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.     

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.

     

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.     

Dependency of Nitrate Reduction on Soluble Carbohydrates in Primary Leaves of Barley under Aerobic Conditions

Nitrate reduction was studied as a function of carbohydrate concentration in detached primary leaves of barley (Hordeum vulgare L. cv Numar) seedlings under aerobic conditions in light and darkness. Seedlings were grown either in continuous light for 8 days or under a regimen of 16-hour light and 8-hour dark for 8 to 15 days. Leaves of 8-day-old seedlings grown in continuous light accumulated 4 times more carbohydrates than leaves of plants grown under a light and dark regimen. When detached leaves from these seedlings were supplied with NO₃⁻ in darkness, those with the higher levels of carbohydrates reduced a greater proportion of the NO₃⁻ that was taken up. In darkness, added glucose increased the percentage of NO₃⁻ reduced up to 2.6-fold depending on the endogenous carbohydrate status of the leaves. Both NO₃⁻ reduction and carbohydrate content of the leaves increased with age. Fructose and sucrose also increased NO₃⁻ reduction in darkness to the same extent as glucose. Krebs cycle intermediates, citrate and succinate, did not increase NO₃⁻ reduction, whereas malate slightly stimulated it in darkness.

In light, 73 to 90% of the NO₃⁻ taken up was reduced by the detached leaves; therefore, an exogenous supply of glucose had little additional effect on NO₃⁻ reduction. The results indicate that in darkness the rate of NO₃⁻ reduction in primary leaves of barley depends upon the availability of carbohydrates.

     

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 photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves

The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO₃⁻ and NO₂⁻ assimilation were studied using detached barley (Hordeum vulgare L. cv Numar) leaves in which only endogenous NO₃⁻ or NO₂⁻ were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO₃⁻ reduction in both light and darkness, while photosynthetic inhibitors did not.

The NO₂⁻ concentration in the control leaves was very low in both light and darkness; 98% or more of the NO₂⁻ formed from NO₃⁻ was further assimilated in control leaves. More NO₂⁻ accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO₂⁻, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO₂⁻ reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors.

Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO₂⁻ formed from NO₃⁻ was further assimilated, whereas in light, 95% or more of the NO₂⁻ was further assimilated.

     

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.     

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.     

The Influence of Nitrate and Chloride Uptake on Expressed Sap pH, Organic Acid Synthesis, and Potassium Accumulation in Higher Plants

The influence of NO₃⁻ uptake and reduction on ionic balance in barley seedlings (Hordeum vulgare, cv. Compana) was studied. KNO₃ and KCl treatment solutions were used for comparison of cation and anion uptake. The rate of Cl⁻ uptake was more rapid than the rate of NO₃⁻ uptake during the first 2 to 4 hours of treatment. There was an acceleration in rate of NO₃⁻ uptake after 4 hours resulting in a sustained rate of NO₃⁻ uptake which exceeded the rate of Cl⁻ uptake. The initial (2 to 4 hours) rate of K⁺ uptake appeared to be independent of the rate of anion uptake. After 4 hours the rate of K⁺ uptake was greater with the KNO₃ treatment than with the KCl treatment, and the solution pH, cell sap pH, and organic acid levels with KNO₃ increased, relative to those with the KCl treatment. When absorption experiments were conducted in darkness, K⁺ uptake from KNO₃ did not exceed K⁺ uptake from KCl. We suggest that the greater uptake and accumulation of K⁺ in NO₃⁻-treated plants resulted from (a) a more rapid, sustained uptake and transport of NO₃⁻ providing a mobile counteranion for K⁺ transport, and (b) the synthesis of organic acids in response to NO₃⁻ reduction increasing the capacity for K⁺ accumulation by providing a source of nondiffusible organic anions.     

Nitrate 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.     

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.     

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.

     

Root respiration associated with ammonium and nitrate absorption and assimilation by barley

We examined nitrate assimilation and root gas fluxes in a wild-type barley (Hordeum vulgare L. cv Steptoe), a mutant (nar1a) deficient in NADH nitrate reductase, and a mutant (nar1a;nar7w) deficient in both NADH and NAD(P)H nitrate reductases. Estimates of in vivo nitrate assimilation from excised roots and whole plants indicated that the nar1a mutation influences assimilation only in the shoot and that exposure to NO₃⁻ induced shoot nitrate reduction more slowly than root nitrate reduction in all three genotypes. When plants that had been deprived of nitrogen for several days were exposed to ammonium, root carbon dioxide evolution and oxygen consumption increased markedly, but respiratory quotient—the ratio of carbon dioxide evolved to oxygen consumed—did not change. A shift from ammonium to nitrate nutrition stimulated root carbon dioxide evolution slightly and inhibited oxygen consumption in the wild type and nar1a mutant, but had negligible effects on root gas fluxes in the nar1a;nar7w mutant. These results indicate that, under NH₄⁺ nutrition, 14% of root carbon catabolism is coupled to NH₄⁺ absorption and assimilation and that, under NO₃⁻ nutrition, 5% of root carbon catabolism is coupled to NO₃⁻ absorption, 15% to NO₃⁻ assimilation, and 3% to NH₄⁺ assimilation. The additional energy requirements of NO₃⁻ assimilation appear to diminish root mitochondrial electron transport. Thus, the energy requirements of NH₄⁺ and NO₃⁻ absorption and assimilation constitute a significant portion of root respiration.     

Sodium Stimulates Growth of Amaranthus tricolor L. Plants through Enhanced Nitrate Assimilation

Effects of Na application on the capacity of NO₃⁻ assimilation were studied in Na-deficient Amaranthus tricolor L. cv Tricolor plants. On day 30 after germination, Na-deficient A. tricolor plants received either 0.5 millimolar NaCl or KCl. The level of nitrate reductase activity doubled within 24 hours by the addition of Na and the enhanced level was maintained thereafter. When the plants were exposed to 2 millimolar ¹⁵NO₃⁻, total ¹⁵N taken up by the plants was greater in the Na-treated plants than in the K-treated plants within 24 hours of the Na treatment. Incorporation of ¹⁵N into the 80% ethanol-insoluble nitrogen fraction of the Na-treated plants in the light period was about 260% of those of the K-treated plants indicating greater capacity of NO₃⁻ assimilation in the Na-treated plants. From these results, it was demonstrated that Na application to the Na-deficient A. tricolor plants promoted NO₃⁻ reduction and its subsequent assimilation into protein, resulting in growth enhancement.     

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.     

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.     

Effect of ammonium on nitrate utilization by roots of dwarf bean

The effect of exogenous NH₄⁺ on NO₃⁻ uptake and in vivo NO₃⁻ reductase activity (NRA) in roots of Phaseolus vulgaris L. cv Witte Krombek was studied before, during, and after the apparent induction of root NRA and NO₃⁻ uptake. Pretreatment with NH₄Cl (0.15-50 millimolar) affected neither the time pattern nor the steady state rate of NO₃⁻ uptake.

When NH₄⁺ was given at the start of NO₃⁻ nutrition, the time pattern of NO₃⁻ uptake was the same as in plants receiving no NH₄⁺. After 6 hours, however, the NO₃⁻ uptake rate (NUR) and root NRA were inhibited by NH₄⁺ to a maximum of 45% and 60%, respectively.

The response of the NUR of NO₃⁻-induced plants depended on the NH₄Cl concentration. Below 1 millimolar NH₄⁺, the NUR declined immediately and some restoration occurred in the second hour. In the third hour, the NUR became constant. In contrast, NH₄⁺ at 2 millimolar and above caused a rapid and transient stimulation of NO₃⁻ uptake, followed again by a decrease in the first, a recovery in the second, and a steady state in the third hour. Maximal inhibition of steady state NUR was 50%. With NO₃⁻-induced plants, root NRA responded less and more slowly to NH₄⁺ than did NUR.

Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, relieved the NH₄⁺ inhibition of the NUR of NO₃⁻-induced plants. We conclude that repression of the NUR by NH₄⁺ depends on NH₄⁺ assimilation. The repression by NH₄⁺ was least at the lowest and highest NH₄⁺ levels tested (0.04 and 25 millimolar).

     

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.     

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.     

Early effects of salinity on nitrate assimilation in barley seedlings

The effect of NaCl and Na₂SO₄ salinity on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO₃⁻ transporter was affected very little; the major effect of the salts was on its activity. Both Cl⁻ and SO₄²⁻ salts severely inhibited uptake of NO₃⁻. When compared on the basis of osmolality of the uptake solutions, Cl⁻ salts were more inhibitory (15-30%) than SO₄²⁻ salts. At equal concentrations, SO₄²⁻ salts inhibited NO₃⁻ uptake 30 to 40% more than did Cl⁻ salts. The absolute concentrations of each ion seemed more important as inhibitors of NO₃⁻ uptake than did the osmolality of the uptake solutions. Both K⁺ and Na⁺ salts inhibited NO₃⁻ uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.

Unlike NO₃⁻ uptake, NO₃⁻ reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO₃⁻ in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the `anaerobic' in vivo assay, was always greater than the actual in situ rate of NO₃⁻ reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO₃⁻ transporter, rather than that of the NO₃⁻ reduction system, may be a critical factor to plant survival during salt stress.