Regulation of NO(3) Assimilation by Anion Availability in Excised Soybean Leaves

The regulation of NO₃⁻ assimilation by xylem flux of NO₃⁻ was studied in illuminated excised leaves of soybean (Glycine max L. Merr. cv Kingsoy). The supply of exogenous NO₃⁻ at various concentrations via the transpiration stream indicated that the xylem flux of NO₃⁻ was generally rate-limiting for NO₃⁻ reduction. However, NO₃⁻ assimilation rate was maintained within narrow limits as compared with the variations of the xylem flux of NO₃⁻. This was due to considerable remobilization and assimilation of previously stored endogenous NO₃⁻ at low exogenous NO₃⁻ delivery, and limitation of NO₃⁻ reduction at high xylem flux of NO₃⁻, leading to a significant accumulation of exogenous NO₃⁻. The supply of ¹⁵NO₃⁻ to the leaves via the xylem confirmed the labile nature of the NO₃⁻ storage pool, since its half-time for exchange was close to 10 hours under steady state conditions. When the xylem flux of ¹⁵NO₃⁻ increased, the proportion of the available NO₃⁻ which was reduced decreased similarly from nearly 100% to less than 50% for both endogenous ¹⁴NO₃⁻ and exogenous ¹⁵NO₃⁻. This supports the hypothesis that the assimilatory system does not distinguish between endogenous and exogenous NO₃⁻ and that the limitation of NO₃⁻ reduction affected equally the utilization of NO₃⁻ from both sources. It is proposed that, in the soybean leaf, the NO₃⁻ storage pool is particularly involved in the short-term control of NO₃⁻ reduction. The dynamics of this pool results in a buffering of NO₃⁻ reduction against the variations of the exogenous NO₃⁻ delivery.     

Charge Balance in NO(3)-Fed Soybean: Estimation of K and Carboxylate Recirculation

Soybeans (Glycine max L. Merr., cv Kingsoy) were grown on media containing NO₃⁻ or urea. The enrichments of shoots in K⁺, NO₃⁻, and total reduced N (N_r), relative to that in Ca²⁺, were compared to the ratios K⁺/Ca²⁺,NO₃⁻/Ca²⁺, and N_r/Ca²⁺ in the xylem saps, to estimate the cycling of K⁺, and N_r. The net production of carboxylates (R⁻) was estimated from the difference between the sums of the main cations and inorganic anions. The estimate for shoots was compared to the theoretical production of R⁻ associated with NO₃⁻ assimilation in these organs, and the difference was attributed to export of R⁻ to roots. The net exchange rates of H⁺ and OH⁻ between the medium and roots were monitored. The shoots were the site of more than 90% of total NO₃⁻ reduction, and N_r was cycling through the plants at a high rate. Alkalinization of the medium by NO₃⁻-fed plants was interrupted by stem girdling, and not restored by glucose addition to the medium. It was concluded that the majority of the base excreted in NO₃⁻ medium originated from R⁻ produced in the shoots, and transported to the roots together with K⁺. As expected, cycling of K⁺ and reduced N was favoured by NO₃⁻ nutrition as compared to urea nutrition.     

Effects of Altered Carbohydrate Availability on Whole-Plant Assimilation of NO(3)

An experiment was conducted to investigate the relative changes in NO₃⁻ assimilatory processes which occurred in response to decreasing carbohydrate availability. Young tobacco plants (Nicotiana tabacum [L.], cv NC 2326) growing in solution culture were exposed to 1.0 millimolar ¹⁵NO₃⁻ for 6 hour intervals during a normal 12 hour light period and a subsequent period of darkness lasting 42 hours. Uptake of ¹⁵NO₃⁻ decreased to 71 to 83% of the uptake rate in the light during the initial 18 hours of darkness; uptake then decreased sharply over the next 12 hours of darkness to 11 to 17% of the light rate, coincident with depletion of tissue carbohydrate reserves and a marked decline in root respiration. Changes also occurred in endogenous ¹⁵NO₃⁻ assimilation processes, which were distinctly different than those in ¹⁵NO₃⁻ uptake. During the extended dark period, translocation of absorbed ¹⁵N out of the root to the shoot varied rhythmically. The adjustments were independent of ¹⁵NO₃⁻ uptake rate and carbohydrate status, but were reciprocally related to rhythmic adjustments in stomatal resistance and, presumably, water movement through the root system. Whole plant reduction of ¹⁵NO₃⁻ always was limited more than uptake. The assimilation of ¹⁵N into insoluble reduced-N in roots remained a constant proportion of uptake throughout, while assimilation in the shoot declined markedly in the first 18 hours of darkness before stabilizing at a low level. The plants clearly retained a capacity for ¹⁵NO₃⁻ reduction and synthesis of insoluble reduced-¹⁵N even when ¹⁵NO₃⁻ uptake was severely restricted and minimal carbohydrate reserves remained in the tissue.     

Exogenous NO(3) Influx and Endogenous NO(3) Efflux by Two Maize (Zea mays L.) Inbreds during Nitrogen Deprivation

The influence of nitrogen stress on net nitrate uptake resulting from concomitant ¹⁵NO₃⁻ influx and ¹⁴NO₃⁻ efflux was examined in two 12-day-old inbred lines of maize. Plants grown on ¹⁴NO₃⁻ were deprived of nitrogen for up to 72 hours prior to the 12th day and then exposed for 0.5 hour to 0.15 millimolar nitrate containing 98.7 atom% ¹⁵N. The nitrate concentration of the roots declined from approximately 100 to 5 micromolar per gram fresh weight during deprivation, and ¹⁴NO₃⁻ efflux was linearly related to root nitrate concentration. Influx of ¹⁵NO₃⁻ was suppressed in nitrogen-replete plants and increased with nitrogen deprivation up to 24 hours, indicating a dissipation of factors suppressing influx. Longer periods of nitrogen-deprivation resulted in a decline in ¹⁵NO₃⁻ influx from its maximal rate. The two inbreds differed significantly in the onset and extent of this decline, although their patterns during initial release from influx suppression were similar. Except for plants of high endogenous nitrogen status, net nitrate uptake was largely attributable to influx, and genetic variation in the regulation of this process is implied.     

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.

     

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.     

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.     

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.     

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

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

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): I. Control of Net Uptake by NO(3) Efflux

A computer-controlled multichannel data acquisition system was employed to obtain continuous measurements of net nitrate or chlorate uptake by roots of intact barley plants (Hordeum vulgare cv Betzes) using nitrate-specific electrodes. Plants, previously grown in solutions maintained at 10 or 200 micromolar NO₃⁻ (low N or high N conditions, respectively), were provided with 200 micromolar NO₃⁻ or ClO₃⁻ during the uptake period. Initial rates of NO₃⁻ uptake were several times higher in low N plants than in high N plants. Within 10 min, uptake in the former plants declined to a new steady rate which was sustained for the remainder of the experiment. No such time-dependent changes were evident in the high N plants. Rates and patterns of net chlorate uptake exhibited almost identical dependence upon previous nitrate provision. NO₃⁻ (³⁶ClO₃⁻) influx, by contrast, appeared to be independent of NO₃⁻ pretreatment prior to influx determination. Nitrate efflux, estimated by several different methods, was strongly correlated with internal nitrate concentration of the roots.     

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.     

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

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

Assimilation and Transport of Nitrogen in Nonnodulated (NO(3)-grown) Lupinus albus L

The response of nonnodulated white lupin (Lupinus albus L. cv. Ultra) plants to a range of NO₃ levels in the rooting medium was studied by in vitro assays of extracts of plant parts for NO₃ reductase (EC 1.6.6.1) activity, measurements of NO₃-N in plant organs, and solute analyses of root bleeding (xylem) sap and phloem sap from stems and petioles. Plants were grown for 65 days with 5 millimolar NO₃ followed by 10 days with 1, 5, 15, or 30 millimolar NO₃. NO₃ reductase was substrate-induced in all tissues. Roots contained 76, 68, 62 and 31% of the total NO₃ reductase activity of plants fed with 1, 5, 15, and 30 millimolar NO₃, respectively. Stem, petioles, and leaflets contained virtually all of the NO₃ reductase activity of a shoot, the activity in extracts of fruits amounting to less than 0.3% of the total enzyme recovered from the plant. Xylem sap from NO₃-grown nonnodulated plants contained the same organic solutes as from nodulated plants grown in the absence of combined N. Asparagine accounted for 50 to 70% and glutamine 10 to 20% of the xylem-borne N. The level of NO₃ in xylem sap amounted to 4, 13, 12, and 17% of the total xylem N at 1, 5, 15, and 30 millimolar NO₃, respectively. Xylem to phloem transfer of N appeared to be quantitatively important in supplying fruits and vegetative apices with reduced N, especially at low levels of applied NO₃. NO₃ failed to transfer in any quantity from xylem to phloem, representing less than 0.3% of the phloem-borne N at all levels of applied NO₃. Shoot organs were ineffective in storing NO₃. Even when NO₃ was supplied in great excess (30 millimolar level) it accounted for only 8% of the total N of stem and petioles, and only 2 and 1% of the N of leaflets and fruits, respectively.     

Nitrate (15NO3) limitation affects nitrogen partitioning between metabolic and storage sinks and nitrogen reserve accumulation in chicory (Cichorium intybus L.)

In chicory, we examined how NO₃ ⁻ supply affected NO₃ ⁻ uptake, N partitioning between shoot and root and N accumulation in the tuberized root throughout the vegetative period. Plants were grown at two NO₃ ⁻ concentrations: 0.6 and 3 mM. We used ¹⁵N-labelling/chase experiments for the quantification of N fluxes between shoot and root and for determining whether N stored in the tuberized root originates from N remobilized from the shoot or from recently absorbed NO₃ ⁻. The rate of ¹⁵NO₃ ⁻ uptake was decreased by low NO₃ ⁻ availability at all stages of growth. In young plants (10–55 days after sowing; DAS), in both NO₃ ⁻ treatments the leaves were the strongest sink for ¹⁵N. In mature (tuberizing) plants, (55–115 DAS), the rate of ¹⁵NO₃ ⁻ uptake increased as well as the amount of exogenous N allocated to the root. In N-limited plants, N allocation to the tuberized root relied essentially on recent N absorption, while in N-replete plants, N remobilized from the shoot contributed more to N-reserve accumulation in the root. In senescing plants (115–170 DAS) the rate of ¹⁵NO₃ ⁻ uptake decreased mainly in N-replete plants whereas it remained almost unchanged in N-limited plants. In both NO₃ ⁻ treatments the tuberized root was the strongest sink for recently absorbed N. Remobilization of previously absorbed N from shoot to tuberized root increased greatly in N-limited plants, whereas it increased slightly in N-replete plants. As a consequence, accumulation of the N-storage compounds vegetative storage protein (VSP) and arginine was delayed until later in the vegetative period in N-limited plants. Our results show that although the dynamics of N storage was affected by NO₃ ⁻ supply, the final content of total N, VSP and arginine in roots was almost the same in N-limited and N-replete plants. This indicates that chicory is able to build up a store of available N-reserves, even when plants are grown on low N. We also suggest that in tuberized roots there is a maximal capacity for N accumulation, which was reached earlier (soon after 100 DAS) in N-replete plants. This hypothesis is supported by the fact that in N-replete plants despite NO₃ ⁻ availability, N accumulation ceased and significant amounts of N were lost due to N efflux. Received: 14 October 1996 / Accepted: 4 February 1997     

Studies of the Uptake of Nitrate in Barley: I. Kinetics of NO(3) Influx

¹³NO₃⁻ was used to investigate patterns of NO₃⁻ influx into roots of barley plants (Hordeum vulgare L. cv Klondike) previously grown with (`induced') or without (`uninduced') a source of external NO₃⁻ ([NO₃⁻]₀). In both induced and uninduced plants, ¹³NO₃⁻ influx was biphasic in the range from 0.005 to 50 moles per cubic meter [NO₃⁻]₀. In the low concentration range (<1 mole per cubic meter for induced plants and <0.3 mole per cubic meter for uninduced plants), influx was saturable and V_max and K_m values for influx either increased or decreased according to NO₃⁻ pretreatment. By contrast, ¹³NO₃⁻ influx in the high concentration range revealed a strictly linear concentration dependence. These fluxes appeared to be mediated by a constitutive, rather than an inducible, transport system.     

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.     

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.     

Organic Acids and Ionic Balance in Xylem Exudate of Wheat during Nitrate or Sulfate Absorption

Experiments were designed to study the importance of organic acids as counterions for K⁺ translocation in the xylem during excess cation uptake. A comparison was made of xylem exudate from wheat seedlings treated 72 hours with either 1.0 millimolar KNO₃ or 0.5 millimolar K₂SO₄, both in the presence of 0.2 millimolar CaSO₄. Exudation from KNO₃ plants had twice the volume and twice the K⁺ and Ca²⁺ fluxes or rate of delivery to shoots, as K₂SO₄ plants. Malate flux was 25% higher in K₂SO₄ than in KNO₃ exudate. Malate was the principal anion accompanying K⁺ or Ca²⁺ in K₂SO₄ treatment, while in the KNO₃ treatment, NO₃⁻ was the principal anion. The contribution of SO₄²⁻ was negligible in both treatments. In a second experiment, exudate was collected every 4 hours during the daytime throughout a 72-hour treatment with KNO₃. Malate was the only anion present in exudate at first, just after the CaSO₄ pretreatment had ended. Malate concentration decreased and NO₃⁻ concentration increased with time and these concentrations were negatively correlated. By 62 hours, NO₃⁻ represented 80% of exudate anions. K⁺ and NO₃⁻ concentrations in exudate were strongly correlated with K⁺ and NO₃⁻ uptake, respectively. The first 36 hours of absorption from KNO₃ solution resembled the continuous absorption of K₂SO₄, in that malate was the principal counterion for translocation of K⁺.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O

A more sensitive analytical method for NO₃⁻ was developed based on the conversion of NO₃⁻ to N₂O by a denitrifier that could not reduce N₂O further. The improved detectability resulted from the high sensitivity of the ⁶³Ni electron capture gas chromatographic detector for N₂O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO₃⁻ to N₂O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO₃⁻ N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO₃⁻ concentrations of <2 ppb of NO₃⁻ N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of ¹⁵N in NO₃⁻. This method avoids the incomplete reduction and contamination of the NO₃⁻ -N by the NH₄⁺ and N₂ pools which can occur by the conventional method of ¹⁵NO₃⁻ analysis. N₂O-producing denitrifier strains were also used to measure the apparent K_m values for NO₃⁻ use by these organisms. Analysis of N₂O production by use of a progress curve yielded K_m values of 1.7 and 1.8 μM NO₃⁻ for the two denitrifier strains studied.     

Phosphorus stress effects on assimilation of nitrate

An experiment was conducted to investigate alterations in uptake and assimilation of NO₃⁻ by phosphorus-stressed plants. Young tobacco plants (Nicotiana tabacum [L.], cv NC 2326) growing in solution culture were deprived of an external phosphorus (P) supply for 12 days. On selected days, plants were exposed to ¹⁵NO₃⁻ during the 12 hour light period to determine changes in NO₃⁻ assimilation as the P deficiency progressed. Decreased whole-plant growth was evident after 3 days of P deprivation and became more pronounced with time, but root growth was unaffected until after day 6. Uptake of ¹⁵NO₃⁻ per gram root dry weight and translocation of absorbed ¹⁵NO₃⁻ out of the root were noticeably restricted in −P plants by day 3, and effects on both increased in severity with time. Whole-plant reduction of ¹⁵NO₃⁻ and ¹⁵N incorporation into insoluble reduced-N in the shoot decreased after day 3. Although the P limitation was associated with a substantial accumulation of amino acids in the shoot, there was no indication of excessive accumulation of soluble reduced-¹⁵N in the shoot during the 12 hour ¹⁵NO₃⁻ exposure periods. The results indicate that alterations in NO₃⁻ transport processes in the root system are the primary initial responses limiting synthesis of shoot protein in P-stressed plants. Elevated amino acid levels evidently are associated with enhanced degradation of protein rather than inhibition of concurrent protein synthesis.