Nitrate alleviates ammonium toxicity in Brassica napus by coordinating rhizosphere and cell pH and ammonium assimilation.

In natural and agricultural situations, ammonium ( NH 4 + ) is a preferred nitrogen (N) source for plants, but excessive amounts can be hazardous to them, known as NH 4 + toxicity. Nitrate ( NO 3 - ) has long been recognized to reduce NH 4 + toxicity. However, little is known about Brassica napus, a major oil crop that is sensitive to high NH 4 + . Here, we found that NO 3 - can mitigate NH 4 + toxicity by balancing rhizosphere and intracellular pH and accelerating ammonium assimilation in B. napus. NO 3 - increased the uptake of NO 3 - and NH 4 + under high NH 4 + circumstances by triggering the expression of NO 3 - and NH 4 + transporters, while NO 3 - and H+ efflux from the cytoplasm to the apoplast was enhanced by promoting the expression of NO 3 - efflux transporters and genes encoding plasma membrane H+ -ATPase. In addition, NO 3 - increased pH in the cytosol, vacuole, and rhizosphere, and down-regulated genes induced by acid stress. Root glutamine synthetase (GS) activity was elevated by NO 3 - under high NH 4 + conditions to enhance the assimilation of NH 4 + into amino acids, thereby reducing NH 4 + accumulation and translocation to shoot in rapeseed. In addition, root GS activity was highly dependent on the environmental pH. NO 3 - might induce metabolites involved in amino acid biosynthesis and malate metabolism in the tricarboxylic acid cycle, and inhibit phenylpropanoid metabolism to mitigate NH 4 + toxicity. Collectively, our results indicate that NO 3 - balances both rhizosphere and intracellular pH via effective NO 3 - transmembrane cycling, accelerates NH 4 + assimilation, and up-regulates malate metabolism to mitigate NH 4 + toxicity in oilseed rape.

Evidence for bidirectional formic acid translocation in vivo via the Escherichia coli formate channel FocA.

Pentameric FocA permeates either formate or formic acid bidirectionally across the cytoplasmic membrane of anaerobically growing Escherichia coli. Each protomer of FocA has its own hydrophobic pore, but it is unclear whether formate or neutral formic acid is translocated in vivo. Here, we measured total and dicyclohexylcarbodiimide (DCCD)-inhibited proton flux out of resting, fermentatively grown, stationary-phase E. coli cells in dependence on FocA. Using a wild-type strain synthesizing native FocA, it was shown that using glucose as a source of formate, DCCD-independent proton efflux was ∼2.5 mmol min-1, while a mutant lacking FocA showed only DCCD-inhibited, FOF1-ATPase-dependent proton-efflux. A strain synthesizing a chromosomally-encoded FocAH209N variant that functions exclusively to translocate formic acid out of the cell, showed a further 20 % increase in FocA-dependent proton efflux relative to the parental strain. Cells synthesizing a FocAT91A variant, which is unable to translocate formic acid out of the cell, showed only DCCD-inhibited proton efflux. When exogenous formate was added, formic acid uptake was shown to be both FocA- and proton motive force-dependent. By measuring rates of H2 production, potassium ion flux and ATPase activity, these data support a role for coupling between formate, proton and K+ ion translocation in maintaining pH and ion gradient homeostasis during fermentation. FocA thus plays a key role in maintaining this homeostatic balance in fermenting cells by bidirectionally translocating formic acid.

R125H, W240S, C386R, and V507I SLC4A11 mutations associated with corneal endothelial dystrophy affect the transporter function but not trafficking in PS120 cells.

SLC4A11 mutations are associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH3 sensitive, electrogenic H+ transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH4Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of NaK ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH4Cl when compared to wild type, indicating a decreased H+ permeability in mutants. All mutants exhibited significantly reduced H+ currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH4Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H+ flux properties.

Fluorometric determination of Vibrio parahaemolyticus using an F0F1-ATPase-based aptamer and labeled chromatophores.

An F0F1-ATPase-based aptasensor is described for the fluorometric determination of Vibrio parahaemolyticus. Chromatophores containing F0F1-ATPases were first prepared from Rhodospirillum rubrum cells. Then, an aptamer-functionalized chromatophore acts as the capture probe, and a chromatophore labeled with the pH probe fluorescein acts as the signalling probe. In the presence of V. parahaemolyticus, the rotation rate of F0F1-ATPase is decreased due to the formation of the aptamer-chromatophore complex. This leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and the fluorescence of the pH probe F1300 is accordingly decreased. The relative fluorescence varies linearly over the 15 to 1.5 × 106 cfu·mL-1 Vibrio parahaemolyticus concentration range, and the limit of detection is 15 cfu·mL-1. The method was applied to analyze artificially contaminated salmon samples where it showed excellent perfomance. Graphical abstract In this assay, aptamer functionalized chromatophores act as a capture probe, and the fluoresce in labeled chromatophores as signalling probe. The formation of aptamer-chromatophore complex leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and fluorescence intensity is accordingly decreased.

Arabidopsis plasma membrane H+-ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress.

Phosphorus deficiency in soil is one of the major limiting factors for plant growth. Plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the plant response to low-phosphorus stress (LP). However, few details are known regarding the action of PM H+-ATPase in mediating root proton (H+) flux and root growth under LP. In this study, we investigated the involvement and function of different Arabidopsis PM H+-ATPase genes in root H+ flux in response to LP. First, we examined the expressions of all Arabidopsis PM H+-ATPase gene family members (AHA1-AHA11) under LP. Expression of AHA2 and AHA7 in roots was enhanced under this condition. When the two genes were deficient in their respective Arabidopsis mutant plants, root growth and responses of the mutants to LP were highly inhibited compared with the wild-type plant. AHA2-deficient plants exhibited reduced primary root elongation and lower H+ efflux in the root elongation zone. AHA7-deficient plants exhibited reduced root hair density and lower H+ efflux in the root hair zone. The modulation of H+ efflux by AHA2 or AHA7 was affected by the action of 14-3-3 proteins and/or auxin regulatory pathways in the context of root growth and response to LP. Our results suggest that under LP conditions, AHA2 acts mainly to modulate primary root elongation by mediating H+ efflux in the root elongation zone, whereas AHA7 plays an important role in root hair formation by mediating H+ efflux in the root hair zone.

Extrabranchial mechanisms of systemic pH recovery in hagfish (Eptatretus stoutii).

This study investigates the role of branchial and extrabranchial processes in acid-base regulation in the Pacific Hagfish (Eptatretus stoutii). Hagfish were injected with one of the following solutions: acid saline (250mM HCl [pH=0.60], 250mM NaCl), alkaline saline (250mM NaHCO3, 250mM NaCl, [pH≈8.43]) or control saline (500mM NaCl) in order to achieve an acid/alkaline/saline load of 6000µmol·kg(-1). Using a custom designed hagfish compartmentalizing flux chamber, we partitioned flux of net acid or base equivalents and ammonia into the anterior (gill+skin) and posterior (skin+intestinal/renal/cloacal) components. We found that Pacific hagfish excrete H(+) primarily via branchial mechanisms but base excretion occurs through extrabranchial mechanisms located in the posterior region. In addition, we demonstrate that hagfish are able to excrete ammonia via the skin although this flux was not involved in compensation from an acid-base disturbance.

The joint action of yeast eisosomes and membraneless organelles in response to ethanol stress.

Elevated ethanol concentrations in yeast affect the plasma membrane. The plasma membrane in yeast has many lipid-protein complexes, such as Pma1 (MCP), Can1 (MCC), and the eisosome complex. We investigated the response of eisosomes, MCPs, and membraneless structures to ethanol stress. We found a correlation between ethanol stress and proton flux with quick acidification of the medium. Moreover, ethanol stress influences the symporter expression in stressed cells. We also suggest that acute stress from ethanol leads to increases in eisosome size and SG number: we hypothesized that eisosomes may protect APC symporters and accumulate an mRNA decay protein in ethanol-stressed cells. Our findings suggest that the joint action of these factors may provide a protective effect on cells under ethanol stress.

Coral reef calcification: carbonate, bicarbonate and proton flux under conditions of increasing ocean acidification.

Data on calcification rate of coral and crustose coralline algae were used to test the proton flux model of calcification. There was a significant correlation between calcification (G) and the ratio of dissolved inorganic carbon (DIC) to proton concentration ([DIC] : [H(+)] ratio). The ratio is tightly correlated with [CO3(2-)] and with aragonite saturation state (Ωa). An argument is presented that correlation does not prove cause and effect, and that Ωa and [CO3(2-)] have no basic physiological meaning on coral reefs other than a correlation with [DIC] : [H(+)] ratio, which is the driver of G.

Root-Apex Proton Fluxes at the Centre of Soil-Stress Acclimation.

Proton (H+) fluxes in plant roots play critical roles in maintaining root growth and facilitating plant responses to multiple soil stresses, including fluctuations in nutrient supply, salt infiltration, and water stress. Soil mining for nutrients and water, rates of nutrient uptake, and the modulation of cell expansion all depend on the regulation of root H+ fluxes, particularly at the root apex, mediated primarily by the activity of plasma membrane (PM) H+-ATPases. Here, we summarize recent findings on the regulatory mechanisms of H+ fluxes at the root apex under three abiotic stress conditions - phosphate deficiency, salinity stress, and water deficiency - and present an integrated physiomolecular view of the functions of H+ fluxes in maintaining root growth in the acclimation to soil stress.

Coral-algae metabolism and diurnal changes in the CO2-carbonate system of bulk sea water.

Precise measurements were conducted in continuous flow seawater mesocosms located in full sunlight that compared metabolic response of coral, coral-macroalgae and macroalgae systems over a diurnal cycle. Irradiance controlled net photosynthesis (P net), which in turn drove net calcification (G net), and altered pH. P net exerted the dominant control on [CO3 (2-)] and aragonite saturation state (Ωarag) over the diel cycle. Dark calcification rate decreased after sunset, reaching zero near midnight followed by an increasing rate that peaked at 03:00 h. Changes in Ωarag and pH lagged behind G net throughout the daily cycle by two or more hours. The flux rate P net was the primary driver of calcification. Daytime coral metabolism rapidly removes dissolved inorganic carbon (DIC) from the bulk seawater and photosynthesis provides the energy that drives G net while increasing the bulk water pH. These relationships result in a correlation between G net and Ωarag, with Ωarag as the dependent variable. High rates of H(+) efflux continued for several hours following mid-day peak G net suggesting that corals have difficulty in shedding waste protons as described by the Proton Flux Hypothesis. DIC flux (uptake) followed P net and G net and dropped off rapidly following peak P net and peak G net indicating that corals can cope more effectively with the problem of limited DIC supply compared to the problem of eliminating H(+). Over a 24 h period the plot of total alkalinity (AT ) versus DIC as well as the plot of G net versus Ωarag revealed a circular hysteresis pattern over the diel cycle in the coral and coral-algae mesocosms, but not the macroalgae mesocosm. Presence of macroalgae did not change G net of the corals, but altered the relationship between Ωarag and G net. Predictive models of how future global changes will effect coral growth that are based on oceanic Ωarag must include the influence of future localized P net on G net and changes in rate of reef carbonate dissolution. The correlation between Ωarag and G net over the diel cycle is simply the response of the CO2-carbonate system to increased pH as photosynthesis shifts the equilibria and increases the [CO3 (2-)] relative to the other DIC components of [HCO3 (-)] and [CO2]. Therefore Ωarag closely tracked pH as an effect of changes in P net, which also drove changes in G net. Measurements of DIC flux and H(+) flux are far more useful than concentrations in describing coral metabolism dynamics. Coral reefs are systems that exist in constant disequilibrium with the water column.