Relationships between Root Temperature and the Transport of Ammonium and Nitrate Ions by Italian and Perennial Ryegrass (Lolium multiflorum and Lolium perenne)

At root temperature below 14 C the absorption of ¹⁵N from NH₄⁺ greatly exceeded that from NO₂⁻ by tillers of Lolium multiflorum and Lolium perenne under conditions where pH, external concentration, plant N status, and pretreatment temperature were varied. There was a marked increase in the temperature sensitivity of NO₃⁻ transport below 14 C, irrespective of the temperature at which plants were grown previously. A marked increase in the temperature sensitivity was also seen for NH₄⁺ transport, but this occurred at the lower temperature of 10 C. Pretreatment of roots at 8 C lowered this still further to 5 C. Above and below these transition temperatures the Q₁₀ values for NO₃⁻ and NH₄⁺ transport were similar. Thus, the increased absorption of NH₄⁺ relative to NO₃⁻ at low temperatures seems to be related primarily to the difference in transition temperatures.     

Studies of the Regulation of Nitrate Influx by Barley Seedlings Using NO(3)

Using ¹³NO₃⁻, effects of various NO₃⁻ pretreatments upon NO₃⁻ influx were studied in intact roots of barley (Hordeum vulgare L. cv Klondike). Prior exposure of roots to NO₃⁻ increased NO₃⁻ influx and net NO₃⁻ uptake. This `induction' of NO<sub>3</sub><sup>−</sup> uptake was dependent both on time and external NO<sub>3</sub><sup>−</sup> concentration ([NO<sub>3</sub><sup>−</sup>]). During induction influx was positively correlated with root [NO<sub>3</sub><sup>−</sup>]. In the postinduction period, however, NO<sub>3</sub><sup>−</sup> influx declined as root [NO<sub>3</sub><sup>−</sup>] increased. It is suggested that induction and negative feedback regulation are independent processes: Induction appears to depend upon some critical cytoplasmic [NO<sub>3</sub><sup>−</sup>]; removal of external NO<sub>3</sub><sup>−</sup> caused a reduction of <sup>13</sup>NO<sub>3</sub><sup>−</sup> influx even though mean root [NO<sub>3</sub><sup>−</sup>] remained high. It is proposed that cytoplasmic [NO<sub>3</sub><sup>−</sup>] is depleted rapidly under these conditions resulting in `deinduction' of the NO₃⁻ transport system. Beyond 50 micromoles per gram [NO₃⁻], ¹³NO₃⁻ influx was negatively correlated with root [NO₃⁻]. However, it is unclear whether root [NO₃⁻] per se or some product(s) of NO₃⁻ assimilation are responsible for the negative feedback effects.     

Nitrate Reduction in Roots as Affected by the Presence of Potassium and by Flux of Nitrate through the Roots

Dark-grown, detopped corn seedlings (cv. Pioneer 3369A) were exposed to treatment solutions containing Ca(NO₃)₂, NaNO₃, or KNO₃; KNO₃ plus 50 or 100 millimolar sorbitol; and KNO₃ at root temperatures of 30, 22, or 16 C. In all experiments, the accelerated phase of NO₃⁻ transport had previously been induced by prior exposure to NO₃⁻ for 10 hours. The experimental system allowed direct measurements of net NO₃⁻ uptake and translocation, and calculation of NO₃⁻ reduction in the root. The presence of K⁺ resulted in small increases in NO₃⁻ uptake, but appreciably stimulated NO₃⁻ translocation out of the root. Enhanced translocation was associated with a marked decrease in the proportion of absorbed NO₃⁻ that was reduced in the root. When translocation was slowed by osmoticum or by low root temperatures, a greater proportion of absorbed NO₃⁻ was reduced in the presence of K⁺. Results support the proposition that NO₃⁻ reduction in the root is reciprocally related to the rate of NO₃⁻ transport through the root symplasm.     

Cyclic variations in nitrogen uptake rate in soybean plants

Uptake of NO₃⁻ by nonnodulated soybean plants (Glycine max L. Merr. cv Ransom) growing in flowing hydroponic culture at 22 and 14°C root temperatures was measured daily during a 31-day growth period. Ion chromatography was used to determine removal of NO₃⁻ from solution during each 24-hour period. At both root-zone temperatures, rate of NO₃⁻ uptake per plant oscillated with a periodicity of 3 to 5 days. The rate of NO₃⁻ uptake per plant was consistently lower at 14°C than 22°C. The lower rate of NO₃⁻ uptake at 14°C during the initial 5 to 10 days was caused by reduced uptake rates per gram root dry weight, but with time uptake rates per gram root became equal at 14 and 22°C. Thereafter, the continued reduction in rate of NO₃⁻ uptake per plant at 14°C was attributable to slower root growth.     

Endogenous NO(3) in the Root as a Source of Substrate for Reduction in the Light

An experiment was conducted to investigate the reduction of endogenous NO₃⁻, which had been taken up by plants in darkness, during the course of the subsequent light period. Vegetative, nonnodulated soybean plants (Glycine max [L]. Merrill, `Ransom') were exposed to 1.0 millimolar <sup>15</sup>NO<sub>3</sub><sup>−</sup> for 12 hours in darkness and then returned to a solution containing 1.0 millimolar <sup>14</sup>NO<sub>3</sub><sup>−</sup> for the 12 hours `chase' period in the light. Another set of plants was exposed to ¹⁵NO₃⁻ during the light period to allow a direct comparison of contributions of substrate from the endogenous and exogenous sources. At the end of the ¹⁵NO₃⁻ exposure in the dark, 70% of the absorbed ¹⁵NO₃⁻ remained unreduced, and 83% of this unreduced NO₃⁻ was retained in roots. The pool of endogenous ¹⁵NO₃⁻ in roots was depleted at a steady rate during the initial 9 hours of light and was utilized almost exclusively in the formation of insoluble reduced-N in leaves. Unlabeled endogenous NO₃⁻, which had accumulated in the root prior to the previous dark period, also was depleted in the light. When exogenous ¹⁵NO₃⁻ was supplied during the light period, the rate of assimilation progressively increased, reflecting an increased rate of uptake and decreased accumulation of NO₃⁻ in the root tissue. The dark-absorbed endogenous NO₃⁻ in the root was the primary source of substrate for whole-plant NO₃⁻ reduction in the first 6 hours of the light period, and exogenous NO₃⁻ was the primary source of substrate thereafter. It is concluded that retention of NO₃⁻ in roots in darkness and its release in the following light period is an important whole-plant regulatory mechanism which serves to coordinate delivery of substrate with the maximal potential for NO₃⁻ assimilation in photosynthetic tissues.     

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.     

Temperature-dependent water and ion transport properties of barley and sorghum roots : I. Relationship to leaf growth

Root temperature strongly affects shoot growth, possibly via “nonhydraulic messengers” from root to shoot. In short-term studies with barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) seedlings, the optimum root temperatures for leaf expansion were 25° and 35°C, respectively. Hydraulic conductance (L_p) of both intact plants and detached exuding roots of barley increased with increasing root temperature to a high value at 25°C, remaining high with further warming. In sorghum, the L_p of intact plants and of detached roots peaked at 35°C. In both species, root temperature did not affect water potentials of the expanded leaf blade or the growing region despite marked changes in L_p. Extreme temperatures greatly decreased ion flux, particularly K⁺ and NO₃⁻, to the xylem of detached roots of both species. Removing external K⁺ did not alter short-term K⁺ flux to the xylem in sorghum but strongly inhibited flux at high temperature in barley, indicating differences in the sites of temperature effects. Leaf growth responses to root temperature, although apparently “uncoupled” from water transport properties, were correlated with ion fluxes. Studies of putative root messengers must take into account the possible role of ions.     

Intercellular localization of nitrate reductase in roots

Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO₃⁻ for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO₃⁻ likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO₃⁻, the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO₃⁻ occurs during uptake and reduction of exogenous NO₃⁻. Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO₃⁻. It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO₃⁻, (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO₃⁻ is taken up by cells of the cortex and stele, and/or (c) NO₃⁻ is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO₃⁻ reduction.     

In Vivo Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) Seedlings in Light and Darkness

In vivo NO₃⁻ reduction in roots and shoots of intact barley (Hordeum vulgare L. var Numar) seedlings was estimated in light and darkness. Seedlings were placed in darkness for 24 hours to make them carbohydrate-deficient. During darkness, the leaves lost 75% of their soluble carbohydrates, whereas the roots lost only 15%. Detached leaves from these plants reduced only 7% of the NO₃⁻ absorbed in darkness. By contrast, detached roots from the seedlings reduced the same proportion of absorbed NO₃⁻, as did roots from normal light-grown plants. The rate of NO₃⁻ reduction in the roots accounted for that found in the intact dark-treated carbohydrate-deficient seedlings. The rates of NO₃⁻ reduction in roots of intact plants were the same for approximately 12 hours, both in light and darkness, after which the NO₃⁻ reduction rate in roots of plants placed in darkness slowly declined. In the dark, approximately 40% of the NO₃⁻ reduction occurred in the roots, whereas in light only 20% of the total NO₃⁻ reduction occurred in roots. A lesser proportion was reduced in roots because the leaves reduced more nitrate in light than in darkness.     

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.     

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.     

Root respiration associated with nitrate assimilation by cowpea

Nitrate uptake by roots of cowpea (Vigna unguiculata) was measured using ¹⁵NO₃⁻, and the energy cost to the root was estimated by respirometry. Roots of 8-day-old cowpea seedlings respired 0.6 to 0.8 milligram CO₂ per plant per hour for growth and maintenance. Adding 10 millimolar NO₃⁻ to the root medium increased respiration by 20 to 30% during the following 6 hours. This increase was not observed if the shoots were in the dark. Removal of NO₃⁻ from the root medium slowed the increase of root respiration. The ratios of additional respiration to the total nitrogen uptake and reduced nitrogen content in roots were 0.4 gram C per gram N and 2.3 grams C per gram N, respectively. The latter value is close to theoretical estimates of nitrate assimilation, and is similar to estimates of 1 to 4 grams C per gram N for the respiratory cost of symbiotic N₂ fixation.     

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.

     

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

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

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.     

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

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

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

     

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.     

Assimilation of NO(3) Taken Up by Plants in the Light and in the Dark

An experiment was conducted to determine the extent that NO₃⁻ taken up in the dark was assimilated and utilized differently by plants than NO₃⁻ taken up in the light. Vegetative, nonnodulated soybean plants (Glycine max L. Merrill, `Ransom') were exposed to ¹⁵NO₃⁻ throughout light (9 hours) or dark (15 hours) phases of the photoperiod and then returned to solutions containing ¹⁴NO₃⁻, with plants sampled subsequently at each light/dark transition over 3 days. The rates of ¹⁵NO₃⁻ absorption were nearly equal in the light and dark (8.42 and 7.93 micromoles per hour, respectively); however, the whole-plant rate of ¹⁵NO₃⁻ reduction during the dark uptake period (2.58 micromoles per hour) was 46% of that in the light (5.63 micromoles per hour). The lower rate of reduction in the dark was associated with both substantial retention of absorbed ¹⁵NO₃⁻ in roots and decreased efficiency of reduction of ¹⁵NO₃⁻ in the shoot. The rate of incorporation of ¹⁵N into the insoluble reduced-N fraction of roots in darkness (1.10 micromoles per hour) was somewhat greater than that in the light (0.92 micromoles per hour), despite the lower rate of whole-plant ¹⁵NO₃⁻ reduction in darkness.

A large portion of the ¹⁵NO₃⁻ retained in the root in darkness was translocated and incorporated into insoluble reduced-N in the shoot in the following light period, at a rate which was similar to the rate of whole-plant reduction of ¹⁵NO₃⁻ acquired during the light period. Taking into account reduction of NO₃⁻ from all endogenous pools, it was apparent that plant reduction in a given light period (~13.21 micromoles per hour) exceeded considerably the rate of acquisition of exogenous NO₃⁻ (8.42 micromoles per hour) during that period. The primary source of substrate for NO₃⁻ reduction in the dark was exogenous NO₃⁻ being concurrently absorbed. In general, these data support the view that a relatively small portion (<20%) of the whole-plant reduction of NO₃⁻ in the light occurred in the root system.

     

Comparative studies on the induction and inactivation of nitrate reductase in corn roots and leaves

A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH₂-nitrate reductase and NO₃⁻-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO₃⁻ was supplied. WO₄⁼ suppressed the induction of NADH- and FMNH₂-nitrate reductase activities in root tips and leaves. The NO₃⁻-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO₄⁼. In leaves, on the other hand, there was a marked superinduction of the NO₃⁻-induced NADH-cytochrome c reductase in the presence of WO₄⁼.     

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

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