Depletion of stromal P(i) induces high 'energy-dependent' antenna exciton quenching (q(E)) by decreasing proton conductivity at CF(O)-CF(1) ATP synthase.

This work tests two models to account for the effects of depletion of stromal inorganic phosphate (P(i)), which results in down-regulation of light capture via the exciton quenching (q(E)) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon P(i) depletion. In one model, an imbalance of ATP or NADPH activates cyclic electron transfer around photosystem I (CEF1), increasing proton influx to the lumen. In the second, the effective conductivity of the CF(O)-CF(1) ATP synthase to protons (g(H)(+)) is decreased, retarding proton efflux from the lumen. Sequestering of P(i) by mannose infiltration increased sensitivities of q(E) and pmf to LEF. The effects were attributable to decreases in g(H)(+), but not to CEF1 and were largely reversed by subsequent P(i) feeding. Rapid recovery of g(H)(+) in the dark suggested that dark-labile metabolic pools are responsible for regulation of the ATP synthase. Overall, these results support models where accumulation of Benson-Calvin cycle intermediates or lowering of stromal P(i) below its K(M)at the ATP synthase, retards proton efflux from the lumen, leading to build-up of pmf and subsequent down-regulation of photosynthetic light capture.     

The long-term responses of the photosynthetic proton circuit to drought

Proton motive force (pmf) across thylakoid membranes is not only for harnessing solar energy for photosynthetic CO₂ fixation, but also for triggering feedback regulation of photosystem II antenna. The mechanisms for balancing these two roles of the proton circuit under the long-term environmental stress, such as prolonged drought, have been poorly understood. In this study, we report on the response of wild watermelon thylakoid 'proton circuit' to drought stress using both in vivo spectroscopy and molecular analyses of the representative photosynthetic components. Although drought stress led to enhanced proton flux via a ∼34% increase in cyclic electron flow around photosystem I (PS I), an observed ∼fivefold decrease in proton conductivity, g_H⁺, across thylakoid membranes suggested that decreased ATP synthase activity was the major factor for sustaining elevated q_E. Western blotting analyses revealed that ATP synthase content decreased significantly, suggesting that quantitative control of the complex plays a pivotal role in down-regulation of g_H⁺. The expression level of cytochrome b ₆ f complex – another key control point in photosynthesis – also declined, probably to prevent excess-reduction of PS I electron acceptors. We conclude that plant acclimation to long-term environmental stress involves global changes in the photosynthetic proton circuit, in which ATP synthase represents the key control point for regulating the relationship between electron transfer and pmf.     

Inorganic Pi Increases Neuronal Survival in the Acute Early Phase Following Excitotoxic/Oxidative Insults

Abstract: Inorganic phosphate (P_i) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high-energy phosphate concentrations, and modulation of cytosolic free [Ca²⁺]. Cultured fetal rat cortical neurons constitutively import P_i, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular P_i is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular P_i dose-dependently enhanced survival of cortical neurons after exposure to NMDA at early (≤6 h) time points after termination of the insult. P_i similarly increased neuronal survival after exposure to kainic acid or H₂O₂. P_i-exposed neurons had higher basal intracellular [P_i], [ATP], and [GSH], and slightly lower cytosolic free [Ca²⁺], compared with P_i-deprived neurons. P_i-exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H₂O₂, compared with P_i-deprived neurons. These findings demonstrate that changes in extracellular and intracellular P_i can affect neuronal survival after excitotoxic or oxidative insults.     

Effect of darkness and CO2 starvation on NH4+ and NO3− assimilation in the unicellular alga Cyanidium caldarium

Cyanidium caldarium (Tilden) Geitler, a non-vacuolate unicellular alga, resuspended in medium flushed with air enriched with 5% CO₂, assimilated NH₄⁺ at high rates both in the light and in the dark. The assimilation of NO₃⁻, by contrast, was inhibited by 63% in the dark. In cell suspensions flushed with CO₂-free air, NH₄⁺ assimilation decreased with time both in the light and in the dark and ceased almost completely after 90 min. The addition of CO₂ completely restored the capacity of the alga to assimilate NH₄⁺. NO₃⁻ assimilation, by contrast, was 33% higher in the absence of CO₂ and was linear with time. It is suggested that NO₃⁻ and NH₄⁺ metabolism in C. caldarium are differently controlled in response to the light and carbon conditions of the cell.     

Growth, photosynthesis, and respiration of Chlorella sorokiniana after N-starvation. Interactions between light, CO2 and NH4+ supply

N-sufficient cells of Chlorella sorokiniana Shihira and Krauss, strain 211/8k, absorbed NH₄⁺ under light plus CO₂ conditions, when growth occurred, but not in darkness or in the absence of CO₂, when growth was inhibited. N-sufficient cells subjected to conditions of N-starvation for a 24-h period showed a marked loss of photosynthetic activity. Upon supply of NH₄⁺, N-starved cells sufflated with CO₂ air exhibited a time-dependent recovery of photosynthetic activity, both when suspended in light and in darkness. By contrast, growth only occurred in cells suspended in light. N-starved cells absorbed NH₄⁺ in darkness, but at a lower rate than in light. All of these data suggest that dark NH₄⁺ uptake is driven by N assimilation to recover from N-starvation and that the light-dependent NH₄⁺ uptake is driven by growth, being then influenced by conditions that affect recovery or growth. Unlike CO₂ conditions, in a CO₂-free atmosphere, absorption of NH₄⁺ by N-starved cells occurred at a higher rate in darkness than in light. Accordingly, resumption of photosynthetic potential after NH₄⁺ supply occurred in darkened cells, but not in illuminated cells. Respiratory activity of N-starved cells was enhanced up to 3-fold by NH₄⁺ and 2-fold by methylammonium, with different patterns, suggesting that respiratory enzymes were affected by N-metabolism, especially through short-term control mechanisms triggered by the expenditure of metabolic energy involved in N-metabolism.     

The role of Mg2+ in proton transport by the tonoplast pyrophosphatase in Riccia fluitans vacuoles

The Mg²⁺-dependent activity of the tonoplast pyrophosphatase (PPase) was investigated by measuring proton transport and by using the acridine orange technique on intact vacuoles of the aquatic liverwort Riccia fluitans L. In solutions with both Mg²⁺ and pyrophosphate present, a number of complexes are formed, which could all influence the enzymatic and hence the transport activity of the PPase. Therefore, the individual concentrations of these complexes were calculated and their contributions to proton transport across the tonoplast were tested. From these experiments we conclude that Mg²⁺ has three different roles: (i) Mg²⁺ stimulates transport activity of the PPase. (ii) Mg₂PP_i inhibits PPase-mediated H⁺ transport, (iii) MgPP_i* (= MgPP_i^2-+ MgHPP_i^-) is the substrate with an apparent K_1/2= 5–10 μM, with no discrimination between MgPP_i^2- and MgHPP_i^-.     

DNA ploidy and pS2 protein expression in breast cancer

The DNA content of ductal breast carcinomas of varying histological grade was measured using static image cytometry and correlated with pS₂ expression in the tumour cells. Our study was performed on imprint of surgical biopsies of 60 women with ductal breast cancer. A statistically significant difference was observed between pS₂⁺ expression and grade of malignancy ( P <0.001). The percentage of euploid tumours significantly decreased from grade I to grade II to grade III ( P =0.01). The percentage of aneuploid tumours increased from pS₂⁺ to pS₂⁻ breast tumours ( P <0.001). These findings may be indicative of pS₂ and DNA ploidy alterations and tumour aggressiveness.     

K+ATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria

The role of a recently identified K⁺_ATP channel in preventing H₂O₂ formation was examined in isolated pea stem mitochondria. The succinate-dependent H₂O₂ formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H₂O₂ formation was not influenced. The succinate-sustained H₂O₂ generation was also unaffected by nigericin (a H⁺/K⁺ exchanger), but completely prevented by valinomycin (a K⁺ ionophore). In addition, cyclosporin A (a K⁺_ATP channel opener) inhibited this H₂O₂ formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K⁺ channel faces the intermembrane space. Finally, the succinate-dependent H₂O₂ formation was partially prevented by phenylarsine oxide (a thiol oxidant).     

Functional asymmetry of the F0 motor in bacterial ATP synthases

F₁F₀ ATP synthases use the electrochemical potential of H⁺ or Na⁺ across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F₀ parts of a Na⁺- and H⁺-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of ∼100 times higher Na⁺ or H⁺ concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F₀ parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo . In Escherichia coli , we observed respiratory chain-driven ATP production at pH 7–8, while P -site pH values < 6.5 were required for ATP synthesis in vitro . This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.     

ATP-dependent H+-pumping of a low-density fraction of pea stem microsomes

A low-density fraction of pea ( Pisum sativum L. cv. Alaska) stem microsomes, obtained from a discontinuous sucrose gradient, possessed an H⁺-ATPase able to generate a proton gradient and an electrical potential. The proton pumping was insensitive to monovalent cations, to vanadate and oligomycin, required a permeant anion and was inhibited by nitrate, N, N'-dicyclohexylcarbodiimide and diethylstilbestrol. The H⁺-ATPase had a pH optimum around 6.0–6.5 and was saturable with respect to the substrate Tris-ATP (K_m≅ 0.4 m M ). Ca²⁺ (0.05–1 m M ) induced a dissipation of the ATP-generated δpH without affecting ATPase activity. At physiological concentrations (1–5 m M ), nitrate caused an initial slight increase of the ATP-generated proton gradient followed by a complete dissipation after 2–3 min. The dissipating effect was not caused by inhibition of ATPase activity, since ATP prevented the nitrate-induced collapse of δpH. On the other hand, ATPase activity, evaluated as release of P_i, was not inhibited by concentrations lower than 20 m M KNO₃. These results indicate that nitrate entered the vesicles in response to an electrical potential and then could exit in symport with protons, while Ca²⁺ entered in exchange for protons (antiport).     

A comparison of NH4+ and NO3– net fluxes along roots of rice and maize

Net fluxes of NH₄⁺ and NO₃^– along adventitious roots of rice ( Oryza sativa L.) and the primary seminal root of maize ( Zea mays L.) were investigated under nonperturbing conditions using ion-selective microelectrodes. The roots of rice contained a layer of sclerenchymatous fibres on the external side of the cortex, whereas this structure was absent in maize. Net uptake of NH₄⁺ was faster than that of NO₃^– at 1 mm behind the apex of both rice and maize roots when these ions were supplied together, each at 0·1 mol m^–3. In rice, NH₄⁺ net uptake declined in the more basal regions, whereas NO₃^– net uptake increased to a maximum at 21 mm behind the apex and then it also declined. Similar patterns of net uptake were observed when NH₄⁺ or NO₃^– was the sole nitrogen source, although the rates of NO₃^– net uptake were faster in the absence of NH₄⁺. In contrast to rice, rates of NH₄⁺ and NO₃^– net uptake in the more basal regions of maize roots were similar to those near the root apex. Hence, the layer of sclerenchymatous fibres may have limited ion absorption in the older regions of rice roots.     

Dopamine D2 Receptor-Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum

Abstract : Presynaptic D₂ dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D₂ DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D₂^+/+), one (D₂^+/-), or no (D₂^-/-) functional copies of the gene coding for the D₂ DA receptor. In vivo microdialysis studies demonstrated that basal and K⁺-evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D₂^-/- mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D₂^+/+ mice. In D₂^+/+ mice, but not D₂^-/- mice, local application of the D₂-like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [³H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D₂ DA receptor subtype modulates activity of the striatal DAT.     

Promoting effect of fusicoccin on Na+ efflux in barley roots: evidence for a Na+−H+ antiport

Abstract. The effect of fusicoccin (FC) on the K⁺stimulated Na⁺ efflux in root cells of Na⁺ loaded barley roots was studied. FC (0.02 mM) stimulated Na⁺ efflux in the presence of K⁺ and its effect was synergistic with that of K⁺, in a similar way as its effect on proton extrusion. Decreasing the pH of the elution medium promoted Na⁺ efflux and partially replaced the effect of FC. As FC is known to increase the electrochemical proton gradient at the plasmalemma level, these results are consistent with the hypothesis that Na⁺ is extruded in exchange for H⁺. A further support to this view came from the finding that Na⁺ efflux was also promoted by a lipophilic cation, tributylbenzylammonium (TBBA ⁺), which stimulates H ⁺ extrusion and is generally accepted not to enter the cells by means of the same carrier as K ⁺.     

SO2 and assimilatory sulfate reduction in beech leaves

The effect of SO₂ on the extractable activity of ATP sulfurylase (EC 2.7.7.4.). adenosine 5'-phosphosulfate sulfotransferase, ribulosebisphosphate carboxylase, chlorophyll, protein, sulfate, and amino acids was examined in leaves of potted grafts of beech ( Fagus sylvatica L.) treated in outdoor fumigation chambers. Addition of 0.025 and 0.075 μl SO₂ 1⁻¹ to unfiltered ambient air caused a decrease in the extractable activity of adenosine 5'-phosphosulfate sulfotransferase to about 20 to 30% of the controls. Neither the extractable activity of ATP sulfurylase and ribulosebisphosphate carboxylase nor the content in chlorophyll, total amino acids and protein were significantly affected by SO₂, but there was an increase in the sulfate content. Leaves treated with 0.075 μl SO₂ 1⁻¹ contained more alanine and cysteine and less serine than the controls. After transfer of the SO2-treated beech trees to control chambers there was an increase in adenosine 5'-phosphosulfate sulfotransferase activity, but no significant decrease in SO₂₋⁴-sulfur.     

An alternative explanation for the post-disturbance NO3– flush in some forest ecosystems

The appearance of soil NO₃^– after forest disturbance is commonly ascribed to a higher availability of NH₄⁺ to autotrophic nitrifiers, or to a reduction in available-C resulting in lower microbial assimilation of NO₃^–. Alternatively, it has been proposed that increasing NH₄⁺ pools following disturbance could increase net nitrification by reducing microbial assimilation of NO₃^–. Forest floor material was collected from shelterwood harvest plots which displayed both low available-C and low NH₄⁺ pools, and where previous experiments had suggested the prevalence of heterotrophic nitrification. Subsamples were amended with incremental rates of glucose-C or NH₄⁺, and gross NO₃^– transformation rates were measured by isotope dilution. Glucose-C additions had little effect on the net difference between gross NO₃^– production and consumption rates. On the other hand, NH₄⁺ additions caused gross NO₃^– consumption processes to decrease sharply, while gross NO₃^– production processes remained constant. The results suggest that NH₄⁺ can have an immediate positive effect on net nitrification rates by suppressing NO₃^– assimilation and uptake systems.     

Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

Effects of 2-iodobenzanilide on potassium uptake, H+ extrusion and K+-stimulated ATPase of corn roots

The carboxanilide systemic fungicide 2-iodobenzanilide (2-IB) after 2 h pretreatment at 0.25 m M inhibited K⁺ and SO₄^2- uptake by excised corn roots ( Zea mays L., cv. Dekalb 342) up to ca 70 and 40%, respectively. Proton extrusion from corn roots was also reduced by ca 50% after 1 h contact, and the microsomal K⁺-stimulated ATPase activity from corn roots and pea stems ( Pisum sativum L., cv. Alaska) inhibited by 50 and 72%, respectively. In contrast, the Mg²⁺-ATPase activities of microsomes and mitochondria at pH 6.0 and 8.7, respectively, were unaffected. After 2 h of preincubation with 0.25 m M 2-IB, O₂ consumption by corn roots and pea stems was inhibited by 12 and 18%, respectively. ATP content of corn roots was not altered by 2-IB treatment. Therefore, energy availability "in vivo" was unaffected and the primary effect on corn roots is suggested to be at the plasmalemma ATPase which forms the proton gradient.
With isolated pea stem mitochondria, 0.25 m M 2-IB inhibited O₂ consumption by ca 60% when NADH or malate plus pyruvate were added as substrates; when succinate was used O₂ consumption was unaffected. The mode of action on isolated mitochondria was different from that shown for carboxin and also formerly attributed to the whole class of carboxanilide fungicides.     

Translocation of NH4+ in oilseed rape plants in relation to glutamine synthetase isogene expression and activity

Translocation of NH₄⁺ was studied in relation to the expression of three glutamine synthetase (GS, EC 6.3.1.2) isogenes and total GS activity in roots and leaves of hydroponically grown oilseed rape ( Brassica napus ). The concentration of NH₄⁺ in the stem xylem sap of NO₃⁻-fed plants was 0.55–0.70 m M , which was ≈60% higher than that in plants deprived of external nitrogen for 2 days. In NH₄⁺-fed plants, xylem NH₄⁺ concentrations increased linearly both with time of exposure to NH₄⁺ and with increasing external NH₄⁺ concentration. The maximum xylem NH₄⁺ concentration was 8 m M , corresponding to 11% of the nitrogen translocated in the xylem. In the leaf apoplastic solution, the NH₄⁺ concentration increased from 0.03 m M in N-deprived plants to 0.20 m M in N-replete plants. The corresponding values for leaf tissue water were 0.33 and 1.24 m M , respectively. The addition of either NO₃⁻ or NH₄⁺ to N-starved plants induced both cytosolic gs isogene expression and GS activity in the roots. In N-replete plants, gs isogene expression and GS activity were repressed, probably due to carbon limitations, thereby protecting the roots against the excessive drainage of photosynthates. Repressed gs isogene expression and GS activity under N-replete conditions caused enhanced NH₄⁺ translocation to the shoots.     

The controlling influence of cell-surface electrical potential on the uptake and toxicity of selenate (SeO42–)

Root elongation in wheat seedlings ( Triticum aestivum L. cv. Atlas 66) was inhibited by micromolar activities of SeO₄^2–. SeO₄^2– inhibition was enhanced by supplementation of the rooting medium with CaCl₂, MgCl₂, SrCl₂, or the reduction of pH. These solute treatments, as well as the addition of tris (ethylenediamine)cobalt³⁺, enhanced the uptake of Se by the roots. The results are interpreted to reflect an elevated PM-surface activity of SeO₄^2– caused by solute-induced reductions of plasma membrane (PM) surface negativity. (PM-surface electrical potential is sometimes measured electrophoretically as the zeta potential.) This study complements an extensive literature documenting the suitability of an electrostatic model (Gouy-Chapman-Stern), based almost entirely upon experiments with cations rather than anions. The close correspondence among uptake, intoxication, and model-computed SeO₄^2– activity at the PM surface adds credibility to the model and its evaluated parameters. The model may be useful for the interpretation of other plant-anion interactions, and phosphate and sulphate nutrition in acidic soils are considered as examples.