The case for cytosolic NO3– heterostasis: a critique of a recently proposed model

A model recently proposed by Siddiqi & Glass (Plant, Cell, and Environment 25, 1211–1217, 2002) attempts to reconcile discrepancies in the measurement of cytosolic nitrate concentrations ([NO₃^–]_cyt) in plant root cells, specifically between low (～ 4 mm ) homeostatic values reported in studies using ion‐specific microelectrodes on the one hand, and wide fluctuations in [NO₃^–]_cyt reported in other studies, especially those using compartmental analysis by tracer efflux (CATE). Although Siddiqi & Glass concede that cytosolic NO₃^– homeostasis, as determined by microelectrodes, is at odds with certain experimental observations, they nevertheless promote a model that takes microelectrode readings at face value, and assert that the variations seen using CATE methodology are artefacts attributable to contributions from substantial, rapidly exchanging, and highly variable NO₃^– pools putatively residing in organelles such as plastids and the endoplasmic reticulum. We show here that such a model is not tenable, drawing upon experimental evidence from previous studies, and from a more comprehensive model that examines the characteristics and consequences of subcompartmented cytoplasmic exchange in root cells.     

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.     

Induction of nitrate uptake and nitrate reductase activity in trembling aspen and lodgepole pine

¹³NO₃^– influx into the roots and in vivo nitrate reductase activity (NRA) in the roots and leaves have been measured in trembling aspen (Populus tremuloides Michx.) and lodgepole pine (Pinus contorta Dougl.) seedlings after exposure to either 0·1 or 1·5 mol m^–3 NO₃^– for varying periods up to 20 d. Both NO₃^– influx and NRA were inducible in these species and, in trembling aspen, peak induction of nitrate influx and NRA were achieved within 12 h, compared to 2–4 d for influx and 4–12 d for NRA in lodgepole pine. In trembling aspen, ≈ 30% of the total ¹³N absorbed during a 10 min influx period followed by 2 min of desorption was translocated to the shoot. In lodgepole pine, by contrast, translocation of ¹³N to the shoot was undetectable during the same time period. Root NRA as well as NO₃^– influx from 0·1 mol m^–3 NO₃^– were substantially higher in trembling aspen than in lodgepole pine at all stages of NO₃^– exposure, i.e. during the uninduced, the peak induction, and steady-state stages. In order to examine whether the lower rates of NO₃^– influx and NRA were related to proportionately fewer young (unsuberized) roots in lodgepole pine, we determined these parameters in young and old (suberized) roots of this species separately. Induction of influx and NRA were initially greater in young roots but at steady-state there were only minor differences between the young and the old roots. However, even the elevated initial rates in the young roots of lodgepole pine were substantially lower than those of aspen. In pine, influx at 1·5 mol m^–3 NO₃^– was ~ 6-fold higher than at 0·1 mol m^–3 NO₃^– and appeared to be mostly via a constitutive system. By contrast, in aspen, steady-state influxes at 0·1 and 1·5 mol m^–3 were not significantly different, being similar to the rate attained by pine at only the higher [NO₃^–]. In aspen, leaf NRA was ~ 2-fold higher than that of roots. In lodgepole pine NRA of the needles was below the detection limit. These results show that trembling aspen seedlings are better adapted for NO₃^– acquisition and utilization than lodgepole pine seedlings.     

A comparative study of fluxes and compartmentation of nitrate and ammonium in early-successional tree species

¹³NO₃^– and ¹³NH₄⁺ compartmental analyses were carried out in seedling roots of trembling aspen (Populus tremuloides Michx.), lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) and interior Douglas-fir (Pseudotsuga menziesii var. glauca [Beissn.] Franco) at 0·1 and 1·5 mol m^–3 external NO₃^– or NH₄⁺ concentrations ([NO₃^–]_o or [NH₄⁺]_o, respectively). At the lower [NO₃^–]_o, the capacities and efficiencies of acquisition and accumulation of NO₃^–, based upon NO₃^– fluxes and cytoplasmic NO₃^– concentrations ([NO₃^–]_c), were in the order aspen >> Douglas-fir > pine. At 1·5 mol m^–3[NO₃^–]_o, the NO₃^– influx increased 18-fold in pine, four-fold in Douglas-fir and approximately 1·4-fold in aspen; in fact, at 1·5 mol m^–3[NO₃^–]_o, the NO₃^– influx in pine was higher than in aspen. However, at high [NO₃^–]_o, efflux also increased in the two conifers to a much greater extent than in aspen. In aspen, at both [NO₃^–]_o, approximately 30% of the ¹³N absorbed was translocated to the shoot during 57 min of ¹³N loading and elution, compared with less than 10% in the conifers. At 0·1 mol m^–3[NH₄⁺]_o, influx and net flux were in the order: aspen > pine > Douglas-fir but the differences were much less than in NO₃^– fluxes. At 1·5 mol m^–3[NH₄⁺]_o, NH₄⁺ influx, efflux and [NH₄⁺]_c greatly increased in aspen and Douglas-fir and, to a much lesser extent, in pine. In aspen, 29 and 12% of the ¹³N absorbed was translocated to the shoot at 0·1 and 1·5 mol m^–3[NH₄⁺]_o, respectively, compared with 5 to 7% in the conifers at either [NH₄⁺]_o. These patterns of nitrogen (N) uptake, particularly in the case of NO₃^–, and the observed concentration responses of NO₃^– uptake, reflect the availability of N in the ecological niches, to which these species are adapted.     

Studies of the uptake of nitrate in barley : v. Estimation of root cytoplasmic nitrate concentration using nitrate reductase activity-implications for nitrate influx

The cytoplasmic NO₃⁻ concentration ([NO₃⁻]_c) was estimated for roots of barley (Hordeum vulgare L. cv Klondike) using a technique based on measurement of in vivo nitrate reductase activity. At zero external NO₃⁻ concentration ([NO₃⁻]_o), [NO₃⁻]_c was estimated to be 0.66 mm for plants previously grown in 100 μm NO₃⁻. It increased linearly with [NO₃⁻]_o between 2 and 20 mm, up to 3.9 mm at 20 mm [NO₃⁻]_o. The values obtained are much lower than previous estimates from compartmental analysis of barley roots. These observations support the suggestion (MY Siddiqi, ADM Glass, TJ Ruth [1991] J Exp Bot 42: 1455-1463) that the nitrate reductase-based technique and compartmental analysis determine [NO₃⁻]_c for two separate pools; an active, nitrate reductase-containing pool (possibly located in the epidermal cells) and a larger, slowly metabolized storage pool (possibly in the cortical cells), respectively. Given the values obtained for [NO₃⁻]_c and cell membrane potentials of −200 to −300 mV (ADM Glass, JE Schaff, LV Kochian [1992] Plant Physiol 99: 456-463), it is very unlikely that passive influx of NO₃⁻ is possible via the high-concentration, low-affinity transport system for NO₃⁻. This conclusion is consistent with the suggestion by Glass et al. that this system is thermodynamically active and capable of transporting NO₃⁻ against its electrochemical potential gradient.     

Short-term regulation of cytosolic Ca2+, cytosolic pH and vacuolar pH under NaCl stress in the charophyte alga Nitellopsis obtusa

Isolated characean internodal cells of Nitellopsis obtusa can be stored in artificial pond water for many days, but they cannot survive in 100mol m^?3 NaCl solution unless more than several mol m^?3 Ca²⁺ is added. Short-term effects of NaCl stress on the cytosolic concentration of Ca²⁺ ([Ca²⁺]_c), cytosolic pH (pH_c) and vacuolar pH (pH_v) were studied in relation to the external concentration of Ca²⁺ ([Ca²⁺]_e). Changes in [Ca²⁺]_c were measured with light emission from a Ca²⁺-sensitive photoprotein, semisynthetic fch-aequorin which had been injected into the cytosol. Both pH_c and pH_v were measured with double-barrelled pH-sensitive microelectrodes. When internodal cells were treated with 100 mol m^?3 NaCl (0–1 mol m^?3 NaCl (0.1 mol m^?3 [Ca²⁺]_e), [Ca²⁺]_c increased and then recovered to the original level within 60 min. The time course of the transient change in [Ca²⁺]_c was not influenced by the level of [Ca²⁺]_c (0.1 and 10 mol m^?3). In some cases, the transient increase in [Ca²⁺]_c was induced only by increasing external osmotic pressure with sorbitol. In response to treatment with 100 mol m^?3 NaCl (0.1 mol m^?3 [Ca²⁺]_c), pH_c decreased by 0.1–0.2 units after 10min but recovered after 30–60 min, while pH_v increased by 0.4–0.5 units after 2–50 min and tended to recover after 60 min. The initial changes in both pH_c and pH_v were suppressed when [Ca²⁺]_e was raised from 0.1 to 10mol m^?3. These results show that the charophyte alga Nitellopsis can regulate [Ca²⁺]_c, pH_c and pH_v under NaCl stress in the short term and that the protective effect of Ca²⁺ on salinity stress is apparently unrelated to perturbation of Ca²⁺ and pH homeostasis.     

Nitrate reductase and nitrate accumulation in relation to nitrate toxicity in Boronia megastigma

Moderate levels of N were toxic to the native Australian plant boronia (Boronia megastigma Nees). As NO^-₃ is the major N form available for plants under cultivated conditions, NO^-₃ reduction and accumulation patterns in boronia were examined following the supply of various levels of NO^-₃ to understand the physiological basis of this toxicity. At a low level of supplied NO^-₃ [15 mmol (plant)^-1], NO^-₃ was reduced without any detectable accumulation and without nitrate reductase activity (NRA) reaching its maximum capacity. When higher NO^-₃ levels [≥25 mmol (plant)^-1] were supplied, both NRA and NO^-₃ accumulation increased further. However, NRA increased to a maximum of ca 500 nmol NO^-₃ (g fresh weight)^-1 h^-1, both in the roots and leaves, irrespective of a 4-fold difference in the levels of supplied NO^-₃, whereas NO^-₃ continued to accumulate in proportion to the level of supplied NO^-₃. Chlorotic toxicity symptoms appeared on the leaves at an accumulation of ca 32 μmol NO^-₃ (g fresh weight)^-1. High endogenous NO^-₃ concentrations inhibited NRA. The low level of NRA in boronia was not limited by NO^-₃ or electron donor availability. It is concluded that the low NR enzyme activity is a genetic adaptation to the low NO^-₃ availability in the native soils of boronia. Thus, when NO^-₃ supply is high, the plat cannot reduce it at high rates, leading to large and toxic accumulations of the ion in the leaf tissues.     

Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells

We recently reported that nitrogen dioxide (NO₂), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO₂ resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO₂ exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO₂ for 1–24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p < 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO₂-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar¹ Ile⁸] Ang II. Exposure of 5 ppm NO₂ for 1–24 hr provoked rapid increases in [³H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO₂ increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.     

Time-course of Nitrate Uptake and Nitrate Reductase Activity in Nitrogen-depleted Dwarf Bean

The rate of nitrate uptake by N-depleted French dwarf bean (Phaseolus vulgaris L. cv. Witte Krombek) increased steadily during the first 6 h after addition of NO₃ -After this initial phase the rale remained constant for many hours. Detached root systems showed the same time-course of uptake as roots of intact plants. In vivo nitrate reductase activity (NRA) was assayed with or without exogenous NO₃^- in the incubation medium and the result ing activities were denoted potential and actual level, respectively. In roots the difference between actual and potential NRA disappeared within 15 min after addition of nitrate, and NRA increased for about 15 h. Both potential and actual NRA were initially very low. In leaves, however, potential NRA was initially very high and was not affected by ambient nitrate (0.1–5 mol m^-3) for about 10 h. Actual and potential leaf NRA became equal after the same period of time. In the course of nitrate nutrition, the two nitrate reductase activities in leaves were differentially inhibited by cycloheximide (3.6 mmol m^-3) and tungstate (1 mol m^-3). We suggest that initial potential NRA reflects the activity of pre-existing enzyme, whereas actual NRA depends on enzyme assembly during NO₃^- supply. Apparent induction of nitrate uptake and most (85%) of the actual in vivo NRA occurred in the root system during the first 6 h of nitrate utilization by dwarf bean.     

Nitrogen balance for wheat canopies (Triticum aestivum cv. Veery 10) grown under elevated and ambient CO2 concentrations

We examined the hypothesis that elevated CO₂ concentration would increase NO₃^– absorption and assimilation using intact wheat canopies (Triticum aestivum cv. Veery 10). Nitrate consumption, the sum of plant absorption and nitrogen loss, was continuously monitored for 23 d following germination under two CO₂ concentrations (360 and 1000 μmol mol^–1 CO₂) and two root zone NO₃^– concentrations (100 and 1000 mmol m³ NO₃^–). The plants were grown at high density (1780 m^–2) in a 28 m³ controlled environment chamber using solution culture techniques. Wheat responded to 1000 μmol mol^–1 CO₂ by increasing carbon allocation to root biomass production. Elevated CO₂ also increased root zone NO₃^– consumption, but most of this increase did not result in higher biomass nitrogen. Rather, nitrogen loss accounted for the greatest part of the difference in NO₃^– consumption between the elevated and ambient [CO₂] treatments. The total amount of NO₃^–-N absorbed by roots or the amount of NO₃^–-N assimilated per unit area did not significantly differ between elevated and ambient [CO₂] treatments. Instead, specific leaf organic nitrogen content declined, and NO₃^– accumulated in canopies growing under 1000 μmol mol^–1 CO₂. Our results indicated that 1000 μmol mol^–1 CO₂ diminished NO₃^– assimilation. If NO₃^– assimilation were impaired by high [CO₂], then this offers an explanation for why organic nitrogen contents are often observed to decline in elevated [CO₂] environments.     

Seasonal changes in nitrate use by three woody species: the importance of the leaf-expansion period

Seasonal changes in plant NO₃ ⁻-N use were investigated by measuring leaf nitrate reductase activity (NRA), leaf N concentration, and leaf expansion in one evergreen woody species (Quercus glauca Thunb.) and two deciduous woody species [Acer palmatum Thunb. and Zelkova serrata (Thunb.) Makino]. Leaf N concentration was highest at the beginning of leaf expansion and decreased during the expansion process to a steady state at the point of full leaf expansion in all species. The leaf NRA of all species was very low at the beginning of leaf expansion, followed by a rapid increase and subsequent decrease. The highest leaf NRA was observed in the middle of the leaf-expansion period, and the lowest leaf NRA occurred in summer for all species. Significant positive correlations were detected between leaf NRA and leaf expansion rates, while leaf N concentrations were negatively correlated with leaf area. In the evergreen Q. glauca, the N concentration in current buds increased before leaves opened; concurrently, the N concentration in 1-year-old leaves decreased by 25%. Our results show that the leaf-expansion period is the most important period for NO₃ ⁻-N assimilation by broadleaf tree species, and that decreases in leaf N concentration through the leaf-expansion period are at least partly compensated for by newly assimilated NO₃ ⁻-N in current leaves.     

Growth,chemical composition,and nitrate reductase activity of Rumex species in relation to form and level of N supply

Growth, chemical composition, and nitrate reductase activity (NRA) of hydroponically cultured Rumex crispus, R. palustris, R. acetosa, and R. maritimus were studied in relation to form (NH₄ ⁺, NO₃ ^-, or both) and level of N supply (4 mM N, and zero-N following a period of 4mM N). A distinct preference for either NH₄ ⁺ or NO₃ ^- could not be established. All species were characterized by a very efficient uptake and utilization of N, irrespective of N source, as evident from high concentrations of organic N in the tissues and concurrent excessive accumulations of free NO₃ ^- and free NH₄ ⁺. Especially the accumulation of free NH₄ ⁺ was unusually large. Generally, relative growth rate (RGR) was highest with a combination of NH₄ ⁺ and NO₃ ^-. Compared to mixed N supply, RGR of NO₃ ^-- and NH₄ ⁺-grown plants declined on average 3% and 9%, respectively. Lowest RGR with NH₄ ⁺ supply probably resulted from direct or indirect toxicity effects associated with high NH₄ ⁺ and/or low Ca²⁺ contents of tissues. NRA in NO₃ ^- and NH₄NO₃ plants was very similar with maxima in the leaves of ca 40 μmol NO₂ ^- g^-1 DW h^-1. ‘Basal’ NRA levels in shoot tissues of NH₄ ⁺ plants appeared relatively high with maxima in the leaves of ca 20 μmol NO₂ ^- g^-1 DW h^-1. Carboxylate to organic N ratios, (C-A)/N_org, on a whole plant basis varied from 0.2 in NH₄ ⁺ plants to 0.9 in NO₃ ^- plants. After withdrawal of N, all accumulated NO₃ ^- and NH₄ ⁺ was assimilated into organic N and the organic N redistributed on a large scale. NRA rapidly declined to similar low levels, irrespective of previous N source. Shoot/root ratios of -N plants were 50–80% lower than those from +N plants. In comparison with +N, RGR of -N plants did not decline to a large extent, decreasing by only 15% in -NH₄ ⁺ plants due to very high initial organic-N contents. N-deprived plants all exhibited an excess cation over anion uptake (net proton efflux), and whole-plant (C-A)/N_org ratios increased to values around unity. Possible difficulties in interpreting the (C-A)/N_org ratio and NRA of plants in their natural habitats are briefly discussed.     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

Assessing crop N status of fertigated vegetable crops using plant and soil monitoring techniques

Evaluation of crop N status will assist optimal N management of intensive vegetable production. Simple procedures for monitoring crop N status such as petiole sap [NO₃^?–N], leaf N content and soil solution [NO₃^?] were evaluated with indeterminate tomato and muskmelon. Their sensitivity to assess crop N status throughout each crop was evaluated using linear regression analysis against nitrogen nutrition index (NNI) and crop N content. NNI is the ratio between the actual and the critical crop N contents (critical N content is the minimum N content necessary to achieve maximum growth), and is an established indicator of crop N status. Nutrient solutions with four different N concentrations (treatments N1–N4) were applied throughout each crop. Average applied N concentrations were 1, 5, 13 and 22 mmol L^?1 in tomato, and 2, 7, 13 and 21 mmol L^?1 in muskmelon. Respective rates of N were 23, 147, 421 and 672 kg N ha^?1 in tomato, and 28, 124, 245 and 380 kg N ha^?1 in muskmelon. For each N treatment in each crop, petiole sap [NO₃^?–N] was relatively constant throughout the crop. During both crops, there were very significant (P < 0.001) linear relationships between both petiole sap [NO₃^?–N] and leaf N content with NNI and with crop N content. In indeterminate tomato, petiole sap [NO₃^?–N] was very strongly linearly related to NNI (R² = 0.88–0.95, P < 0.001) with very similar slope and intercept values on all dates. Very similar relationships were obtained from published data of processing tomato. A single linear regression (R² = 0.77, P < 0.001) described the relationship between sap [NO₃^?–N] and NNI for both indeterminate and processing tomato, each grown under very different conditions. A single sap [NO₃^?–N] sufficiency value of 1050 mg N L^?1 was subsequently derived for optimal crop N nutrition (at NNI = 1) of tomato grown under different conditions. In muskmelon, petiole sap [NO₃^?–N] was strongly linearly related to NNI (R² = 0.75 – 0.88, P < 0.001) with very similar slope and intercept values for much of the crop (44–72 DAT, days after transplanting). A single linear relationship between sap [NO₃^?–N] and NNI (R² = 0.77, P < 0.001) was derived for this period, but sap sufficiency values could not be derived for muskmelon as NNI values were >1. Relationships between petiole sap [NO₃^?–N] with crop N content, and leaf N content with both NNI and crop N content had variable slopes and intercept values during the indeterminate tomato and the muskmelon crops. Soil solution [NO₃^?] in the root zone was not a sensitive indicator of crop N status. Of the three systems examined for monitoring crop/soil N status, petiole sap [NO₃^?–N] is suggested to be the most useful because of its sensitivity to crop N status and because it can be rapidly analysed on the farm.     

Delivery of N2 to bacteroids in simulated soybean nodule cells: consideration of gradients of concentration of dissolved N2 in cell walls,cytoplasm, and symbiosomes

Summary The previously published simulation of physiological functions occurring in infected cells of soybean nodules has been extended to include consideration of the diffusion of N₂ from the outside of a nodule to the nitrogen-fixing bacteroids, in relation to published values for the apparentK _m(N₂) for nitrogen fixation in the soybean nodule system. Nitrogen fixation is driven by bacteroid respiration, so increases in the average relative oxygenation (Y) of cytoplasmic leghaemoglobin lead to increased bacteroid respiration, increased nitrogen fixation, and greater differences in concentration of dissolved N₂ between the cell surface and the innermost bacteroids (d[N₂]). Over the range ofY considered, values for d[N₂] were from 5.2- to 6.2-fold greater than the corresponding values for d[O₂], because of facilitation of O₂ flux by cytoplasmic leghaemoglobin. Gradients of [N₂] within symbiosomes are small relative to cytoplasmic values and at the symbiosome surface [N₂] was greater than 0.4 mol/m³ at the greatest rates of nitrogen fixation calculated. Therefore, it is unlikely that values for [N₂] anywhere in the infected cell are low enough to affect rates of nitrogen fixation significantly, unless low external atmospheric N₂ pressures are used experimentally.Abbreviations Lb leghaemoglobin - LbO₂ oxyleghaemoglobin - [O₂], [N₂ concentrations of free, dissolved oxygen and nitrogen - Y fractional oxygenation of leghaemoglobin     

Nitrate reductase activity and nitrogen compounds in xylem exudate of Juglans nigra seedlings: relation to nitrogen source and supply

Nitrogen (N) limits plant productivity and its uptake and assimilation may be regulated by N source, N availability, and nitrate reductase activity (NRA). Knowledge of how these factors interact to affect N uptake and assimilation processes in woody angiosperms is limited. We fertilized 1-year-old, half-sib black walnut (Juglans nigra L.) seedlings with ammonium (NH₄ ⁺) [as (NH₄)₂SO₄], nitrate (NO₃ ⁻) (as NaNO₃), or a mixed N source (NH₄NO₃) at 0, 800, or 1,600 mg N plant⁻¹ season⁻¹. Two months following final fertilization, growth, in vivo NRA, plant N status, and xylem exudate N composition were assessed. Specific leaf NRA was higher in NO₃ ⁻-fed and NH₄NO₃-fed plants compared to observed responses in NH₄ ⁺-fed seedlings. Regardless of N source, N addition increased the proportion of amino acids (AA) in xylem exudate, inferring greater NRA in roots, which suggests higher energy cost to plants. Root total NRA was 37% higher in NO₃ ⁻-fed than in NH₄ ⁺-fed plants. Exogenous NO₃ ⁻ was assimilated in roots or stored, so no difference was observed in NO₃ ⁻ levels transported in xylem. Black walnut seedling growth and physiology were generally favored by the mixed N source over NO₃ ⁻ or NH₄ ⁺ alone, suggesting NH₄NO₃ is required to maximize productivity in black walnut. Our findings indicate that black walnut seedling responses to N source and level contrast markedly with results noted for woody gymnosperms or herbaceous angiosperms.     

Studies of the Uptake of Nitrate in Barley : II. Energetics

Q₁₀ values for ¹³NO₃⁻ influx were determined in `uninduced' (NO<sub>3</sub><sup>−</sup>-starved) and `induced' (NO₃⁻-pretreated) roots of barley (Hordeum vulgare L.) plants at various concentrations of external NO₃⁻ ([NO₃⁻]₀). At 0.02 mole per cubic meter [NO₃⁻]₀, Q₁₀ values for influx were from 3 to 4 between 5 and 10°C. As [NO₃⁻]₀ increased Q₁₀ values decreased, reaching values of 1.2 and 2.0, respectively, at 20 moles per cubic meter in uninduced and induced plants. The metabolic dependence of ¹³NO₃⁻ influx at low and high [NO₃⁻]₀ (0.1 and 20.0 moles per cubic meter, respectively) in uninduced and induced plants was probed by the use of various inhibitors. These experiments confirmed the findings of the Q₁₀ studies, demonstrating that at low [NO₃⁻]₀¹³NO₃⁻ influx was extremely sensitive to metabolic inhibition. By contrast, at high [NO₃⁻]₀, influx was relatively insensitive to the presence of inhibitors.     

Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH₄⁺ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO₃⁺ in the presence of 0, 1 or 10 mM NH₄⁺, Exogenous NH₄⁺ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO₃⁺, In contrast, the induction of NiRA was inhibited by NH₄⁺ at all NO₃⁺ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH₄⁺ Pre-treatment with NH₄⁺ had no effect on the subsequent induction of NRA in the absence of additional NH₄⁺ whereas the induction of NiRA in NH₄⁺-pretreated roots was inhibited in the absence of NH₄⁺ At 10 mM NO₃⁺ L-methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH₄⁺ was present. In contrast, the induction of NiRA was inhibited by L-methionine sulfoximine irrespective of NH₄⁺ supply. During the postinduction phase, exogenous NH₄⁺ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH₃⁺ whereas, NiRA was unaffected by NH₄⁺ at either substrate concentration. The results indicate that exogenous NH₄⁺ regulates the induction of NRA in roots by limiting the availability of NO₃⁺. Conversely, it has a direct effect, independent of the availability of NO₃⁺, on the induction of NiRA. The lack of an NH₄⁺ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.     

A dynamic whole-plant model of integrated metabolism of nitrogen and carbon. 1. Comparative ecological implications of ammonium-nitrate interactions

The dynamics of ammonium (NH₄ ⁺) and nitrate (NO₃ ^-) concentrations in the soil solution is an important determinant of the species composition of natural vegetation. A mathematical model of uptake, assimilation and translocation of NH₄ ⁺ and NO₃ ^- is presented to assess the performance of species with respect to NO₃ ^-/NH₄ ⁺ feeding characterised by physiologically defined parameters. Nitrate efflux is explicitly considered. The capacities of NO₃ ^-, [U _NM], and NH₄ ⁺ influx, [U _AM], and NO₃ ^- reduction, [A _NM], appear sufficient to characterise whole-plant N metabolism including NO₃ ^- translocation. The parameter space made up by these parameters is represented by 276 parameter combinations (`species'). Simulated total net N uptake rate and C costs for uptake and assimilation per mole total net N taken up are used to decide on how a species profits or suffers from NO<sub>3</sub> <sup>-</sup>+NH<sub>4</sub> <sup>+</sup> mixtures relative to pure N forms with similar total N concentration for external concentrations up to 1.6 mM. Five response categories were identified and contrasted with categories defined by Bogner (1968) on the basis of experimental results on forest plants. The largest category comprises species that respond positively to NO<sub>3</sub> <sup>-</sup> and positively or indifferently to NH<sub>4</sub> <sup>+</sup>. These species have intermediate to high [U <sub>NM</sub>] and [A <sub>NM</sub>] and variable [U <sub>AM</sub>] and correspond to woodland edge species and forest plants on rich soil including typical `nitrophilic' species. This category fades into a group of species that respond positively to NO₃ ^- and negatively to NH₄ ⁺. These species have high [U _NM] and low [U _AM] and [A _NM]; several species from oak-hornbeam woodland (Carpinion) belong to this group. Many parameter combinations were found that responded positively to NH₄ ⁺ and indifferently to NO₃ ^-: low to medium [U _NM], medium to high [U _AM] and variable [A _NM]. This category includes all heathland species. No species were found which responded negatively to NO₃ ^-. The physiological background of differences between the categories is explained with respect to the equilibrium NO₃ ^- concentration in roots, influx, efflux, translocation and assimilation of NO₃ ^- and uptake and assimilation of NH₄ ⁺. The relationship between NO₃ ^- accumulation capacity and morphology is discussed. Some slow-growing species with high [U _NM] and low [A _NM] use NO₃ ^- mainly as an osmotic solute. Respiratory costs in roots of inherently slow-growing species are discussed with respect to patterns in NH₄ ⁺ and NO₃ ^- availabilities of their habitat. This revised version was published online in June 2006 with corrections to the Cover Date.