Effect of pH and calcium on short-term NO3- fluxes in roots of barley seedlings

The effect of pH and Ca2+ on net NO3- uptake, influx, and efflux by intact roots of barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with NO3- or NO2-. Net NO3- uptake and efflux, respectively, were determined by following its depletion from, and accumulation in, the external solution. Since roots of both uninduced and NO2(-)-induced seedlings contain little internal NO3- initial net uptake rates are equivalent to influx (M. Aslam, R.L. Travis, R.C. Huffaker [1994] Plant Physiol 106: 1293-1301). NO3-, uptake (influx) by these roots was little affected at acidic pH. In contrast, in NO3(-)-induced roots, which accumulate NO3-, net uptake rates decreased in response to acidic pH. Under these conditions, NO3- efflux was stimulated and was a function of root NO3- concentration. Conversely, at basic pH, NO3- uptake by NO3- and NO2(-)-induced and uninduced roots decreased, apparently because of the inhibition of influx. Calcium had little effect on NO3- uptake (influx) by NO2(-)-induced roots at either pH 3 or 6. However, in NO3(-)-induced roots, lack of Ca2+ at pH 3 significantly decreased net NO3- uptake and stimulated efflux. The results indicate that at acidic pH the decrease in net NO3- uptake is due to the stimulation of efflux, whereas at basic pH, it is due to the inhibition of influx.     

Stimulation of Nitrate and Nitrite Efflux by Ammonium in Barley (Hordeum vulgare L.) Seedlings

The inhibitory effect of NH4+ on net NO3- uptake has been attributed to an enhancement of efflux and, recently, to an inhibition of influx. To study this controversy, we devised treatments to distinguish the effects of NH4+ on these two processes. Roots of intact barley (Hordeum vulgare L.) seedlings, uninduced or induced with NO3- or NO2-, were used. Net uptake and efflux, respectively, were determined by following the depletion and accumulation in the external solutions. In roots of both uninduced and NO2- -induced seedlings, NO3- efflux was negligible; hence, the initial uptake rates were equivalent to influx. Under these conditions, NH4+ had little effect on NO3- uptake (influx) rates by either the low- or high-Km uptake systems. In contrast, in plants preloaded with NO3-, NH4+ and its analog CH3NH3+ decreased net uptake, presumably by enhancing NO3- efflux. The stimulatory effect of NH4+ on NO3- efflux was a function of external NH4+ and internal NO3- concentration. These results were corroborated by the absence of any effect of NH4+ on NO2- uptake unless the roots were preloaded with NO2-. In this case NH4+ increased efflux and decreased net uptake. Hence, the main effect of NH4+ on net NO3- and NO2- uptake appears to be due to enhancement of efflux and not to inhibition of influx.     

Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.     

Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation

Soybean [Glycine max (L.) Merrill] plants that had been subjected to 15 d of nitrogen deprivation were resupplied for 10 d with 1.0 mol m-3 nitrogen provided as NO3-, NH4+, or NH4(+) + NO3- in flowing hydroponic culture. Plants in a fourth hydroponic system received 1.0 mol m-3 NO3- during both stress and resupply periods. Concentrations of soluble carbohydrates and organic acids in roots increased 210 and 370%, respectively, during stress. For the first day of resupply, however, specific uptake rates of nitrogen, determined by ion chromatography as depletion from solution, were lower for stressed than for non-stressed plants by 43% for NO3- resupply, by 32% for NH4(+) + NO3- resupply, and 86% for NH4+ resupply. When specific uptake of nitrogen for stressed plants recovered to rates for non-stressed plants at 6 to 8 d after nitrogen resupply, carbohydrates and organic acids in their roots had declined to concentrations lower than those of non-stressed plants. Recovery of nitrogen uptake capacity of roots thus does not appear to be regulated simply by the content of soluble carbon compounds within roots. Solution concentrations of NH4+ and NO3- were monitored at 62.5 min intervals during the first 3 d of resupply. Intermittent 'hourly' intervals of net influx and net efflux occurred. Rates of uptake during influx intervals were greater for the NH4(+)-resupplied than for the NO3(-)-resupplied plants. For NH4(+)-resupplied plants, however, the hourly intervals of efflux were more numerous than for NO3(-)-resupplied plants. It thus is possible that, instead of repressing NH4+ influx, increased accumulation of amino acids and NH4+ in NH4(+)-resupplied plants inhibited net uptake by stimulation of efflux on NH4+ absorbed in excess of availability of carbon skeletons for assimilation. Entry of NH4+ into root cytoplasm appeared to be less restricted than translocation of amino acids from the cytoplasm into the xylem.     

Induction and Turnover of Nitrate Reductase in Zea mays (Influence of NO3-)

Zea mays (cv W64A x W182E) was used to investigate the induction and turnover of nitrate reductase (NR). In our system, 5 or 10 mM KNO3 gave the best growth over a 6-d growing period. With these NO3- levels, NR reached steady-state levels after 24 h. For the turnover experiments, the seedlings were transferred to a NO3--free medium after a 24-h induction. Shoot NR was less sensitive to the removal of NO3- than root NR, which declined almost as soon as NO3- was removed when the seedlings were induced with 5 or 10 mM NO3-. With 1 mM NO3-, however, removal of NO3- from medium resulted in declines in both NR activity and NO3- in shoot and root. Although there was a delay in the degradation of NR protein relative to the loss of NR activity, this protein was not reactivated when NO3- was resupplied. These results indicate that NO3- regulates NR by influencing the de novo synthesis of the NR protein and not by a reversible activation-inactivation of that protein.     

Comparative Induction of Nitrate and Nitrite Uptake and Reduction Systems by Ambient Nitrate and Nitrite in Intact Roots of Barley (Hordeum vulgare L.) Seedlings

The induction by ambient NO3- and NO2- of the NO3- and NO2- uptake and reduction systems in roots of 8-d-old intact barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with concentrations of NaNO3 or NaNO2 ranging from 0.25 to 1000 [mu]M. Uptake was determined by measuring the depletion of either NO3- or NO2- from uptake solutions. Enzyme activities were assayed in vitro using cell-free extracts. Uptake and reduction systems for both NO3- and NO2- were induced by either ion. The Km values for NO3- and NO2- uptake induced by NO2- were similar to those for uptake induced by NO3-. Induction of both the uptake and reduction systems was detected well before any NO3- or NO2- was found in the roots. At lower substrate concentrations of both NO3- and NO2- (5-10 [mu]M), the durations of the lag periods preceding induction were similar. Induction of uptake, as a function of concentration, proceeded linearly and similarly for both ions up to about 10 [mu]M. Then, while induction by NO3- continued to increase more slowly, induction by NO2- sharply decreased between 10 and 1000 [mu]M, apparently due to NO2- toxicity. In contrast, induction of NO3- reductase (NR) and NO2- reductase (NiR) by NO2- did not decrease above 10 [mu]M but rather continued to increase up to a substrate concentration of 1000 [mu]M. NO3- was a more effective inducer of NR than was NO2-; however, both ions equally induced NiR. Cycloheximide inhibited the induction of both uptake systems as well as NR and NiR activities whether induced by NO3- or NO2-. The results indicate that in situ NO3- and NO2- induce both uptake and reduction systems, and the accumulation of the substrates per se is not obligatory.     

Influence of exogenous sugars and polyols on C1-influx and efflux by the ascomycete Neocosmospora vasinfecta.

Glucose and other transportable sugars and polyols inhibited Cl- influx very soon after addition to mycelium in the process of Cl- accumulation. Under the usual experimental conditions (0.1 mM KCl, glucose greater than or equal to 2 mM) the mean percentage of inhibition of Cl- influx by glucose was 54.1 +/- 8.0 (+/- standard error; N = 26). Transport of the exogenous carbohydrate was necessary for inhibition of Cl- influx. Thus, the estimated Ki for glucose inhibition of Cl- influx (28 muM) was close to the Km for glucose transport; glycerol did not inhibit Cl- influx unless it was itself transported, and the degree of inhibition exerted by various carbohydrates correlated with their uptake rates. Inhibition was not caused by the accumulated sugar itself, as high levels (ca. 60 mM) of intramycelial 3-O-methylglucose gave rise to a stimulation of Cl- influx when the exogenous sugar was removed. It is suggested that interaction of Cl- and carbohydrate transport arises from competition for a common energy-coupling mechanism in the cell membrane. Both glucose and 3-O-methylglucose elicited Cl- efflux, but the maximal Cl- efflux rates were observed only after 40 min of incubation and only in the presence of the readily metabolizable glucose. Removal of the exogenous glucose, even after maximal Cl- efflux had been established, resulted in the rapid cessation of efflux. Studies under anaerobic conditions gave further evidence that glucose uptake was necessary and that efflux was not due to temporary depletion of energy reserves. It is proposed that glucose-induced leakage of Cl- is due to reversal of the Cl- uptake system, even though the Km for efflux is much greater than that for influx.     

Nitrate transport and signalling

Physiological measurements of nitrate (NO(3)(-)) uptake by roots have defined two systems of high and low affinity uptake. In Arabidopsis, genes encoding both of these two uptake systems have been identified. Most is known about the high affinity transport system (HATS) and its regulation and yet measurements of soil NO(3)(-) show that it is more often available in the low affinity range above 1 mM concentration. Several different regulatory mechanisms have been identified for AtNRT2.1, one of the membrane transporters encoding HATS; these include feedback regulation of expression, a second component protein requirement for membrane targeting and phosphorylation, possibly leading to degradation of the protein. These various changes in the protein may be important for a second function in sensing NO(3)(-) availability at the surface of the root. Another transporter protein, AtNRT1.1 also has a role in NO(3)(-) sensing that, like AtNRT2.1, is independent of their transport function. From the range of concentrations present in the soil it is proposed that the NO(3)(-)-inducible part of HATS functions chiefly as a sensor for root NO(3)(-) availability. Two other key NO(3)(-) transport steps for efficient nitrogen use by crops, efflux across membranes and vacuolar storage and remobilization, are discussed. Genes encoding vacuolar transporters have been isolated and these are important for manipulating storage pools in crops, but the efflux system is yet to be identified. Consideration is given to how well our molecular and physiological knowledge can be integrated as well to some key questions and opportunities for the future.     

A comparison of ammonium, nitrate and proton net fluxes along seedling roots of Douglas-fir and lodgepole pine grown and measured with different inorganic nitrogen sources

Significant spatial variability in NH4+, NO3- and H+ net fluxes was measured in roots of young seedlings of Douglas-fir (Pseudotsuga menziesii) and lodgepole pine (Pinus contorta) with ion-selective microelectrodes. Seedlings were grown with NH4+, NO3-, NH4NO3 or no nitrogen (N), and were measured in solutions containing one or both N ions, or no N in a full factorial design. Net NO3- and NH4+ uptake and H+ efflux were greater in Douglas-fir than lodgepole pine and in roots not exposed to N in pretreatment. In general, the rates of net NH4+ uptake were the same in the presence or absence of NO3-, and vice versa. The highest NO3- influx occurred 0-30 mm from the root apex in Douglas-fir and 0-10 mm from the apex in lodgepole pine. Net NH4+ flux was zero or negative (efflux) at Douglas-fir root tips, and the highest NH4+ influx occurred 5-20 mm from the root tip. Lodgepole pine had some NH4+ influx at the root tips, and the maximum net uptake 5 mm from the root tip. Net H+ efflux was greatest in the first 10 mm of roots of both species. This study demonstrates that nutrient uptake by conifer roots can vary significantly across different regions of the root, and indicates that ion flux profiles along the roots may be influenced by rates of root growth and maturation.     

Nitrate transport across the tonoplast of Cucumis sativus L. root cells

Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cucumber roots grown in nitrate. The addition of NO3- ions into the tonoplast with ATP-generated transmembrane proton gradient caused the dissipation of delta pH, indicating the NO3(-)-induced proton efflux from vesicles. NO3(-)-dependent H+ efflux was almost insensitive to the transmembrane electrical potential difference, suggesting the presence of an electroneutral NO3-/H+ antiporter in the tonoplast. Apart from saturation kinetics, with respect to nitrate ions, NO3(-)-linked H+ efflux from the tonoplast of cucumber roots showed other characteristics expected of substrate-specific transporters. Experiments employing protein modifying reagents (NEM, pCMBS, PGO and SITS) indicated that a crucial role in the activity of tonoplast nitrate/proton antiporter is played by lysine residues (strong inhibition of NO3-/H+ antiport by SITS). None of the ion-channel inhibitors (NIF, ZnSO4 and TEA-Cl) used in the experiments had a direct effect on the nitrate transport into tonoplast membranes. On the other hand, every protein reagent, as well as NIF and ZnSO4, significantly affected the ATP-dependent proton transport in vesicles. Only TEA-Cl, the potassium channel blocker, had no effect on the vacuolar proton pumping activity.     

Regulation of nitrate reductase in excised barley roots

When excised barley roots (Hordeum distichum L.) are appropriately pretreated, the level of nitrate reductase in the roots increases upon exposure to nitrate. Relatively low levels of nitrate (10 mum) gave maximum induction of nitrate reductase. This increase was inhibited by inhibitors of protein and RNA synthesis, indicating that de novo protein synthesis is probably involved. Induction of nitrate reductase by nitrate is partially prevented by the inclusion of ammonium, an eventual product of nitrate reduction, in the incubation medium. Under the experimental conditions used, ammonium did not inhibit the uptake of nitrate by excised barley roots. It is concluded, therefore, that ammonium, or a product of ammonium metabolism, has a direct effect on the synthesis of nitrate reductase in this tissue.     

Protein-carbohydrate interaction. A turbidimetric study of the interaction of concanavalin A with amylopectin and glycogen and some of their enzymic and chemical degradation products

1. RNA and protein synthesis was studied during the incubation of excised radish cotyledons in nitrate, conditions that induced nitrate reductase activity in the tissue. 2. Synthesis of total RNA and protein, as measured by the incorporation of radioactive precursor, was significantly stimulated in the presence of nitrate (compared with chloride control), but was decreased in the presence of ammonium nitrate, which induced higher enzyme activity. 3. Synthesis of RNA and protein was required for induction of enzyme activity, as determined by using the inhibitors actinomycin D, puromycin and cycloheximide. 4. On the basis of 5-fluorouracil inhibition, the synthesis of only DNA-like RNA was required for induction, but no differences, either quantitative or qualitative, were observed in DNA-like RNA synthesis in the presence or absence of induction. 5. A 100-fold purification of the nitrate reductase activity showed no increase in nitrate reductase protein, nor any increased incorporation of radioactive precursor into nitrate reductase protein in the induced versus the control system. Such results suggested that the protein synthesis required for induction may be for a protein other than nitrate reductase.     

Effects of fluoride and vanadate on K+ transport across the erythrocyte membrane of Rana temporaria

K-Cl cotransport activity in frog erythrocytes was estimated as a Cl- -dependent component of K+ efflux from cells incubated in Cl- - or NO3- -containing medium at 20 degrees C. Decreasing the osmolality of the medium resulted in an increase in K+ efflux from the cells in a Cl- medium but not in an NO3- medium. Treatment of red cells with 5 mM NaF caused a significant decrease (approximately 50%) in K+ loss from the cells in iso- and hypotonic Cl- media but only a small decrease in K+ loss in isotonic NO3- medium. Addition of 1 mM vanadate to an isotonic Cl- medium also led to a significant reduction in K+ efflux. Similar inhibitory effects of NaF and vanadate on K+ efflux in a Cl- medium, but not in an NO3- medium were observed when the incubation temperature was decreased from 20 to 5 degrees C. Thus, under various experimental conditions, NaF and vanadate inhibited about 50% of Cl- -dependent K+ efflux from frog red cells probably due to inhibition of protein phosphatases. Cl- -dependent K+ (86Rb) influx into frog erythrocytes was nearly completely blocked (approximately 94%) by 5 mM NaF. In a NO3- medium, K+ influx was mainly mediated by the Na+,K+ pump and was unchanged in the presence of 5 mM NaF, 0.03 mM Al3+ or their combination. These data indicate that G proteins or cAMP are not involved in the regulation of Na+,K+ pump activity which is activated by catecholamines and phosphodiesterase blockers in these cells.     

Magnesium ameliorates aluminum rhizotoxicity in soybean by increasing citric acid production and exudation by roots

Superior effectiveness of Mg over Ca in alleviating Al rhizotoxicity cannot be accounted for by predicted changes in plasma membrane Al3+ activity. The influence of Ca and Mg on the production and secretion of citrate and malate, and on Al accumulation by roots was investigated with soybean genotypes Young and PI 416937 which differ in Al tolerance. In the presence of a solution Al3+ activity of 4.6 microM, citrate and malate concentrations of tap root tips of both genotypes increased with additions of either Ca up to 3 mM or Mg up to 50 microM. Citrate efflux rate from roots exposed to Al was only enhanced with Mg additions and exceeded malate efflux rates by as much as 50-fold. Maximum citrate release occurred within 12 h after adding Mg to solution treatments. Adding 50 microM Mg to 0.8 mM CaSO4 solutions containing Al3+ activities up to 4.6 microM increased citrate concentration of tap root tips by 3- to 5-fold and root exudation of citrate by 6- to 9-fold. Plants treated with either 50 microM Mg or 3 mM Ca had similar reductions in Al accumulation at tap root tips, which coincided with the respective ability of these ions to relieve Al rhizotoxicity. Amelioration of Al inhibition of soybean root elongation by low concentrations of Mg in solution involved Mg-stimulated production and efflux of citrate by roots.     

Cyclic variations in nitrogen uptake rate of soybean plants: effects of external nitrate concentration

Net uptake of NO3- by non-nodulated soybean plants [Glycine max (L.) Merr. cv. Ransom] growing in flowing hydroponic cultures containing 0.5, 1.0 and 10.0 mol m-3 NO3- was measured daily during a 24-d period of vegetative development to determine if amplitude of maximum and minimum rates of net NO3- uptake are responsive to external concentrations of NO3-. Removal of NO3- from the replenished solutions during each 24-h period was determined by ion chromatography. Neither dry matter accumulation nor the periodicity of oscillations in net uptake rate was altered by the external NO3- concentrations. The maxima of the oscillations in net uptake rate, however, increased nearly 3-fold in response to external NO3- concentrations. The maxima and minima, respectively, changed from 4.0 and 0.6 mmol NO3- per gram root dry weight per day at an external solution level of 0.5 mol m-3 NO3- to 15.2 and -2.7 mmol NO3- per gram root dry weight per day at an external solution level of 10.0 mol m-3 NO3-. The negative values for minimum net uptake rate from 10.0 mol m-3 NO3- solutions show that net efflux was occurring and indicate that the magnitude of the efflux component of net uptake was responsive to external concentration of NO3-.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Enhancing phytoremediative ability of Pisum sativum by EDTA application

The aim of our research was to demonstrate how the presence of EDTA affects resistance of pea plants to Pb and Pb-EDTA presence, and to show the effectivity of lead ions accumulation and translocation. It was determined that EDTA not only increased the amount of Pb taken up by plants but also Pb ion transport through the xylem and metal translocation from roots to stems and leaves. It can be seen in the presented research results that addition of the chelator with Pb limited metal phytotoxicity. We also demonstrated a significant effect of EDTA not only on Pb accumulation and metal transport to the aboveground parts but also on the profile and amount of thiol compounds: glutathione (GSH), homoglutathione (hGSH) or phytochelatins (PCs), synthesized by the plants. We observed a significant effect of the synthetic chelator on increasing the level of Pb accumulation in roots of plants treated with Pb including EDTA (0.5 and 1 mM). Pisum sativum plants treated only with 1 mM Pb(NO3)2 accumulated over 50 mg Pb x g(-1) dry wt during 4 days of cultivation. Whereas in roots of pea plants exposed to Pb+0.5 mM EDTA 35% more Pb was observed. When 1 mM EDTA was applied roots of pea accumulated over 67% more metal. The presence of EDTA also increased metal uptake and transport to the aboveground parts. In pea plants treated only with 1 mM lead nitrate less than 3 mg Pb x g(-1) dry wt was transported, whereas in P. sativum treated with Pb-EDTA doubled amount of Pb was observed in stems and leaves.