Effect of oxygen concentration on intracellular pH, glucose-6-phosphate and NTP content in rice (Oryza sativa) and wheat (Triticum aestivum) root tips: in vivo 31P-NMR study

Hypoxic pretreatment is known to induce anoxia tolerance in plant species sensitive to oxygen deprivation. However, we still do not have detailed information on changes in cytoplasmic and vacuolar pH (pH_cyt and pH_vac) in plants under low-oxygen availability (hypoxia) and under anoxia. To investigate this, we have studied the influence of hypoxia and anoxia on pH_cyt and pH_vac, glucose-6-phosphate (Glc-6-P) and nucleotide triphosphate (NTP) contents in rice ( Oryza sativa L.) root tips in comparison with those of wheat ( Triticum aestivum L.) with in vivo ³¹P-nuclear magnetic resonance. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units after 6 h). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt= 10^−pHcyt) for both hypoxic and anoxic conditions. Glc-6-P content increased in rice under anoxia and hypoxia. In wheat, Glc-6-P was not detectable under anoxia but increased under hypoxia. In this study, rice root tips were shown to behave as anoxia tolerant tissues. Our results suggest that the initial cytoplasmic acidification and subsequent pH_cyt are differently regulated in anoxia tolerant and intolerant plants and depend on the external oxygen concentration.     

Anoxia-induced elevation of cytosolic Ca<Superscript>2+</Superscript> concentration depends on different Ca<Superscript>2+</Superscript> sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, was investigated in plants differing in tolerance to hypoxia. The [Ca²⁺]_cyt was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca²⁺]_cyt in rice leaf protoplasts. A tenfold drop in the external Ca²⁺ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca²⁺]_cyt shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La³⁺ and EGTA) and intracellular membrane Ca²⁺-channel antagonists (Li⁺, ruthenium red and cyclosporine A) reduced the anoxic Ca²⁺-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca²⁺]_cyt. In wheat leaf protoplasts, the amplitude of the Ca²⁺-shift little depended on the external level of Ca²⁺. Wheat root protoplasts were characterized by a small shift of [Ca²⁺]_cyt under anoxia. Plasmalemma Ca²⁺-channel blockers had little effect on the elevation of cytosolic Ca²⁺ in wheat protoplasts. Intact rice seedlings absorbed Ca²⁺ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca²⁺. Verapamil abolished the Ca²⁺ influx in rice roots and Ca²⁺ efflux from wheat roots. Anoxia-induced [Ca²⁺]_cyt elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca²⁺ stores are important for anoxic [Ca²⁺]_cyt elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca²⁺]_cyt rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.     

Kinetic studies of the variations of cytoplasmic pH, nucleotide triphosphates (31P-NMR) and lactate during normoxic and anoxic transitions in maize root tips

We have followed the dynamic evolution of intracellular pH and of the intracellular concentration of nucleotides (NDP, NTP), Pi and lactate in maize root tips during the course of normoxia and anoxia transition. The intracellular pH, determined from the 31P-NMR chemical shift of the cytoplasmic P1 peak, dropped from 7.5 to 6.9 during the first few minutes after anaerobiosis. It increased again, then settled to a steady-state value of 7.1-7.2, 25 min after the beginning of the anoxic treatment. Following oxygenation, the chemical shift of the cytoplasmic Pi peak drifted gradually to its initial value. The cytoplasmic pH followed an oscillatory time course which was almost identical to the time course of NTP. Intracellular lactate accumulated steadily during the first 30 min after anaerobiosis, then its intracellular concentration remained almost constant. Following oxygenation, the intracellular concentration of lactate decreased slowly. The cytoplasmic pH followed a time course which was not identical to the time course of lactate. Following hypoxia, the pH dropped to low values long before the intracellular lactate concentration reached a steady-state equilibrium. Conversely, subsequent to oxygenation, the pH returned to normal values long before lactate. These results do not agree with the statement that cytoplasmic acidification in hypoxic maize root tips is necessarily associated with lactic acid synthesis.     

Manipulating cytoplasmic pH under anoxia: A critical test of the role of pH in the switch from aerobic to anaerobic metabolism

Ethanol production by maize (Zea mays L.) root tips, measured by an enzymic assay of the suspending medium, was correlated with changes in the cytoplasmic pH, determined by in-vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy, following the onset of anoxia. Strong evidence for the role of the cytoplasmic pH in triggering the switch to ethanol production under anoxia was obtained by: (i) varying the pH of the suspending medium between pH 4 and pH 10; and (ii) using the permeant weak base methylamine to combat the acidification of the cytoplasm induced by the anoxic conditions. Experimentally, it proved to be much easier to manipulate the cytoplasmic pH under anoxia after the pH had stabilised, rather than during the initial rapid acidification that occurred following the onset of anoxia, and in the presence of methylamine, it was possible to impose a normal aerobic cytoplasmic pH value on tissue that was metabolising anaerobically. By this means it was possible to demonstrate the reversibility of the pH effect on ethanol production under anoxia and thus to provide good evidence in support of the biochemical pH-stat model of the anoxic response. The NMR measurement of the cytoplasmic pH in the presence of methylamine was achieved by using a manganese pretreatment technique to eliminate interference between the cytoplasmic and vacuolar P_i signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.Abbreviations Caps 3-(cyclohexylamino)-1-propane sulphonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - NMR nuclear magnetic resonance - Pi inorganic phosphate We acknowledge the financial support of the Agricultural and Food Research Council and G.G.F. acknowledges the receipt of a Research Fellowship from the Royal Commission for the Exhibition of 1851.     

Response to Anoxia in Rice and Wheat Seedlings: Changes in the pH of Intracellular Compartments, Glucose-6-Phosphate Level, and Metabolic Rate

³¹P nuclear magnetic resonance spectroscopy was used to measure intracellular pH in living tissues. Oxygen deprivation caused fast cytoplasmic acidification from pH 7.4 to 7.0 in shoots of rice, Oryza sativa L. var arborio, a species highly resistant to anoxia. Acidification was complete after 10 minutes of anoxia. Alkalinization of both cytosplasm and vacuole followed thereafter. In the anoxia intolerant wheat shoots, Triticum aestivum L. var MEK, the same treatment caused a sharper cytoplasmic acidification, from pH 7.4 to 6.6, which occurred during a period of 2 hours. Cytoplasmic acidification continued with progress of anoxia and there was no vacuolar alkalinization comparable to the one observed in rice. In wheat oxyen, withdrawal also caused the reduction of both glucose-6-phosphate level and of metabolic rate. It also induced heavy losses of inorganic phosphate from tissues. Conversely, in rice, glucose-6-phosphate level and metabolic rate were increased and inorganic phosphate leakage from tissues was completely absent. These results are discussed in relation to the mechanisms of plant resistance to anoxia.     

Activity of biochemical pH-stat enzymes in cereal root tips under oxygen deficiency

The activity of the enzymes of alcoholic and lactic-acid fermentation: pyruvate decarboxylase (PDC, EC 4.1.1.1), alcohol dehydrogenase (ADH, EC 1.1.1.1), lactate dehydrogenase (LDH, EC 1.1.1.27) and the enzymes of malic acid metabolism: phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.23), NAD-dependent malate dehydrogenase (NAD-MDH, EC 1.1.1.37), and NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) involved in the operation of biochemical pH-stat was investigated in the root tips of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) under hypoxia and anoxia. Exposures lasted for 6, 12, and 18 h. The most pronounced response was detected for the enzymes of alcoholic fermentation. The activation of ADH and PDC in wheat occurred only under hypoxia, whereas in rice it was detected both under hypoxia and anoxia. The activation of LDH in wheat occurred under hypoxia, and in rice, the activity of this enzyme was slightly enhanced. The activity of the enzymes of malic acid metabolism did not change except in wheat root tips under hypoxia when PEPC activity decreased and NADP-ME activity simultaneously rose. The role of biochemical pH-stat in the regulation of cytoplasmic pH in plant cells under oxygen deficit and the mechanisms for regulating the activities of enzymes involved in biochemical pH-stat are discussed as well as the interaction between biochemical pH-stat and other mechanisms maintaining pH of plant cells. The results are analyzed within a context of intracellular pH regulation.     

Effect of anoxia and hypoxia on brain lipid metabolism

Lipid metabolism in rat brain was investigated in mild hypoxia (5–7% O₂ in nitrogen), which is associated with no apparent change in energy metabolism, and in severe anoxic conditions (ischemic anoxia), which are associated with a rapid decrease in ATP and oxygen content in brain. When brain slices were incubated with labeled glucose or acetate, the amount of labeled CO₂ produced was no different in experimental and control conditions, but the incorporation of radioactivity into brain lipids was decreased in all hypoxic and anoxic conditions. Interestingly, the incorporation of label from [¹⁴C]glucose into phosphatidylinositols was specifically inhibited by both hypoxic conditions but not by conditions associated with anoxia. The incorporation of the same labeled precursor, i.e., [¹⁴C]glucose, into fatty acids was elevated in ischemic anoxia but reduced after mild hypoxia. Because of the obvious differences in oxygen utilization in brain in anoxic and hypoxic conditions, we believe that the observed disturbances in lipid metabolism may be due to factors other than those that arise from oxygen deficiency alone.     

Effects of external pH,fusicoccin and butyrate on the cytoplasmic pH in barley root tips measured by 31P-nuclear magnetic resonance spectroscopy

³¹P-Nuclear magnetic resonance spectroscopy was used to measure the cytoplasmic pH (pH_c) in barley (Hordeum vulgare L.) root tips. As the external pH was raised from 4–10, pH_c was found to increase from 7.44 to 7.75. The sensitivity of pH_c to changes in external pH decreased with increasing external pH. Metabolic inhibition by sodium azide caused pH_c to fall by 0.3 units. Addition of 10 mM butyrate resulted in a gradual decline in pH_c, by approx. 0.3 units over 90 min. At a concentration of 1 mM, butyrate had no effect on pH_c even after 2 h. Fusicoccin caused pH_c to rise by 0.1–0.2 units. In maize (Zea mays L.) root tips, pH_c was shown to have a similar sensitivity to fusicoccin. The results are discussed in relation to the regulation of pH_c and the possible role of pH_c in determining transmembrane electrical potential differences.Abbreviations and symbols FC Fusicoccin - NMR nuclear magnetic resonance - p.d. membrane electrical potential difference - pH_c cytoplasmic pH - P1 inorganic phosphate - chemical shift     

Further Evidence that Cytoplasmic Acidosis Is a Determinant of Flooding Intolerance in Plants

We present two pieces of evidence that regulation of cytoplasmic pH near neutrality is a prerequisite for survival of root tips during hypoxia. First, blackeye peas and navy beans show earlier cytoplasmic acidosis under hypoxia than soybeans or pumpkin or maize, and die earlier. Second, when cytoplasmic acidosis in maize root tips is greatly retarded by treatment with 25 millimolar Ca(NO₃)₂, they remain viable under hypoxia for a much longer period of time than untreated hypoxic root tips. We also show that viability of maize root tips is unaffected by the supply of exogenous sugar (and so on the rate of ethanolic fermentation) for at least 16 hours of hypoxia.     

Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy

³¹P-, ¹³C-, and ¹⁵N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and γ-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitrogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gln, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from Glc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the patterns of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.     

Anoxic Responses of Seedling Roots of Rice Cultivars Varying in Tolerance to Submergence

Differences in tolerance to submergence and anoxia exhibited by cultivar-specific rice (Oryza sativa L.) extend to the primary root tips and axes of 3-day-old seedlings. This paper considers the physiological mechanisms which might account for rice root intolerance to anoxia, particularly those implicated in pH regulation and sugar metabolism in relation to hypoxic acclimation. Hypoxic treatment and the presence of glucose during anoxia did not permit root tips and axes of intolerant cultivars to survive 24-h anoxia. The absence of typical glycolytic and fermentative enzyme induction together with no improvement of ethanol production and energy status during anoxia suggest that intolerant cultivars are not capable of hypoxic acclimation at the level of energy and sugar metabolism. However, root tip survival was enhanced in buffered medium after hypoxic treatment, suggesting a relationship between hypoxic treatment and improved pH regulation.     

Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant

Summary In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pH_cyt) were monitored in mesophyll protoplasts from the CAM plantKalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pH_cyt indicator. At the beginning of the light phase, pH_cyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pH_cyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring ΔpH_cyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H⁺-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pH_cyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pH_cyt. A diurnal oscillation in pH_cyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.     

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells

Anoxia induces a rapid elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca²⁺ release is important for gene activation and survival in O₂-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca²⁺ to the anoxic [Ca²⁺]_cyt elevation in maize cells. Imaging of intramitochondrial Ca²⁺ levels showed that a majority of mitochondria released their Ca²⁺ in response to anoxia and took up Ca²⁺ upon reoxygenation. We also investigated whether the mitochondrial Ca²⁺ release contributed to the increase in [Ca²⁺]_cyt under anoxia. Analysis of the spatial association between anoxic [Ca²⁺]_cyt changes and the distribution of mitochondrial and other intracellular Ca²⁺ stores revealed that the largest [Ca²⁺]_cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca²⁺]_cyt under aerobic conditions but prevented a [Ca²⁺]_cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca²⁺]_cyt and participate in the signaling of O₂ deprivation.     

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.     

Nucleotide Levels Do Not Critically Determine Survival of Maize Root Tips Acclimated to a Low-Oxygen Environment

We tested the hypothesis that ATP levels and energy charge determine the resistance of maize (Zea mays) root tips to anoxia. We focused on root tips of whole maize seedlings that had been acclimated to low O2 by exposure to an atmosphere of 3% (v/v) O2 in N2. Acclimated anoxic root tips characteristically have higher ATP levels and energy charge and survive longer under anoxia than nonacclimated tips. We poisoned intact, acclimated root tips with either fluoride or mannose, causing decreases in ATP and energy charge to values similar to or, in most cases, below those found in nonacclimated anoxic tips. With the exception of the highest fluoride concentration used, the poisoned, acclimated tips remained much more tolerant of anoxia than nonacclimated root tips. We conclude that high ATP and energy charge are not components critical for the survival of acclimated root tips during anoxia. The reduced nucleotide status in poisoned, acclimated root tips had little effect on cytoplasmic pH regulation during anoxia. This result indicates that in anoxic, acclimated root tips either cytoplasmic pH regulation is not dominated by ATP-dependent processes or these processes can continue in vivo largely independently of any changes in ATP levels in the physiological range. The role of glycolytic flux in survival under anoxia is discussed.     

Metabolic Acclimation to Anoxia Induced by Low (2-4 kPa Partial Pressure) Oxygen Pretreatment (Hypoxia) in Root Tips of Zea mays

Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1⁻ mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of NO₃⁻. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO₃⁻ to NO₂⁻. These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl⁻ mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours.     

Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels

Activity of voltage-gated potassium (Kv) channels controls membrane potential, which subsequently regulates cytoplasmic free calcium concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMCs). Acute hypoxia inhibits Kv channel function in PASMCs, inducing membrane depolarization and a rise in [Ca²⁺ ]_cyt that triggers vasoconstriction. Prolonged hypoxia inhibits expression of Kv channels and reduces Kv channel currents in PASMCs. The consequent membrane depolarization raises [Ca²⁺]_cyt, thus stimulating PASMC proliferation. The present review discusses recent evidence for the involvement of Kv channels in initiation of hypoxic pulmonary vasoconstriction and in chronic hypoxia-induced pulmonary hypertension.     

Calcium supply effects on wheat cultivars differing in salt resistance with special reference to leaf cytosol ion homeostasis

Salinity causes changes in cytosolic Ca²⁺, [Ca²⁺]_cyt, Na⁺, [Na⁺]_cyt and pH, pH_cyt, which induce specific reactions and signals. Reactions causing a rebalancing of the physiological homeostasis of the cytosol could result in plant resistance and growth. Two wheat cultivars, Triticum aestivum, Seds1 and Vinjett, were grown in nutrient solution for 7 days under moderate salinity (0 and 50 mM NaCl) with and without extra addition of 5 mM CaSO₄ to investigate the seedling‐ion homeostasis under salinity. In the leaf protoplasts [Ca²⁺]_cyt, [Na⁺]_cyt and pH_cyt were detected using acetoxymethyl esters of the ion‐specific dyes, Fura 2, SBFI and BCECF, respectively, and fluorescence microscopy. In addition, both cultivars were grown for 3 weeks at 0, 50 and 125 mM NaCl with, or without, extra addition of 5 mM CaSO₄ to detect overall Na⁺ and Ca²⁺ concentrations in leaves and salinity effects on dry weights. In both cultivars, salinity decreased [Ca²⁺]_cyt, while at extra Ca²⁺ supplied, [Ca²⁺]_cyt increased. The [Ca²⁺]_cyt increase was accompanied by increase in the overall Ca²⁺ concentrations in leaves and decrease in the overall Na⁺ concentration. Moreover, irrespective of Ca²⁺ treatment under salinity, the cultivars reacted in different ways; [Na⁺]_cyt significantly increased only in cv. Vinjett, while pH_cyt increased only in cv. Seds1. Even at rather high total Na⁺ concentrations, the cytosolic concentrations were kept low in both cultivars. It is discussed whether the increase of [Ca²⁺]_cyt and pH_cyt can contribute to salt tolerance and if the cytosolic changes are due to changes in overall Ca²⁺ and Na⁺ concentrations.     

Cytosolic calcium and pH signaling in plants under salinity stress

Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pH_cyt, either individually, or in coordination with changes in cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca²⁺ and protons. The variety and complexity of Ca²⁺ and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca²⁺]_cyt concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca²⁺]_cyt and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca²⁺]_cyt and pH_cyt at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.Key words: cytosolic calcium, ionic toxicity, osmotic stress, pH, salinity stress, salt tolerance, signaling     

Relationship between intracellular pH and N metabolism in maize (Zea mays L.) roots

In vivo ³¹P nuclear magnetic resonance (NMR) was used to characterize the effect of the N form (NO₃ vs. NH₄) and the external pH (4, 6, and 8), on the intracellular pH of root tips (0–5 mm) and root segments (5–30 mm). Ammonium-grown root tips were the most sensitive to changes in the external pH. In vivo ¹⁵N NMR was used to characterize the pathway of primary ammonium assimilation in the ammonium-grown roots and to compare the activity of the apical and more-basal root parts. The kinetics of ¹⁵NH₄ ⁺ incorporation showed that primary assimilation in both root tips and root segments followed the glutamine synthetase (GS) pathway. In agreement with the reported gradient of GS along the seminal root of maize, incorporation of label into glutamine amide was more rapid in tips than in segments. It is suggested that this higher GS activity increases the endogenous proton production and thus contributes to the greater dependence of the cytoplasmic pH on the external pH in the ammonium-treated root tips.