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.     

The influence of ammonium and chloride on potassium and nitrate absorption by barley roots depends on time of exposure and cultivar

Net uptakes of K⁺ and NO₃⁻ were monitored simultaneously and continuously for two barley (Hordeum vulgare) cultivars, Prato and Olli. The cultivars had similar rates of net K⁺ and NO₃⁻ uptake in the absence of NH₄⁺ or Cl⁻. Long-term exposure (over 6 hours) to media which contained equimolar mixtures of NH₄⁺, K⁺, Cl⁻, or NO₃⁻ affected the cultivars very differently: (a) the presence of NH₄⁺ as NH₄Cl stimulated net NO₃⁻ uptake in Prato barley but inhibited net NO₃⁻ uptake in Olli barley; (b) Cl⁻ inhibited net NO₃⁻ uptake in Prato but had little effect in Olli; and (c) NH₄⁺ as (NH₄)₂SO₄ inhibited net K⁺ uptake in Prato but had little effect in Olli. Moreover, the immediate response to the addition of an ion often varied significantly from the long-term response; for example, the addition of Cl⁻ initially inhibited net K⁺ uptake in Olli barley but, after a 4 hour exposure, it was stimulatory. For both cultivars, net NH₄⁺ and Cl⁻ uptake did not change significantly with time after these ions were added to the nutrient medium. These data indicate that, even within one species, there is a high degree of genotypic variation in the control of nutrient absorption.     

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.     

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO₃⁻ uptake by more than 90% but had no effect on NO₂⁻ uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO₃⁻ uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO₃⁻ uptake. The results present the possibility that NO₃⁻ uptake and NO₃⁻ reduction in the PM of barley roots may be related.     

Enhancement of Nitrate Reductase Activity by Benzyladenine in Agrostemma githago

Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO₃⁻ and cytokinins. We asked the question whether cytokinins affected nitrate reductase activity directly or through NO₃⁻, either by amplifying the effect of low endogenous NO₃⁻ levels, or by making NO₃⁻ available for induction from a metabolically inactive compartment. Nitrate reductase activity was enhanced on the average by 50% after 1 hour of benzyladenine treatment. In some experiments, the cytokinin response was detectable as early as 30 minutes after addition of benzyladenine. Nitrate reductase activity increased linearly for 4 hours and began to decay 13 hours after start of the hormone treatment. When embryos were incubated in solutions containing mixtures of NO₃⁻ and benzyladenine, additive responses were obtained. The effects of NO₃⁻ and benzyladenine were counteracted by abscisic acid. The increase in nitrate reductase activity was inhibited at lower abscisic acid concentrations in embryos which were induced with NO₃⁻, as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO₃⁻ was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO₃⁻ and benzyladenine were withdrawn from the medium after maximal enhancement of nitrate reductase activity, the level of the enzyme decreased rapidly. Nitrate reductase activity increasd again as a result of a second treatment with benzyladenine but not with NO₃⁻. At the time of the second exposure to benzyladenine, no NO₃⁻ was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO₃⁻.     

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.     

Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution

The effect of nitrogen form (NH₄-N, NH₄-N + NO₃⁻, NO₃⁻) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO₃⁻ increases, and NH₄⁺ and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO₃⁻, NH₄-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH⁻ and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH⁻ or by the synthesized organic anions rather than by nitrate itself. Addition of HCO₃⁻ to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO₃⁻, like OH⁻, increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO₃⁻, NH₄-N, and amide-N.     

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

Effect of Phloem-Translocated Malate on NO(3) Uptake by Roots of Intact Soybean Plants

In soybean (Glycine max L. Merr. cv Kingsoy), NO₃⁻ assimilation in leaves resulted in production and transport of malate to roots (B Touraine, N Grignon, C Grignon [1988] Plant Physiol 88: 605-612). This paper examines the significance of this phenomenon for the control of NO₃⁻ uptake by roots. The net NO₃⁻ uptake rate by roots of soybean plants was stimulated by the addition of K-malate to the external solution. It was decreased when phloem translocation was interrupted by hypocotyl girdling, and partially restored by malate addition to the medium, whereas glucose was ineffective. Introduction of K-malate into the transpiration stream using a split root system resulted in an enrichment of the phloem sap translocated back to the roots. This treatment resulted in an increase in both NO₃⁻ uptake and C excretion rates by roots. These results suggest that NO₃⁻ uptake by roots is dependent on the availability of shoot-borne, phloem-translocated malate. Shoot-to-root transport of malate stimulated NO₃⁻ uptake, and excretion of HCO₃⁻ ions was probably released by malate decarboxylation. NO₃⁻ uptake rate increased when the supply of NO₃⁻ to the shoot was increased, and decreased when the activity of nitrate reductase in the shoot was inhibited by WO₄²⁻. We conclude that in situ, NO₃⁻ reduction rate in the shoot may control NO₃⁻ uptake rate in the roots via the translocation rate of malate in the phloem.     

Intracellular pH Regulation during NO(3) Assimilation in Shoot and Roots of Ricinus communis

Ricinus communis L. was used to test the Dijkshoorn-Ben Zioni hypothesis that NO₃⁻ uptake by roots is regulated by NO₃⁻ assimilation in the shoot. The fate of the electronegative charge arising from total assimilated NO₃⁻ (and SO₄²⁻) was followed in its distribution between organic anion accumulation and HCO₃⁻ excretion into the nutrient solution. In plants adequately supplied with NO₃⁻, HCO₃⁻ excretion accounted for about 47% of the anion charge, reflecting an excess nutrient anion over cation uptake. In vivo nitrate reductase assays revealed that the roots represented the site of about 44% of the total NO₃⁻ reduction in the plants. To trace vascular transport of ionic and nitrogenous constituents within the plant, the composition of both xylem and phloem saps was thoroughly investigated. Detailed dry tissue and sap analyses revealed that only between 19 and 24% of the HCO₃⁻ excretion could be accounted for from oxidative decarboxylation of shoot-borne organic anions produced in the NO₃⁻ reduction process. The results obtained in this investigation may be interpreted as providing direct evidence for a minor importance of phloem transport of cation-organate for the regulation of intracellular pH and electroneutrality, thus practically eliminating the necessity for the Dijkshoorn-Ben Zioni recycling process.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

Characterization of Anion Effects on the Nitrate-Sensitive ATP-Dependent Proton Pumping Activity of Soybean (Glycine max L.) Seedling Root Microsomes

The ATP-dependent proton-pumping activity of soybean (Glycine max L.) root microsomes is predominantly nitrate sensitive and presumably derived from the tonoplast. We used microsomes to characterize anion effects on proton pumping of the tonoplast vesicles using two distinctly different techniques.

Preincubation of the vesicles with nitrate caused inhibition of proton pumping and ATPase activity, with similar concentration dependence. Fluoride, which preferentially inhibits the plasma membrane ATPase, inhibited ATPase activity strongly at concentrations which did not affect proton pumping activity.

Addition of potassium salts, after a steady-state pH gradient is established in the absence of such salts, caused an increased pH gradient which was due to alleviation of Δ Ψ and subsequent increased influx of H⁺ into these vesicles. This anion-induced increase in the pH gradient could be used as a measure of the relative anion permeabilities, which were of the order Br⁻ = NO₃⁻ > Cl⁻ SO₄²⁻. Phosphate and fluoride caused no increase in the pH gradient. Since the concentration dependence of KCl- and KNO₃-induced quenching exhibited a saturable component, and since H⁺ uptake was increased by only certain anions, the data suggest that there may be a relatively specific anion channel associated with tonoplast-derived vesicles.

     

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.

     

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.     

Characteristics of Injury and Recovery of Net NO3? Transport of Barley Seedlings from Treatments of NaCl

The nature of the injury and recovery of nitrate uptake (net uptake) from NaCl stress in young barley (Hordeum vulgare L, var CM 72) seedlings was investigated. Nitrate uptake was inhibited rapidly by NaCl, within 1 minute after exposure to 200 millimolar NaCl. The duration of exposure to saline conditions determined the time of recovery of NO₃⁻ uptake from NaCl stress. Recovery was dependent on the presence of NO₃⁻ and was inhibited by cycloheximide, 6-methylpurine, and cerulenin, respective inhibitors of protein, RNA, and sterol/fatty acid synthesis. These inhibitors also prevented the induction of the NO₃⁻ uptake system in uninduced seedlings. Uninduced seedlings exhibited endogenous NO₃⁻ transport activity that appeared to be constitutive. This constitutive activity was also inhibited by NaCl. Recovery of constitutive NO₃⁻ uptake did not require the presence of NO₃⁻.     

Dissimilatory Selenate Reduction Potentials in a Diversity of Sediment Types

We measured potential rates of bacterial dissimilatory reduction of ⁷⁵SeO₄²⁻ to ⁷⁵Se⁰ in a diversity of sediment types, with salinities ranging from freshwater (salinity = 1 g/liter) to hypersaline (salinity = 320 g/liter and with pH values ranging from 7.1 to 9.8. Significant biological selenate reduction occurred in all samples with salinities from 1 to 250 g/liter but not in samples with a salinity of 320 g/liter. Potential selenate reduction rates (25 nmol of SeO₄²⁻ per ml of sediment added with isotope) ranged from 0.07 to 22 μmol of SeO₄²⁻ reduced liter⁻¹ h⁻¹. Activity followed Michaelis-Menten kinetics in relation to SeO₄²⁻ concentration (K_m of selenate = 7.9 to 720 μM). There was no linear correlation between potential rates of SeO₄²⁻ reduction and salinity, pH, concentrations of total Se, porosity, or organic carbon in the sediments. However, potential selenate reduction was correlated with apparent K_m for selenate and with potential rates of denitrification (r = 0.92 and 0.81, respectively). NO₃⁻, NO₂⁻, MoO₄²⁻, and WO₄²⁻ inhibited selenate reduction activity to different extents in sediments from both Hunter Drain and Massie Slough, Nev. Sulfate partially inhibited activity in sediment from freshwater (salinity = 1 g/liter) Massie Slough samples but not from the saline (salinity = 60 g/liter) Hunter Drain samples. We conclude that dissimilatory selenate reduction in sediments is widespread in nature. In addition, in situ selenate reduction is a first-order reaction, because the ambient concentrations of selenium oxyanions in the sediments were orders of magnitude less than their K_ms.     

Nitrate Reduction in Response to CO(2)-Limited Photosynthesis : Relationship to Carbohydrate Supply and Nitrate Reductase Activity in Maize Seedlings

The effects of CO₂-limited photosynthesis on ¹⁵NO₃⁻ uptake and reduction by maize (Zea mays, DeKalb XL-45) seedlings were examined in relation to concurrent effects of CO₂ stress on carbohydrate levels and in vitro nitrate reductase activities. During a 10-hour period in CO₂-depleted air (30 microliters of CO₂/ per liter), cumulative ¹⁵NO₃⁻ uptake and reduction were restricted 22 and 82%, respectively, relative to control seedlings exposed to ambient air containing 450 microliters of CO₂ per liter. The comparable values for roots of decapitated maize seedlings, the shoots of which had previously been subjected to CO₂ stress, were 30 and 42%. The results demonstrate that reduction of entering nitrate by roots as well as shoots was regulated by concurrent photosynthesis. Although in vitro nitrate reductase activity of both tissues declined by 60% during a 10-hour period of CO₂ stress, the remaining activity was greatly in excess of that required to catalyze the measured rate of ¹⁵NO₃⁻ reduction. Root respiration and soluble carbohydrate levels in root tissue were also decreased by CO₂ stress. Collectively, the results indicate that nitrate uptake and reduction were regulated by the supply of energy and carbon skeletons required to support these processes, rather than by the potential enzymatic capacity to catalyze nitrate reduction, as measured by in vitro nitrate reductase activity.     

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO₃⁻, Na⁺, Cl⁻, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged. Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na⁺ and Cl⁻. Toxin did not affect uptake of Na₂⁻, K⁺, Ca²⁺, phosphate ion (H₂PO₄⁻ and HPO₄⁻), SO₄⁻, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.     

Quantitative and Physiological Analyses of Chloride Dependence of Growth of Halobacillus halophilus

A quantitative analysis of the Cl⁻ dependence of growth of Halobacillus halophilus was performed. Optimal growth rates were obtained at Cl⁻ concentrations of between 0.5 and 2.0 M, and the final yield was also strictly dependent on the Cl⁻ concentration. Br⁻ but not I⁻, SO₄²⁻, NO₂⁻, SO₂⁻, OCN⁻, SCN⁻, BO₂⁻, or BrO₃⁻ could substitute for Cl⁻. To analyze the function of chloride, chloride concentration was determined. At low external Cl⁻ (Cl_e⁻) concentrations, the growth rate was low and Cl⁻ was excluded from the cytoplasm; increasing the Cl_e⁻ concentration led to an increase in the growth rate and an energy-dependent uptake of Cl⁻, thus decreasing the Cl_e⁻/internal Cl_i⁻ gradient from ≥10 at 0.1 M Cl_e⁻ to a nearly constant value of 2 at Cl_e⁻ concentrations which allowed optimal growth. Two membrane proteins with apparent molecular masses of 31 and 16 kDa which were identified to be specific for Cl⁻-grown cultures are possible candidates for a chloride uptake system.     

Bacterium-Based NO2− Biosensor for Environmental Applications

A sensitive NO₂⁻ biosensor that is based on bacterial reduction of NO₂⁻ to N₂O and subsequent detection of the N₂O by a built-in electrochemical N₂O sensor was developed. Four different denitrifying organisms lacking NO₃⁻ reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO₂⁻ tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO₃⁻ and N₂O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N₂O reductase. The macroscale biosensors constructed exhibited a linear NO₂⁻ response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO₂⁻, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO₂⁻, and interference was observed only with NH₂OH, NO, N₂O, and H₂S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO₂⁻ biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO₂⁻ biosensor were constructed and used to measure NO₂⁻ profiles in marine sediment.