Effects of Al on nitrogen (NH 4 + and NO 3 − ) uptake,nitrate reductase activity and proton release in two sorghum cultivars differing in Al tolerance

After growth for 17 to 36 days on nutrient solutions with NH₄NO₃ as nitrogen source (pH 4.2) dry matter of sorghum genotype SC0283 was much less affected by Al (1.5 and 3.0 ppm) than that of genotype NB9040. In the absence of Al both cultivars released protons into the nutrient solution as a result of an excess of cationic nutrients taken up. When Al was present, this proton efflux per unit dry weight increased drastically, especially with the sensitive genotype NB9040. Chemical analysis of plant material and continuous analyses of NO ₃ ⁻ and NH ₄ ⁺ in the nutrient solution indicated, that the Al-induced shift in H⁺-balance of both genotypes could almost completely be attributed to a decreased NO ₃ ⁻ /NH ₄ ⁺ uptake ratio. In vivo nitrate reductase activity (NRA) was reduced in the shoot of NB9040 and to a lesser degree in SC0283. Al-induced decrease in NRA was accompanied by similar percentual decreases in NO ₃ ⁻ tissue concentrations. Therefore this decrease is interpreted as being indirect,i.e., the consequence of the reduced NO ₃ ⁻ uptake of the plants. A direct repression of NRA by Al seems also unlikely because nitrate reductase activity of the roots (where cellular Al-concentrations should be higher than in shoots) was not affected in Al-treated plants of either genotype.     

Utilization of nitrite and nitrate by dwarf bean

The uptake and conversion of NO ₂ ^- and the effect of NO ₂ ^- on the uptake and reduction of NO ₃ ^- were examined in N-depleted Phaseolus vulgaris L. Nitrite uptake at 0.1 mmol dm^-3 was against an electrochemical gradient and became constant after one or two initial phases. Steadystate uptake declined with increasing ambient NO ₂ ^- concentration (0–0.7 mmol dm^-3). In this concentration range root oxygen consumption was unaffected by NO ₂ ^- , indicating that the decrease of NO ₂ ^- uptake was not related to respiration. After 6 h NO ₂ ^- supply, about one-third of the absorbed NO ₂ ^- had accumulated, mainly in the root system. Oxidation of NO ₂ ^- to NO ₃ ^- was not observed. The apparent induction period for NO ₃ ^- uptake was about 6 h in control plants and 3.5 h in plants that were pretreated for 18 h with NO ₂ ^- . In contrast, the time course of NO ₂ ^- uptake was unaffected by pretreatment with NO ₃ ^- . Steadystate NO ₃ ^- uptake was less affected by NO ₂ ^- than was steady-state NO ₂ ^- uptake by NO ₃ ^- . Nitrate reductase activity (NRA) in leaves and roots was induced by both NO ₃ ^- and NO ₂ ^- . In roots, induction with NO ₂ ^- was faster than with NO ₃ ^- , but there was no difference in NRA after 5 h. Nitrite inhibited NRA in the roots of NO ₃ ^- -induced plants and thus seems to stimulate the induction, but not the activity of induced nitrate reductase. In view of the observed differences in time course and mutual competition, a common uptake mechanism for NO ₂ ^- and NO ₃ ^- seems unlikely. Expression of the NO ₂ ^- effect on the induction of NO ₃ ^- uptake required more time than the induction itself. We therefore conclude that NO ₂ ^- is not the physiological inducer of NO ₃ ^- uptake.Abbreviations NR(A) nitrate reductase (activity) - BM basal medium     

Influx,efflux and net uptake of nitrate in Quercus suber seedlings

Nitrate influx, efflux and net nitrate uptake were measured for the slow-growing Quercus suber L. (cork-oak) to estimate the N-uptake efficiency of its seedlings when grown with free access to nitrate. We hypothesise that nitrate influx, an energetically costly process, is not very efficiently controlled so as to avoid losses through efflux, because Q. suber has relatively high respiratory costs for ion uptake. Q. suber seedlings were grown in a growth room in hydroponics with 1 mM NO₃ ^-. Seedlings were labelled with ¹⁵NO₃ ^- in nutrient solution for 5 min to measure influx and for 2 h for net uptake. Efflux was calculated as the difference between influx and net uptake. Measurements were made in the morning, afternoon and night. The site of nitrate reduction was estimated from the ratio of NO₃ ^- to amino acids in the xylem sap; the observed ratio indicated that nitrate reduction occurred predominantly in the roots. Nitrate influx was always much higher than net acquisition and both tended to be lower at night. High efflux occurred both during the day and at night, although the proportion of ¹⁵NO₃ ^- taken up that was loss through efflux was proportionally higher during the night. Efflux was a significant fraction of influx. We concluded that the acquisition system is energetically inefficient under the conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue

Vacuolar proton pumping pyrophosphatase (H⁺-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP_i hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H⁺-translocation of vacuolar H⁺-PPase in a concentration-dependent manner. Absorbance of vacuolar H⁺-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PP_i hydrolysis activity as well. The pK _a of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H⁺-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H⁺-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H⁺-PPase. Inhibition of vacuolar H⁺-PPase by DEPC changes V _max but not K _m values. Moreover, DEPC inhibition of vacuolar H⁺-PPase could be substantially protected against by its physiological substrate, Mg²⁺-PP_i. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H⁺-PPase by DEPC. Taken together, we suggest that vacuolar H⁺-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.     

A microcomputer method for continuous measurement,recording and control of ion activity during nitrate uptake byLolium perenne

A microprocessor controlled apparatus is described which can measure, control and record nitrate uptake byLolium perenne in nutrient solution, comparing seven selection lines in duplicate. Nutrient solution flowed at 1 min⁻¹, and linear response was found from 10⁻¹ to 10⁻⁴ M NO ₃ ⁻ . Uptake rates for Lolium were between 10⁻⁵ and 10⁻⁴ M NO ₃ ⁻ , plant⁻¹, h⁻¹, which agreed with previous, manually determined, rates, ‘Overshoot’ in nitrate dosing, which was a problem with manual systems, was eliminated. Nitrate concentration was controlled (±3%) in modified Hoagland’s solution.     

Isolation and characterization of a <Emphasis Type="Italic">Mastigocladus</Emphasis> species capable of growth,N<Subscript>2</Subscript>-fixation and N-assimilation at elevated temperature

A Mastigocladus species was isolated from the hot spring of Jakrem (Meghalaya) India. Uptake and utilization of nitrate, nitrite, ammonium and amino acids (glutamine, asparagine, arginine, alanine) were studied in this cyanobacterium grown at different temperatures (25°C, 45°C). There was 2–3 fold increase in the heterocyst formation and nitrogenase activity in N-free medium at higher temperature (45°C). Growth and uptake and assimilation of various nitrogen sources were also 2–3 fold higher at 45°C indicating that it is a thermophile. The extent of induction and repression of nitrate uptake by NO₃ ⁻ and NH₄ ⁺, respectively, differed from that of nitrite. It appeared that Mastigocladus had two independent nitrate/nitrite transport systems. Nitrate reductase and nitrite reductase activitiy was not NO₃ ⁻-inducible and ammonium or amino acids caused only partial repression. Presence of various amino acids in the media partially repressed glutamine synthetase activity. Ammonium (methylammonium) and amino acid uptake showed a biphasic pattern, was energy-dependent and the induction of uptake required de novo protein synthesis. Ammonium transport was substrate (NH₄ ⁺)-repressible, while the amino acid uptake was substrate inducible. When grown at 25°C, the cyanobacterium formed maximum akinetes that remained viable upto 5 years under dry conditions.     

Role of nitrification and denitrification for NO metabolism in soil

Release and uptake of NO was measured in a slightly alkaline (pH 7.8) and an acidic (pH 4.7) cambisol. In the alkaline soil under aerobic conditions, NO release was stimulated by ammonium and inhibited by nitrapyrin. Nitrate accumulated simultaneously and was also inhibited by nitrapyrin.¹⁵NO was released after fertilization with¹⁵NH₄NO₃ but not with NH₄ ¹⁵NO₃. The results indicate that in aerobic alkaline cambisol NO was mainly produced during nitrification of ammonium. The results were different under anaerobic conditions and also in the acidic cambisol. There, NO release was stimulated by nitrate and not by ammonium, and was inhibited by chlorate and not by nitrapyrin indicating that NO production was exclusively due to reduction of nitrate. The results were confirmed by¹⁵NO being released mainly from NH₄ ¹⁵NO₃ rather than from¹⁵NH₄NO₃. The observed patterns of NO release were explained by the NO production processes being stimulated by either ammonium or nitrate in the two different soils, whereas the NO consumption processes being only stimulated by nitrate. NO release was larger than N₂O release, but both were small compared to changes in concentrations of soil ammonium or nitrate.(*request for offprints)     

Induction of a high-capacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism

Roots of nitrate-starved and nitrate-pretreated seedlings of Hordeum vulgare were used to investigate the induction of a high-capacity uptake mechanism for nitrate. When exposed to 0.2 mmol·l^-1KNO₃, nitrate-starved roots took up nitrate at a rate of approx. 1 mol·(g FW)^-1·h^-1; K⁺ was absorbed at a rate ten-times higher. Nitrate uptake accelerated after a lag of about 1 h, until it matched the rate of K⁺ uptake about 4 h later. p-Fluorophenylalanine (FPA), which prevents the synthesis of functioning proteins, suppressed the development of the high-capacity mechanism. Pretreatment of the roots with 0.2 mmol·l^-1 Ca(NO₃)₂ for 24 h established the high-capacity mechanism. Pretreated roots were able to absorb nitrate at high rates immediately upon exposure to 0.2 mmol·l^-1KNO₃, in the absence or presence of FPA. The high-capacity mechanism, once established, appeared to have a protein turnover as slow as that of the low-capacity mechanism or that of the mechanism involved in the uptake of K⁺. In contrast, the mechanisms for the transport of nitrate and K⁺ into the xylem vessels were completely blocked by FPA within 1 h of application, confirming earlier evidence for a rapid turnover of the transport proteins in the xylem parenchyma.Nitrate reduction proceeded at rates which were roughly one-tenth as large as the rates of the respective nitrate-uptake processes, indicating that nitrate-reductase activity was determined by the rate of nitrate uptake and not vice versa.We conclude that the formation of a high-capacity nitrate-uptake mechanism in barley roots occurs in response to nitrate uptake through a constitutive mechanism of low capacity which appears to function as a sensing mechanism for nitrate in the environment of the roots.Abbreviation FPA p-fluorophenylalanine     

The kinetics of NH4 + and NO3 − uptake by Douglas fir from single N-solutions and from solutions containing both NH4 + and NO3 −

The kinetics of NH₄ ⁺ and NO₃ ⁻ uptake in young Douglas fir trees (Pseudotsuga menziesii [Mirb.] Franco) were studied in solutions, containing either one or both N species. Using solutions containing a single N species, the V_max of NH₄ ⁺ uptake was higher than that of NO₃ ⁻ uptake. The K_m of NH₄ ⁺ uptake and K_m of NO₃ ⁻ uptake differed not significantly. When both NH₄ ⁺ and NO₃ ⁻ were present, the V_max for NH₄ ⁺ uptake became slightly higher, and the K_m for NH₄ ⁺ uptake remained in the same order. Under these conditions the NO₃ ⁻ uptake was almost totally inhibited over the whole range of concentrations used (10–1000 μM total N). This inhibition by NH₄ ⁺ occurred during the first two hours after addition. ei]{gnA C}{fnBorstlap}     

The mechanism of nitrate transport across the tonoplast of barley root cells

Nitrate-selective microelectrodes were used to measure not only nitrate activity in the cytoplasm and vacuole of barley (Hordeum vulgare L.) root cells, but also the tonoplast electrical membrane potential. For epidermal cells, the mean cytoplasmic and vacuolar pNO₃ (-log₁₀ [NO₃]) values were 2.3±0.04 (n=19) and 1.41±0.03 (n=35), respectively, while for cortical cells, the mean cytoplasmic and vacuolar nitrate values were 2.58±0.18 (n=4) and 1.17±0.06 (n=13), respectively. These results indicate that the accumulation of nitrate in the vacuole must be an active process. Proton-selective microelectrodes were used to measure the proton gradient across the tonoplast to assess the possibility that nitrate transport into the vacuole is mediated by an H⁺/NO ₃ ^– antiport mechanism. For epidermal cells, the mean cytoplasmic and vacuolar pH values were 7.12±0.06 (n=10) and 4.93±0.11 (n=22), respectively, while for cortical cells, the mean cytoplasmic and vacuolar pH values were 7.24±0.07 (n=3) and 5.09±0.17 (n=7), respectively. Calculations of the energetics for this mechanism indicate that the observed gradient of nitrate across the tonoplast of both epidermal and cortical cells could be achieved by an H⁺/NO ₃ ^– antiport with a 11 stoichiometry.Abbreviations and Symbols G/F free-energy change for H⁺/NO ₃ ^– antiport - F Faraday constant - pH_c cytoplasmic pH - pH_v vacuolar pH - p[NO₃]_c log₁₀ (cytoplasmic [NO ₃ ^– ]) - P[NO₃]_v -log₁₀ (vacuolar [NO₃]) We wish to thank Dr. K. Moore for assistance with statistical analysis.     

Comparative uptake of nitrate by intact seedlings of C3 (barley) and C4 (corn) plants: Effect of light and exogenously supplied sucrose

The effect of light and exogenously supplied sucrose on NO₃ ⁻ uptake was studied in 9-day-old intact C₃ (barley) and C₄ (corn) seedlings. The seedlings used were uninduced for nitrate uptake system (i.e. had never seen nitrogen during germination and growth) and were exposed to continuous light for 3 days to avoid any diurnal variation and to load the seedlings fully with photosynthates. The uptake assay was conducted either in light or in darkness. Prior to assay, seedlings were treated with darkness or light for 24 h. Accordingly, four sets of seedlings, i.e. pretreated with light and assayed in light (LL); pretreated and assayed in darkness (DD); pretreated with light and assayed in darkness (LD); and pretreated with darkness and assayed in light (DL) were formed. Barley exhibited 55% higher NO₃ ⁻ uptake than corn during light (LL) and 91% higher during darkness (DD). Shifting barley seedlings from light to dark (LD) or dark to light (DL) for uptake assay, did not affect NO₃ ⁻ uptake, i.e. in LD the uptake was similar to LL and in DL it was similar to DD. However, in corn, the light conditions during the assay determined the uptake regardless of the conditions during the period preceding the assay. One percent sucrose in the medium increased NO₃ ⁻ uptake by 31% in barley and 70% in corn during light (LL). The corresponding increase during darkness (DD) was 38% in both barley and corn. Removal of the corn residual endosperm decreased NO₃ ⁻ uptake by 40% during darkness. Etiolated seedlings (those having never seen light) of both barley and corn were able to take up significant amount of NO₃ ⁻ during darkness. Externally supplied sucrose in the assay medium of etiolated seedlings increased the NO₃ ⁻ uptake to about 4 and 2 fold in barley and corn, respectively. The data presented here provide evidence that: 1. In intact seedlings, light per se is not obligatory for NO₃ ⁻ uptake and that the carbohydrate supply may mimic light. 2. Light affected the NO₃ ⁻ uptake differently in barley and corn. This revised version was published online in June 2006 with corrections to the Cover Date.     

A field method of determining NH 4 + and NO 3 - uptake kinetics in intact roots: Effects of CO2 enrichment on trees and crop species

Models describing plant and ecosystem N cycles require an accurate assessment of root physiological uptake capacity for NH ₄ ⁺ and NO ₃ ^- under field conditions. Traditionally, rates of ion uptake in field-grown plants are determined by using excised root segments incubated for a short period in an assay solution containing N either as a radioactive or stable isotope tracer (e.g., ³⁶ClO₃ as a NH ₄ ⁺ analogue, ¹⁴CH₃NH₃ as an NO ₃ ^- analogue or ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^- ). Although reliable, this method has several drawbacks. For example, in addition to radioactive safety issues, purchase and analysis of radioactive and stable isotopes is relatively expensive and can be a major limitation. More importantly, because excision effectively interrupts exchange of compounds between root and shoot (e.g., carbohydrate supply to root and N transport to shoot), the assay must be conducted quickly to avoid such complications. Here we present a novel field method for simultaneous measurements of NH ₄ ⁺ and NO ₃ ^- uptake kinetics in intact root systems. The application of this method is demonstrated using two tree species; red maple (Acer rubrum) and sugar maple (Acer saccharum) and two crop species soybean (Glycine max) and sorghum (Sorghum bicolor). Plants were grown in open-top chambers at either ambient or elevated levels of atmospheric CO₂ at two separate US national sites involved in CO₂ research. Absolute values of net uptake rates and the kinetic parameters determined by our method were found to be in agreement with the literature reports. Roots of the crop species exhibited a greater uptake capacity for both N forms relative to tree species. Elevated CO₂ did not significantly affect kinetics of N uptake in species tested except in red maple where it increased root uptake capacity, V, for NH ₄ ⁺ . The application, reliability, advantages and disadvantages of the method are discussed in detail. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimizing environmental factors for large-scale multiplication of chrysanthemum (<Emphasis Type="Italic">Chrysanthemum grandiflorum</Emphasis>) in balloon-type bioreactor culture

Summary We have developed optimum culture conditions for the large-scale propagation of chrysanthemum in balloon-type bioreactors to achieve vigorous growth and quality. The effects of NH ₄ ⁺ /NO ₃ ⁻ ratio, air volume, air temperature, photosynthetic photo flux, and an inoculation density on the growth and quality of plantlets were investigated. The best production conditions were an NH ₄ ⁺ :NO ₃ ⁻ ratio of 20∶40 mM, air exchange of 0.1 vvm min⁻¹, air temperature 25°C, photosynthetic photo flux (PPF) at 100 μmol m⁻² s⁻¹, and an inoculation density of 40 nodes Chrysanthemum grandiflorum. Under each of these conditions, the maximum growth rate reached 279.0, 260,0, 20.0, 23.3, and 94.5 (g-fresh weight per plantlet d⁻¹), respectively, at 12 wk of culture. These results specify the key environmental factors that can be regulated to improve the quality and quantity of flowers and increase yield in large-scale bioreactor cultures of chrysanthemum.     

Phloem loading in Vicia faba leaves: Effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H+ extrusion,K+ and sucrose uptake

The effects of a penetrating (NEM) and a non-penetrating (PCMBS) sulfhydryl-specific reagent on proton extrusion, ⁸⁶Rb and [U-¹⁴C]sucrose uptake by Vicia faba leaves have been studied. Proton extrusion was strongly or completely inhibited by 0.1 mM NEM. ⁸⁶Rb and [U-¹⁴C]sucrose uptake were markedly reduced by NEM concentrations equal to or higher than 0.5 mM. Under our experimental conditions, PCMBS (1 mM) exerted a strong inhibition on [¹⁴C]sucrose uptake but did not inhibit proton extrusion and ⁸⁶Rb uptake. The sensitivity of phloem loading to PCMBS is thought to be a consequence of sugar-carrier blockage and not of inhibition of the proton pump.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DES diethylstilbestrol - DCCD dicyclohexylcarbodiimide - FC Fusicoccin - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid     

Plant and microbial nitrogen use and turnover: Rapid conversion of nitrate to ammonium in soil with roots

Immobilization of ammonium (NH ₄ ⁺ ) by plants and microbes, a controlling factor of ecosystem nitrogen (N) retention, has usually been measured based on uptake of¹⁵NH ₄ ⁺ solutions injected into soil. To study the influence of roots on N dynamics without stimulating consumption of NH ₄ ⁺ , we estimated gross nitrification in the presence or absence of live roots in an agricultural soil. Tomato (Lycopersicon esculentum var. Peto76) plants were grown in microcosms containing root exclosures. When the plants were 7 weeks old,¹⁵N enriched nitrate (NO ₃ ⁻ ) was applied in the 0–150 mm soil layer. After 24 h, > 30 times more¹⁵NH ₄ ⁺ was found in the soil with roots than in the soil of the root exclosures. At least 18% of the NH ₄ ⁺ -N present at this time in the soil with roots had been converted from NO ₃ ⁻ . We estimated rates of conversion of NO ₃ ⁻ to NH ₄ ⁺ , and rates ofNH ₄ ⁺ immobilization by plants and microbes, by simulating N-flow of¹⁴⁺¹⁵N and¹⁵N in three models representing mechanisms that may be underlying the experimental data: Dissimilatory NO ₃ ⁻ reduction to NH ₄ ⁺ (DNRA), plant N efflux, and microbial biomass nitrogen (MBN) turnover. Compared to NO ₃ ⁻ uptake, plant NH ₄ ⁺ uptake was modest. Ammonium immobilization by plants and microbes was equal to at least 35% of nitrification rates. The rapid recycling of NO ₃ ⁻ to NH ₄ ⁺ via plants and/or microbes contributes to ecosystem N retention and may enable plants growing in agricultural soils to capture more NH ₄ ⁺ than generally assumed.     

Comparative study of N uptake and distribution in three lines of common bean (Phaseolus vulgaris L.) at early pod filling stage

Summary The uptake and distribution of¹⁵NH ₄ ⁺ ,¹⁵NO ₃ ⁻ and¹⁵N₂ was studied in greenhouse-grown beans (Phaseolus vulgaris L.) with a commercial cultivar and 2 recombinant inbred backcross lines;¹⁵N was supplied in the nutrient solution at the R3 (50% bloom) stage. Plants were harvested 1, 5 and 10 days after treatment, and were separated into nodules, roots, stems, mature leaflets, immature leaflets, and flowers/fruits. All 3 lines showed rapid increases in the N content of flowers/fruits after the R3 stage. However, the percentage N in these tissues decreased after the R3 stage. One of the recombinant lines showed a greater uptake of NH ₄ ⁺ than the other 2 lines. Rates of¹⁵N₂ fixation and NO ₃ ⁻ uptake were similar for all 3 lines, N₂ fixation estimated from total N content showed the 2 recombinant lines with 24 and 34 percent greater activity than the commercial cultivar. Distribution of¹⁵N at the whole plant level was similar for all 3 lines for a similar N source.¹⁵NO ₃ ⁻ was transported first to leaflets and the label then moved into flowers/fruits. Transport of fixed N₂ was from the nodules to roots, stems and into flowers/fruits; usually less than 10 percent entered the leaflets. This indicates that N₂ fixation furnishes N directly to flowers/fruits with over 50 percent of the fixed N being deposited into flowers/fruits within 5 days after treatment.     

Chronic Atmospheric NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Deposition Does Not Induce NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Use by <Emphasis Type="Italic">Acer saccharum</Emphasis> Marsh.

The ability of an ecosystem to retain anthropogenic nitrogen (N) deposition is dependent upon plant and soil sinks for N, the strengths of which may be altered by chronic atmospheric N deposition. Sugar maple (Acer saccharum Marsh.), the dominant overstory tree in northern hardwood forests of the Lake States region, has a limited capacity to take up and assimilate NO₃⁻. However, it is uncertain whether long-term exposure to NO₃⁻ deposition might induce NO₃⁻ uptake by this ecologically important overstory tree. Here, we investigate whether 10 years of experimental NO₃⁻ deposition (30 kg N ha⁻¹ y⁻¹) could induce NO₃⁻ uptake and assimilation in overstory sugar maple (approximately 90 years old), which would enable this species to function as a direct sink for atmospheric NO₃⁻ deposition. Kinetic parameters for NH₄⁺ and NO₃⁻ uptake in fine roots, as well as leaf and root NO₃⁻ reductase activity, were measured under conditions of ambient and experimental NO₃⁻ deposition in four sugar maple-dominated stands spanning the geographic distribution of northern hardwood forests in the Upper Lake States. Chronic NO₃⁻ deposition did not alter the V _max or K _m for NO₃⁻ and NH₄⁺ uptake nor did it influence NO₃⁻ reductase activity in leaves and fine roots. Moreover, the mean V _max for NH₄⁺ uptake (5.15 μmol ¹⁵N g⁻¹ h⁻¹) was eight times greater than the V _max for NO₃⁻ uptake (0.63 μmol ¹⁵N g⁻¹ h⁻¹), indicating a much greater physiological capacity for NH₄⁺ uptake in this species. Additionally, NO₃⁻ reductase activity was lower than most values for woody plants previously reported in the literature, further indicating a low physiological potential for NO₃⁻ assimilation in sugar maple. Our results demonstrate that chronic NO₃⁻ deposition has not induced the physiological capacity for NO₃⁻ uptake and assimilation by sugar maple, making this dominant species an unlikely direct sink for anthropogenic NO₃⁻ deposition.     

Arginyl and histidyl groups are essential for organic anion exchange in renal brush-border membrane vesicles

The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.     

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle

Summary Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca²⁺ uptake into and Ca²⁺ release from the membrane vesicles were studied by a rapid filtration method, and Ca²⁺ transport properties of the different regions of the stomach were compared. ATP-dependent Ca²⁺ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN₃. Oxalate affected Ca²⁺ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100mm NaCl and then diluted to 1/20 concentration with Na⁺-free medium had significantly higher ATP-independent Ca²⁺ uptake than vesicles preincubated in 100mm KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na⁺-dependent Ca²⁺ uptake, and NaCl enhanced Ca²⁺ efflux from fundus microsome vesicles. The halflife values of Ca²⁺ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca²⁺ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had ro effect on Ca²⁺ release from antrum microsome vesicles.Results suggest two distinct mechanisms of stomach membrane Ca²⁺ transport: (1) ATP-dependent Ca²⁺ uptake and (2) Na⁺–Ca²⁺ exchange; the latter in the fundus only.