of darkness of up to 34 hours. Sulfate accumulated in the vacuoles appears to be trapped there since incubation of D. ligulata for up to 10 days in sulfate-free medium resulted in little loss of either vacuolar sulfate or H⁺. Although the uptake of H₂SO₄ into the vacuole must require energy, the maintenance of the vacuolar H₂SO₄ may be due to the impermeability of the tonoplast, with little necessity for continued expenditure of energy.

     

Long-term effects of SO2 on plants,SO2 metabolism and regulation of intracellular pH

The impact of SO₂ on the ionic balance of plants and its implications for intracellular pH regulation was studied to find explanations for long-term effects of SO₂. When sulphur, taken up as SO₂ by the shoots of plants, is not assimilated in organic compounds, but stored as sulphate, an equivalent amount of H⁺ is produced. These H⁺ ions are not buffered chemically, but removed by metabolic processes.On the basis of knowledge on metabolic buffering mechanisms a conceptual model is proposed for the removal of shoot-generated H⁺ by (i) OH^- ions, produced in the leaves when sulphate and nitrate are assimilated in organic compounds and/or by (ii) OH^- ions produced by decarboxylation of organic anions (a biochemical pH stat mechanism). The form in which nitrogen is supplied largely determines the potential of the plant to neutralize H⁺ in the leaves during SO₂ uptake by the proposed mechanisms.In field experiments with N₂ fixing Vicia faba L. crops, the increase of sulphate in the shoots of SO₂-exposed plants was equivalent in charge to the decrease of organic anion content, calculated as the difference between inorganic cation content (C) and inorganic anion content (A), indicating that H⁺ ions produced in the leaves following SO₂ uptake were partly removed by OH^- from sulphate reduction and partly by decarboxylation of organic anions.The appearance of chronic SO₂ injury (leaf damage) in the field experiment at the end of the growing period is discussed in relation to the impact of SO₂ on the processes involved in regulation of intracellular pH. It is proposed that the metabolic buffering capacity of leaf cells is related to the rates of sulphate and nitrate reduction and the import rate of organic anions, rather than to the organic anion content in the vacuoles of the leaf cells.     

Mesophyll Resistances to SO(2) Fluxes into Leaves

Uptake of label from solutions containing ³⁵SO₂, H³⁵SO₃⁻ and ³⁵SO₃²⁻ into mesophyll protoplasts, vacuoles, and chloroplasts isolated from young barley leaves was measured at different pH values. Uptake was fast at low pH, when the concentration of SO₂ was high, and low at high pH, when the concentration of SO₂ was low. When the resistance (R) of plasmalemma, tonoplast, and chloroplast envelope to the penetration of SO₂ was calculated from rates of uptake of label, comparable values were obtained for the different biomembranes at low pH values. R was close to 8000 seconds per meter and permeability coefficients were close to 1.25 × 10⁻⁴ meters per second. Under these conditions R may describe resistance to SO₂ diffusion across a lipid bilayer. At higher pH values, R decreased. As R was calculated on the assumption that SO₂ is the only penetrating molecular species, the data suggest that carrier-mediated anion transport contributes to the uptake of sulfur at physiological pH values thereby decreasing apparent R_SO2. The contribution of anion transport appeared to be smaller for transfer across the plasmalemma than for transfer across the tonoplast. It was large for transfer across the chloroplast envelope. The phosphate translocator of the chloroplast envelope catalyzed uptake of SO₃²⁻ into chloroplasts at neutral pH. Uptake was decreased in the presence of high levels of phosphate or sulfate and by pyridoxal phosphate. SO₂ transfer into cells leads to the intracellular liberation of one or two protons, depending on pH and oxidizing conditions. When the divalent sulfite anion is exchanged across the chloroplast envelope, bisulfite formation results in proton uptake in the chloroplast stroma, whereas SO₂ uptake into chloroplasts lowers the stroma pH.     

Flux of SO(2) into Leaf Cells and Cellular Acidification by SO(2)

A comparison of fluxes of SO₂ from the atmosphere into leaves with fluxes across biomembranes revealed that, apart from the cuticle, the main barrier to SO₂ entry into leaves are the stomates. SO₂ fluxes into leaves can be calculated with an accuracy sufficient for many purposes on the assumption that the intracellular SO₂ concentration is zero. SO₂ entering green leaf cells is trapped in the cytoplasm. In the light, the products formed in its reaction with water are processed particularly in the chloroplasts. Flux of SO₂ to the acidic central vacuole of leaf cells is insignificant. Intracellular acidification of barley mesophyll protoplasts by SO₂ was measured by the uptake of ¹⁴C-labeled 5,5-dimethyl-oxazolidine-2,4-dione. The measured acidification was similar to the acidification calculated from known buffer capacities and the rate of SO₂ influx when the H⁺/SO₂ ratio was assumed to be 2. A comparison of photosynthesis inhibition by SO₂ with calculated acidification revealed different mechanisms of inhibition at low and at high concentrations of SO₂. At very low concentrations, inhibition by SO₂ was even smaller than expected from calculated acidification. The data suggest that, if acidification cannot be compensated by pH-stabilizing cellular mechanisms, it is a main factor of SO₂ toxicity at low SO₂ levels. At high levels of SO₂, anion toxicity and/or radical formation during oxidation of SO₂ to sulfate may play a large role in inhibition.     

Measurement of the Cytoplasmic and Vacuolar Buffer Capacities in Chara corallina

The cytoplasm and the vacuole were isolated from internodal cells of Chara corallina by using the intracellular perfusion technique, and their buffer capacities (β_i) were determined from the titration curves. The pH of the isolated vacuolar sap was 5.19 ± 0.029 (mean ± standard error). At this pH, β_i was minimal and amounted to 0.933 ± 0.11 millimoles H⁺/pH unit/liter vacuolar sap. The pH of isolated cytoplasm was 7.22 ± 0.028. β_i was minimal in this pH region and amounted to 14.2 ± 0.80 millimoles H⁺/pH unit/liter cytoplasm. When 1% (volume/volume) Triton X-100 was added to the cytoplasmic solution to permeabilize the subcellular organelles, the cytoplasmic pH increased to 7.32 ± 0.026, where β_i was 20.35 ± 2.66 millimoles H⁺/pH unit/liter cytoplasm. This shows that alkaline subcellular compartments exist in the cytoplasm and also that the cytoplasmic pH before adding Triton X-100 may represent the cytosolic pH. These data indicate that the pH values of the cytoplasm and the vacuole are regulated at the values where the β_i values are minimal. This suggests that ATP- and inorganic pyrophosphate-dependent H⁺ pumps in the plasma membrane and the tonoplast could efficiently regulate the pH of both cytoplasm and vacuole in Chara internodal cells.     

Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase : Evidence for a Hydrophobic l-Amino Acid Carrier System

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K_m-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V_{max app} was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 10⁶ vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H⁺-translocating enzyme activities revealed K_{m app} values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH⁺ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.     

Role of apoplast acidification by the H+ pump

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H⁺ extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H⁺ extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO₄). These results support the hypothesis that the apoplastic pH shift caused by H⁺ excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H⁺ excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H⁺ excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H⁺ pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H⁺ transport. Our results shed light on the way by which the strong buffering effect of HCO ₃ ^- in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.     

Chloroplastic ATP synthase optimizes the trade-off between photosynthetic CO2 assimilation and photoprotection during leaf maturation

In the present study, we studied the role of chloroplastic ATP synthase in photosynthetic regulation during leaf maturation. We measured gas exchange, chlorophyll fluorescence, P700 redox state, and the electrochromic shift signal in mature and immature leaves. Under high light, the immature leaves displayed high levels of non-photochemical quenching (NPQ) and P700 oxidation ratio, and higher values for proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes but lower values for the activity of chloroplastic ATP synthase (g_H⁺) than the mature leaves. Furthermore, g_H⁺ was significantly and positively correlated with CO₂ assimilation rate and linear electron flow (LEF), but negatively correlated with pmf and ΔpH. ΔpH was significantly correlated with LEF and the P700 oxidation ratio. These results indicated that g_H⁺ was regulated to match photosynthetic capacity during leaf maturation, and the formation of pmf and ΔpH was predominantly regulated by the alterations in g_H⁺. In the immature leaves, the high steady-state ΔpH increased lumen acidification, which, in turn, stimulated photoprotection for the photosynthetic apparatus via NPQ induction and photosynthetic control. Our results highlighted the importance of chloroplastic ATP synthase in optimizing the trade-off between CO₂ assimilation and photoprotection during leaf maturation.     

pH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves

After opening the stomata in CO₂-free air, darkened leaves of several plant species were titrated with CO₂ at concentrations between 1 and 16%, in air in order to reversibly decrease cellular pH values and to calculate buffer capacities from pH changes and bicarbonate accumulation using both gas-exchange and fluorescence methods for analysis. After equilibration with CO₂ for times ranging between 4.4 and 300 s, fast CO₂ release from bicarbonate indicated catalysis by highly active carbonic anhydrase. Its time constant was below 2.5 s. Additional CO₂ was released with time constants of about 5, 15 and approximately 300 s. With CO₂ as the acidifying agent, calculated buffer capacities depend on assumptions regarding initial pH in the absence of an acid load. At an initial stroma pH of 7.7, the stromal buffer capacity was about 20 mM pH-unit⁻¹ in darkened spinach leaves. At an initial pH of 7.5 it would be only 12 mM pH-unit⁻¹, i.e. not higher than expected solely on the basis of known stromal concentrations of phosphate and phosphate esters, disregarding the contribution of other solutes. At a concentration of 16%, CO₂ reduced the stromal pH by about 1 pH unit. Buffering of the cytosol was measured by the CO₂-dependent quenching of the fluorescence of pyranine which was fed to spinach leaves via the petiole. Brief exposures to high CO₂ minimized interference by effective cytosolic pH regulation. Cytosolic buffering appeared to be similar to or only somewhat higher than chloroplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic pH values. The difference from chloroplast pH values indicates the existence of a pH gradient across the chloroplast envelope even in darkened leaves. Apoplastic buffering was weak as measured by the CO₂-dependent quenching of dextran-conjugated fluorescein isothiocyanate which was infiltrated together with sodium vanadate into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescence quenching were more complex than in its presence, indicating fast apoplastic pH regulation which strongly interfered with the determination of apoplastic buffering capacities. At an apoplastic pH of 6.1 in potato leaves, apoplastic buffering as determined by CO₂ titration with and without added buffer was somewhat below 4 mM pH-unit⁻¹. Thus the apoplastic and cytosolic pH responses to additions of CO₂ indicated that the observed cytoplasmic pH regulation under acid stress involves pumping of protons from the cytosol into the vacuole of leaf cells, but not into the apoplast. Received: 27 November 1998 / Accepted: 22 March 1999     

Chloroplast pH values and buffer capacities in darkened leaves as revealed by CO2 solubilization in vivo

Leaves of the C₃ plants Brassica oleracea L., Datura suaveolens Humb. & Bonpl. ex Willd., Helianthus annuus L. and Nicotiana tabacum L. with open stomata were exposed in a leaf chamber in the dark to CO₂ concentrations varying from 1 to 20% in air. When they were transferred back into CO₂-free air, CO₂ was rapidly released. It originated from dissolved CO₂ and from the bicarbonate in the chloroplast stroma, since vacuoles are acidic and chloroplasts contain carbonic anhydrase which rapidly liberates CO₂ from bicarbonate. The data were fitted to a model which accounts for the CO₂/bicarbonate equilibrium in buffers with different CO₂ concentrations and initial pH values. From this, pH values and CO₂-dependent pH changes in the chloroplast stroma were calculated. The full range of external CO₂ concentration caused acidic shifts up to 1 pH unit. The best fits of the data points were obtained with stromal buffer concentrations ranging from 45 to 65 mM and stromal pH values at low CO₂ between 7.5 and 7.9. Calculated buffer capacities ranged from 23 to 31 mM H⁺ per pH unit. The work shows that measurements of solubilized CO₂ are useful to investigate proton buffering and pH regulation in the chloroplast stroma of intact leaves.Abbreviation Chl chlorophyll This work was supported by Sonderforschungsbereich 251 of the University of Würzburg and the Volkswagenstiftung grant I-67762. We are very grateful to Dr. V. Oja for helpful advice.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Effect of path or sink anoxia on sugar translocation in roots of maize seedlings

The subcellular and developmental distribution of β-cyanoalanine synthase (EC 4.4.1.9), which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, were investigated in barley (Hordeum vulgare) leaves. Total leaf activity was 1.1 micromoles per minute per gram fresh weight. Sucrose density gradients of lysed mesophyll protoplasts of barley revealed the exclusive or predominant localization of β-cyanoalanine synthase in the mitochondria. The enzyme was absent from both vacuole and chloroplast fractions.

β-Cyanoalanine synthase activity was distributed over the entire length of the barley leaf. Activity was dependent on the developmental stage, with a 3.5-fold higher activity in the oldest (apical) compared to the youngest (basal) parts of the leaf. The corresponding difference in activity for mesophyll protoplasts isolated from these parts was 7.5-fold. In younger leaf seagments, the nonchlorophyllous tissues accounted for up to 70% of the total β-cyanoalanine synthase activity. These results are discussed with reference to the formation of HCN as a substrate in barley leaves.

     

A malic Acid permease in isolated vacuoles of a crassulacean Acid metabolism plant

Pine (Pinus silvestris L.) trees subjected to relatively low concentration of SO₂ in the field emit H₂S from the needles, as demonstrated by gas chromatographic analysis after preconcentration on a molecular sieve. H₂S is the only reduced sulfurous compound emitted from SO₂ fumigated leaves. The emission is light and SO₂ concentration dependent. Pine trees in the field and in laboratory experiments continue to emit H₂S several hours after the termination of prolonged SO₂ fumigation. The maximum emission rates observed from pine trees in the field and in laboratory experiments, 14 and 20 nanomoles per milligram chlorophyll per hour respectively, are about the activity expected for the sulfur assimilation pathway in the chloroplasts.     

Sulfur-dioxide fluxes into different cellular compartments of leaves photosynthesizing in a polluted atmosphere

A computer model is used to analyze fluxes of SO₂ from polluted air into leaves and the intracellular distribution of sulfur species derived from SO₂. The analysis considers only effects of acidification and of anion accumulation. (i) The SO₂ flux into leaves is practically exclusively controlled by the boundary-layer resistance of leaves to gas diffusion and by stomatal opening. At constant stomatal opening, flux is proportional to the concentration of SO₂ in air. (ii) The sink capacity of cellular compartments for SO₂ depends on intracellular pH and the intracellular localization of reactions capable of oxidizing or reducing SO₂. In the mesophyll of illuminated leaves, the chloroplasts possess the highest trapping potential for SO₂. (iii) If intracellular ion transport were insignificant, and if bisulfite and sulfite could not be oxidized or reduced, leaves with opened stomata would rapidly be killed both by the accumulation of sulfites and by acidification of chloroplasts and cytosol even if SO₂ levels in air did not exceed concentrations thought to be permissible. Acidification and sulfite accumulation would remain confined largely to the chloroplasts and to the cytosol under these conditions. (iv) Transport of bisulfite and protons produced by hydration of SO₂ into the vacuole cannot solve the problem of cytoplasmic accumulation of bisulfite and sulfite and of cytoplasmic acidification, because SO₂ generated in the acidic vacuole from the bisulfite anion would diffuse back into the cytoplasm. (v) Oxidation to sulfate which is known to occur mainly in the chloroplasts can solve the problem of cytoplasmic sulfite and bisulfite accumulation, but aggravates the problem of chloroplastic and cytosolic acidification. (vi) A temporary solution to the problem of acidification requires the transfer of H⁺ and sulfate into the vacuole. This transport needs to be energized. The storage capacity of the vacuole for protons and sulfate defines the extent to which SO₂ can be detoxified by oxidation and removal of the resulting protons and sulfate anions from the cytoplasm. Calculations show that even at atmospheric levels of SO₂ thought to be tolerable, known vacuolar buffer capacities are insufficient to cope with proton production during oxidation of SO₂ to sulfate within a vegetation period. (vii) A permanent solution to the problem of acidification is the removal of protons. Protons are consumed during the reduction of sulfate to sulfide. Proteins and peptides contain sulfur at the level of sulfide. During photosynthesis in the presence of the permissible concentration of 0.05l·l^-1 SO₂, sulfur may be deposited in plants at a ratio not far from 1/500 in relation to carbon. The content of reduced sulfur to carbon is similar to that ratio only in fast-growing, protein-rich plants. Such plants may experience little difficulty in detoxifying SO₂. In contrast, many trees may contain reduced sulfur at a ratio as low as 1/10 000 in relation to carbon. Excess sulfur deposited in such trees during photosynthesis in polluted air gives rise to sulfate and protons. If detoxification of SO₂ by reduction is inadequate, and if the storage capacity of the vacuoles for protons and sulfate is exhausted, damage is unavoidable. Calculations indicate that trees with a low ratio of reduced S to C cannot tolerate long-term exposure to concentrations of SO₂ as low as 0.02 or 0.03 l·l^-1 which so far have been considered to be non-toxic to sensitive plant species.     

H Uptake and Extrusion by Nitella clavata

Very high rates of H⁺ extrusion by internodal cells of Nitella clavata Kutz were measured after acid loading at pH 4.6. The highest rate observed, 160 picomoles per square centimeter per second, was more than twice the rate of photosynthetic bicarbonate utilization under saturating light. These results are consistent with the recently proposed hypothesis that bicarbonate is not taken in directly but is protonated at the exterior surface; the CO₂ thereby formed diffuses preferentially into the cell because of the asymmetric concentration gradient.

The H⁺ taken up, about 150 nanomoles per square centimeter in 2 hours, was distributed in three fractions: 30% in the cell wall, 40% in the cytoplasm, and 30% in the vacuole. This was concluded from the kinetics of the H⁺ release by intact cells and isolated walls, and from the pH decrease of the vacuolar sap.

The cytoplasmic H⁺ was extruded rapidly, with a half-time of about 2 minutes when the external pH was 5.7 or higher. The extrusion of the vacuolar H⁺ only proceeded at a measurable rate when the [K⁺] in the medium was raised to 20 millimolar; the half-time was about 100 minutes. There was little H⁺ extruded when the external pH was 5.0.

     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

The influence of salinity on cell ultrastructures and photosynthetic apparatus of barley genotypes differing in salt stress tolerance

A hydroponic experiment was conducted to elucidate the difference in growth and cell ultrastructure between Tibetan wild and cultivated barley genotypes under moderate (150 mM NaCl) and high (300 mM NaCl) salt stress. The growth of three barley genotypes was reduced significantly under salt stress, but the wild barley XZ16 (tolerant) was less affected relative to cultivated barley Yerong (moderate tolerant) and Gairdner (sensitive). Meanwhile, XZ16 had lower Na⁺ and higher K⁺ concentrations in leaves than other two genotypes. In terms of photosynthetic and chlorophyll fluorescence parameters, salt stress reduced maximal photochemical efficiency (F _v/F _m), net photosynthetic rate (Pn), stomatal conductance (Gs), and intracellular CO₂ concentration (Ci). XZ16 showed relatively smaller reduction in comparison with the two cultivated barley genotypes. The observation of transmission electron microscopy found that fundamental cell ultrastructure changes happened in both leaves and roots of all barley genotypes under salt NaCl stress, with chloroplasts being most changed. Moreover, obvious difference could be detected among the three genotypes in the damage of cell ultrastructure under salt stress, with XZ16 and Gairdner being least and most affected, respectively. It may be concluded that high salt tolerance in XZ16 is attributed to less Na⁺ accumulation and K⁺ reduction in leaves, more slight damage in cell ultrastructure, which in turn caused less influence on chloroplast function and photosynthesis.