Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes

Neutral carrier-based liquid membrane ion-selective microelectrodes for NH₄⁺ and NO₃⁻ were developed and used to investigate inorganic nitrogen acquisition in two varieties of barley, Hordeum vulgare L. cv Olli and H. vulgare L. cv Prato, originating in cold and warm climates, respectively. In the present paper, the methods used in the fabrication of ammonium- and nitrate-selective microelectrodes are described, and their application in the study of inorganic nitrogen uptake is demonstrated. Net ionic fluxes of NH₄⁺ and NO₃⁻ were measured in the unstirred layer of solution immediately external to the root surface. The preference for the uptake of a particular ionic form was examined by measuring the net flux of the predominant form of inorganic nitrogen, with and without the alternative ion in solution. Net flux of NH₄⁺ into the cold-adapted variety remained unchanged when equimolar concentrations (200 micromolar) of NH₄⁺ and NO₃⁻ were present. Similarly, net flux of NO₃⁻ into the warm-adapted variety was not affected when NH₄⁺ was also present in solution. The high temporal and spatial resolution afforded by ammonium- and nitrate-selective microelectrodes permits a detailed examination of inorganic nitrogen acquisition and its component ionic interactions.     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4)

The influence of NH₄⁺, in the external medium, on fluxes of NO₃⁻ and K⁺ were investigated using barley (Hordeum vulgare cv Betzes) plants. NH₄⁺ was without effect on NO₃⁻ (³⁶ClO₃⁻) influx whereas inhibition of net uptake appeared to be a function of previous NO₃⁻ provision. Plants grown at 10 micromolar NO₃⁻ were sensitive to external NH₄⁺ when uptake was measured in 100 micromolar NO₃⁻. By contrast, NO₃⁻ uptake (from 100 micromolar NO₃⁻) by plants previously grown at this concentration was not reduced by NH₄⁺ treatment. Plants pretreated for 2 days with 5 millimolar NO₃⁻ showed net efflux of NO₃⁻ when roots were transferred to 100 micromolar NO₃⁻. This efflux was stimulated in the presence of NH₄⁺. NH₄⁺ also stimulated NO₃⁻ efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO₃⁻ occur via effects upon efflux. By contrast to the situation for NO₃⁻, net K⁺ uptake and influx of ³⁶Rb⁺-labeled K⁺ was inhibited by NH₄⁺ regardless of the nutrient history of the plants. Inhibition of net K⁺ uptake reached its maximum value within 2 minutes of NH₄⁺ addition. It is concluded that the latter ion exerts a direct effect upon K⁺ influx.     

Differential Inhibition by Ferulic Acid of Nitrate and Ammonium Uptake in Zea mays L

The influence of the allelopathic compound ferulic acid (FA) on nitrogen uptake from solutions containing both NO₃⁻ and NH₄⁺ was examined in 8-day-old nitrogen-depleted corn (Zea mays L.) seedlings. Concurrent effects on uptake of Cl⁻ and K⁺ also were assessed. The presence of 250 micromolar FA inhibited the initial (0-1 hours) rate of NO₃⁻ uptake and also prevented development of the NO₃⁻-inducible accelerated rate. The pattern of recovery when FA was removed was interpreted as indicating a rapid relief of FA-restricted NO₃⁻ uptake activity, followed by a reinitiation of the induction of that activity. No inhibition of NO₃⁻ reduction was detected. Ammonium uptake was less sensitive than NO₃⁻ uptake to inhibition by FA. An inhibition of Cl⁻ uptake occurred as induction of the NO₃⁻ transport system developed in the absence of FA. Alterations of Cl⁻ uptake in the presence of FA were, therefore, a result of a beneficial effect, because NO₃⁻ uptake was restricted, and a direct inhibitory effect. The presence of FA increased the initial net K⁺ loss from the roots during exposure to the low K, ammonium nitrate uptake solution and delayed the recovery to positive net uptake, but it did not alter the general pattern of the response. The implications of the observations are discussed for growth of plants under natural conditions and cultural practices that foster periodic accumulation of allelopathic substances.     

Measurement of Net Fluxes of Ammonium and Nitrate at the Surface of Barley Roots Using Ion-Selective Microelectrodes : II. Patterns of Uptake Along the Root Axis and Evaluation of the Microelectrode Flux Estimation Technique

Net fluxes of NH₄⁺ and NO₃⁻ into roots of 7-day-old barley (Hordeum vulgare L. cv Prato) seedlings varied both with position along the root axis and with time. These variations were not consistent between replicate plants; different roots showed unique temporal and spatial patterns of uptake. Axial scans of NH₄⁺ and NO₃⁻ net fluxes were conducted along the apical 7 centimeters of seminal roots of intact barley seedlings in solution culture using ion-selective microelectrodes in the unstirred layer immediately external to the root surface. Theoretically derived relationships between uptake and concentration gradients, combined with experimental observations of the conditions existing in our experimental system, permitted evaluation of the contribution of bulk water flow to ion movement in the unstirred layer, as well as a measure of the spatial resolution of the microelectrode flux estimation technique. Finally, a method was adopted to assess the accuracy of this technique.     

The Influence of Nitrate and Chloride Uptake on Expressed Sap pH, Organic Acid Synthesis, and Potassium Accumulation in Higher Plants

The influence of NO₃⁻ uptake and reduction on ionic balance in barley seedlings (Hordeum vulgare, cv. Compana) was studied. KNO₃ and KCl treatment solutions were used for comparison of cation and anion uptake. The rate of Cl⁻ uptake was more rapid than the rate of NO₃⁻ uptake during the first 2 to 4 hours of treatment. There was an acceleration in rate of NO₃⁻ uptake after 4 hours resulting in a sustained rate of NO₃⁻ uptake which exceeded the rate of Cl⁻ uptake. The initial (2 to 4 hours) rate of K⁺ uptake appeared to be independent of the rate of anion uptake. After 4 hours the rate of K⁺ uptake was greater with the KNO₃ treatment than with the KCl treatment, and the solution pH, cell sap pH, and organic acid levels with KNO₃ increased, relative to those with the KCl treatment. When absorption experiments were conducted in darkness, K⁺ uptake from KNO₃ did not exceed K⁺ uptake from KCl. We suggest that the greater uptake and accumulation of K⁺ in NO₃⁻-treated plants resulted from (a) a more rapid, sustained uptake and transport of NO₃⁻ providing a mobile counteranion for K⁺ transport, and (b) the synthesis of organic acids in response to NO₃⁻ reduction increasing the capacity for K⁺ accumulation by providing a source of nondiffusible organic anions.     

Early effects of salinity on nitrate assimilation in barley seedlings

The effect of NaCl and Na₂SO₄ salinity on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO₃⁻ transporter was affected very little; the major effect of the salts was on its activity. Both Cl⁻ and SO₄²⁻ salts severely inhibited uptake of NO₃⁻. When compared on the basis of osmolality of the uptake solutions, Cl⁻ salts were more inhibitory (15-30%) than SO₄²⁻ salts. At equal concentrations, SO₄²⁻ salts inhibited NO₃⁻ uptake 30 to 40% more than did Cl⁻ salts. The absolute concentrations of each ion seemed more important as inhibitors of NO₃⁻ uptake than did the osmolality of the uptake solutions. Both K⁺ and Na⁺ salts inhibited NO₃⁻ uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.

Unlike NO₃⁻ uptake, NO₃⁻ reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO₃⁻ in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the `anaerobic' in vivo assay, was always greater than the actual in situ rate of NO₃⁻ reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO₃⁻ transporter, rather than that of the NO₃⁻ reduction system, may be a critical factor to plant survival during salt stress.

     

Effect of ammonium on nitrate utilization by roots of dwarf bean

The effect of exogenous NH₄⁺ on NO₃⁻ uptake and in vivo NO₃⁻ reductase activity (NRA) in roots of Phaseolus vulgaris L. cv Witte Krombek was studied before, during, and after the apparent induction of root NRA and NO₃⁻ uptake. Pretreatment with NH₄Cl (0.15-50 millimolar) affected neither the time pattern nor the steady state rate of NO₃⁻ uptake.

When NH₄⁺ was given at the start of NO₃⁻ nutrition, the time pattern of NO₃⁻ uptake was the same as in plants receiving no NH₄⁺. After 6 hours, however, the NO₃⁻ uptake rate (NUR) and root NRA were inhibited by NH₄⁺ to a maximum of 45% and 60%, respectively.

The response of the NUR of NO₃⁻-induced plants depended on the NH₄Cl concentration. Below 1 millimolar NH₄⁺, the NUR declined immediately and some restoration occurred in the second hour. In the third hour, the NUR became constant. In contrast, NH₄⁺ at 2 millimolar and above caused a rapid and transient stimulation of NO₃⁻ uptake, followed again by a decrease in the first, a recovery in the second, and a steady state in the third hour. Maximal inhibition of steady state NUR was 50%. With NO₃⁻-induced plants, root NRA responded less and more slowly to NH₄⁺ than did NUR.

Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, relieved the NH₄⁺ inhibition of the NUR of NO₃⁻-induced plants. We conclude that repression of the NUR by NH₄⁺ depends on NH₄⁺ assimilation. The repression by NH₄⁺ was least at the lowest and highest NH₄⁺ levels tested (0.04 and 25 millimolar).

     

Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.     

Regulation of NO(3) Influx in Barley : Studies Using NO(3)

Short-term (10 minutes) measurements of plasmalemma NO₃⁻ influx (_oc) into roots of intact barley plants were obtained using ¹³NO₃⁻. In plants grown for 4 days at various NO₃⁻ levels (0.1, 0.2, 0.5 millimolar), _oc was found to be independent of the level of NO₃⁻ pretreatment. Similarly, pretreatment with Cl⁻ had no effect upon plasmalemma ¹³NO₃⁻ influx. Plants grown in the complete absence of ¹³NO₃⁻ (in CaSO₄ solutions) subsequently revealed influx values which were more than 50% lower than for plants grown in NO₃⁻. Based upon the documented effects of NO₃⁻ or Cl⁻ pretreatments on net uptake of NO₃⁻, these observations suggest that negative feedback from vacuolar NO₃⁻ and/or Cl⁻ acts at the tonoplast but not at the plasmalemma. When included in the influx medium, 0.5 millimolar Cl⁻ was without effect upon ¹³NO₃⁻ influx, but NH₄⁺ caused approximately 50% reduction of influx at this concentration.     

Dissimilatory Reduction of NO2− to NH4+ and N2O by a Soil Citrobacter sp.

Dissimilatory reduction of NO₂⁻ to N₂O and NH₄⁺ by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N₂O production by this mechanism. In batch cultures with defined media, NO₂⁻ reduction to NH₄⁺ was favored by high glucose and low NO₃⁻ concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO₃⁻ concentrations. With succinate as the energy source, little or no NO₂⁻ was reduced to NH₄⁺ but N₂O was produced. Resting cell suspensions reduced NO₂⁻ simultaneously to N₂O and free extracellular NH₄⁺. Chloramphenicol prevented the induction of N₂O-producing activity. The K_m for NO₂⁻ reduction to N₂O was estimated to be 0.9 mM NO₂⁻, yet the apparent K_m for overall NO₂⁻ reduction was considerably lower, no greater than 0.04 mM NO₂⁻. Activities for N₂O and NH₄⁺ production increased markedly after depletion of NO₃⁻ from the media. Amendment with NO₃⁻ inhibited N₂O and NH₄⁺ production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH₄⁺ but not of N₂O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N₂O at rates equal to the wild type. These observations suggest that N₂O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO₂⁻ to NH₄⁺.     

Uptake and Assimilation of NO(3) and NH(4) by Nitrogen-Deficient Perennial Ryegrass Turf

Assimilation of NO₃⁻ and NH₄⁺ by perennial ryegrass (Lolium perenne L.) turf, previously deprived of N for 7 days, was examined. Nitrogen uptake rate was increased up to four- to five-fold for both forms of N by N-deprivation as compared to N-sufficient controls, with the deficiency-enhanced N absorption persisting through a 48 hour uptake period. Nitrate, but not NH₄⁺, accumulated in the roots and to a lesser degree in shoots. By 48 hours, 53% of the absorbed NO₃⁻ had been reduced, whereas 97% of the NH₄⁺ had been assimilated. During the early stages (0 to 8 hours) of NO₃⁻ uptake by N-deficient turf, reduction occurred primarily in the roots. Between 8 and 16 hours, however, the site of reduction shifted to the shoots. Nitrogen form did not affect partitioning of the absorbed N between roots (40%) and shoots (60%) but did affect growth. Compared to NO₃⁻, NH₄⁺ uptake inhibited root, but not shoot, growth. Total soluble carbohydrates decreased in both roots and shoots during the uptake period, principally the result of fructan metabolism. Ammonium uptake resulted in greater total depletion of soluble carbohydrates in the root compared to NO₃⁻ uptake. The data indicate that N assimilation by ryegrass turf utilizes stored sugars but is also dependent on current photosynthate.     

Growth and Nitrogen Uptake Characteristics Reveal Outbreak Mechanism of the Opportunistic Macroalga Gracilaria tenuistipitata

Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources'' (NH₄ ⁺, NO₃ ⁻) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH₄ ⁺ than that with NO₃ ⁻ addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH₄ ⁺ and NO₃ ⁻ uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH₄ ⁺ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO₃ ⁻ uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters V_max = 37.2 µM g⁻¹ DM h⁻¹ and K_s = 61.5 µM. Further, based on the isotope ¹⁵N tracer method, we found that ¹⁵N from NH₄ ⁺ accumulated faster and reached an atom% twice than that of ¹⁵N from NO₃ ⁻, suggesting when both NH₄ ⁺ and NO₃ ⁻ were available, NH₄ ⁺ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation.     

Inhibition of Chemoautotrophic Nitrification by Sodium Chlorate and Sodium Chlorite: a Reexamination

The oxidation of NH₄⁺ by Nitrosomonas europaea was insensitive to 10 mM NaClO₃ (sodium chlorate) but was strongly inhibited by NaClO₂ (sodium chlorite; K_i, 2 μM). The oxidation of NO₂⁻ by Nitrobacter winogradskyi was inhibited by both ClO₃⁻ and ClO₂⁻ (K_i for ClO₂⁻, 100 μM). N. winogradskyi reduced ClO₃⁻ to ClO₂⁻ under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO₂⁻ was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH₄⁺ and NO₂⁻ was inhibited in the presence of 10 mM ClO₃⁻ after a 2-h lag period, despite the fact that, under these conditions, ClO₂⁻ was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH₄⁺ oxidation is inhibited by ClO₂⁻ produced by reduction of ClO₃⁻ by the NO₂⁻ oxidizer. The use of ClO₃⁻ inhibition of NO₂⁻ oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH₄⁺ is not affected.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Nitrogen Uptake by Wheat Seedlings,Interactive Effects of Four Nitrogen Sources: NO3?, NO2?, NH4+, and Urea

The net influx (uptake) rates of NO₃⁻, NH₄⁺, NO₂⁻, and urea into roots of wheat (Triticum aestivum cv Yecora Rojo) seedlings from complete nutrient solutions containing all four compounds were monitored simultaneously. Although urea uptake was too slow to monitor, its presence had major inhibitory effects on the uptake of each of the other compounds. Rates of NO₃⁻, NH₄⁺, and NO₂⁻ uptake depended in a complex fashion on the concentration of all four N compounds. Equations were developed which describe the uptake rates of each of the compounds, and of total N, as functions of concentrations of all N sources. Contour plots of the results show the interactions over the range of concentrations employed. The coefficients of these equations provide quantitative values for evaluating primary and interactive effects of each compound on N uptake.     

Active extrusion of protons into deionized water by roots of intact maize plants

The investigations were focussed on the question as to whether roots of intact maize plants (Zea mays L. cv Blizzard) release protons into deionized H₂O. Plants in the six to seven leaf stage depressed the pH of deionized H₂O from 6 to about 4.8 during an experimental period of 4 hours. Only one-third of the protons released could be ascribed to the solvation of CO₂ in H₂O. The main counter anions released were Cl⁻, NO₃⁻, and SO₄²⁻. At low temperature (2°C), the H⁺ release was virtually blocked while a relatively high amount of K⁺ was released. The presence of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the external solution increased the H⁺ secretion significantly. Addition of vanadate to the outer medium inhibited the H⁺ release while fusicoccin had a stimulating effect. Substituting the nutrient solution of deionized H₂O resulted in a substantial increase of the membrane potential difference from −120 to −190 millivolts. The experimental results support the conclusion that the H⁺ release by roots of intact maize plants is an active process driven by a plasmalemmalocated ATPase. Since the net H⁺ release was not associated with a net uptake of K⁺, it is unlikely to originate from a K⁺/H⁺ antiport.     

Comparative Kinetics and Reciprocal Inhibition of Nitrate and Nitrite Uptake in Roots of Uninduced and Induced Barley (Hordeum vulgare L.) Seedlings

Nitrate and NO₂⁻ transport by roots of 8-day-old uninduced and induced intact barley (Hordeum vulgare L. var CM 72) seedlings were compared to kinetic patterns, reciprocal inhibition of the transport systems, and the effect of the inhibitor, p-hydroxymercuribenzoate. Net uptake of NO₃⁻ and NO₂⁻ was measured by following the depletion of the ions from the uptake solutions. The roots of uninduced seedlings possessed a low concentration, saturable, low K_m, possibly a constitutive uptake system, and a linear system for both NO₃⁻ and NO₂⁻. The low K_m system followed Michaelis-Menten kinetics and approached saturation between 40 and 100 micromolar, whereas the linear system was detected between 100 and 500 micromolar. In roots of induced seedlings, rates for both NO₃⁻ and NO₂⁻ uptake followed Michaelis-Menten kinetics and approached saturation at about 200 micromolar. In induced roots, two kinetically identifiable transport systems were resolved for each anion. At the lower substrate concentrations, less than 10 micromolar, the apparent low K_ms of NO₃⁻ and NO₂⁻ uptake were 7 and 9 micromolar, respectively, and were similar to those of the low K_m system in uninduced roots. At substrate concentrations between 10 and 200 micromolar, the apparent high K_m values of NO₃⁻ uptake ranged from 34 to 36 micromolar and of NO₂⁻ uptake ranged from 41 to 49 micromolar. A linear system was also found in induced seedlings at concentrations above 500 micromolar. Double reciprocal plots indicated that NO₃⁻ and NO₂⁻ inhibited the uptake of each other competitively in both uninduced and induced seedlings; however, K_i values showed that NO₃⁻ was a more effective inhibitor than NO₂⁻. Nitrate and NO₂⁻ transport by both the low and high K_m systems were greatly inhibited by p-hydroxymercuribenzoate, whereas the linear system was only slightly inhibited.     

Potassium Transport in Corn Roots : IV. Characterization of the Linear Component

A detailed examination was conducted on the linear, or first-order kinetic component for K⁺(⁸⁶Rb⁺) influx into root segments of both low- and high-salt grown corn seedlings (Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl⁻ in the uptake solution by either SO₄²⁻, H₂PO₄⁻, or NO₃⁻ caused a significant (50-60%) and specific inhibition of the linear component of K⁺ influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl⁻ influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K⁺ uptake system; this inhibition was identical to that observed when Cl⁻ was replaced by other anions in the K⁺ uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K⁺ channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K⁺ influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium.

Our present results indicate that the linear component of K⁺ influx may occur by a passive process involving transmembrane K⁺ channels. Fluxes through these K⁺ channels may be partly coupled to a saturating Cl⁻ influx mechanism.

     

Effect of exogenous and endogenous nitrate concentration on nitrate utilization by dwarf bean

The effect of the exogenous and endogenous NO₃⁻ concentration on net uptake, influx, and efflux of NO₃⁻ and on nitrate reductase activity (NRA) in roots was studied in Phaseolus vulgaris L. cv. Witte Krombek. After exposure to NO₃⁻, an apparent induction period of about 6 hours occurred regardless of the exogenous NO₃⁻ level. A double reciprocal plot of the net uptake rate of induced plants versus exogenous NO₃⁻ concentration yielded four distinct phases, each with simple Michaelis-Menten kinetics, and separated by sharp breaks at about 45, 80, and 480 micromoles per cubic decimeter.

Influx was estimated as the accumulation of ¹⁵N after 1 hour exposure to ¹⁵NO₃⁻. The isotherms for influx and net uptake were similar and corresponded to those for alkali cations and Cl⁻. Efflux of NO₃⁻ was a constant proportion of net uptake during initial NO₃⁻ supply and increased with exogenous NO₃⁻ concentration. No efflux occurred to a NO₃⁻-free medium.

The net uptake rate was negatively correlated with the NO₃⁻ content of roots. Nitrate efflux, but not influx, was influenced by endogenous NO₃⁻. Variations between experiments, e.g. in NO₃⁻ status, affected the values of K_m and V_max in the various concentration phases. The concentrations at which phase transitions occurred, however, were constant both for influx and net uptake. The findings corroborate the contention that separate sites are responsible for uptake and transitions between phases.

Beyond 100 micromoles per cubic decimeter, root NRA was not affected by exogenous NO₃⁻ indicating that NO₃⁻ uptake was not coupled to root NRA, at least not at high concentrations.

     

Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.