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.

     

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.     

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.     

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.

     

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.

     

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.

     

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.     

Relative Content of NO(3) and Reduced N in Xylem Exudate as an Indicator of Root Reduction of Concurrently Absorbed NO(3)

It is unclear if the relative content of NO₃⁻ and reduced N in xylem exudate provides an accurate estimate of the percentage reduction of concurrently absorbed NO₃⁻ in the root. Experiments were conducted to determine whether NO₃⁻ and reduced N in xylem exudate of vegetative, nonnodulated soybean plants (Glycine max [L.] Merr., `Ransom') originated from exogenous recently absorbed ¹⁵NO₃⁻ or from endogenous ¹⁴N pools. Plants either were decapitated and exposed to ¹⁵NO₃⁻ solutions for 2 hours or were decapitated for the final 20 minutes of a 50-minute exposure to ¹⁵NO₃⁻ in the dark and in the light. Considerable amounts of ¹⁴NO₃⁻ and reduced ¹⁴N were transported into the xylem, but almost all of the ¹⁵N was present as ¹⁵NO₃⁻. Dissimilar changes in transport of ¹⁴NO₃⁻, reduced ¹⁴N and ¹⁵NO₃⁻ during the 2 hours of sap collection resulted in large variability over time in the percentage of total N in the exudate which was reduced N. Over a 20-minute period the rate of ¹⁵N transport into the xylem of decapitated plants was only 21 to 36% of the ¹⁵N delivered to the shoot of intact plants. Based on the proportion of total ¹⁵N which was found as reduced ¹⁵N in exudate and in intact plants in the dark, it was estimated that 5 to 17% of concurrently absorbed ¹⁵NO₃⁻ was reduced in the root. This was much less than the 38 to 59% which would have been predicted from the relative content of total NO₃⁻ and total reduced N in the xylem exudate.     

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.     

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.     

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.     

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.     

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.     

Simultaneous Influx and Efflux of Nitrate during Uptake by Perennial Ryegrass

Experiments with intact plants of Lolium perenne previously grown with ¹⁴NO₃⁻ revealed significant efflux of this isotopic species when the plants were transferred to solutions of highly enriched ¹⁵NO₃⁻. The exuded ¹⁴NO₃⁻ was subsequently reabsorbed when the ambient solutions were not replaced. When they were frequently replaced, continual efflux of the ¹⁴NO₃⁻ was observed. Influx of ¹⁵NO₃⁻ was significantly greater than influx of ¹⁴NO₃⁻ from solutions of identical NO₃⁻ concentration. Transferring plants to ¹⁴NO₃⁻ solutions after a six-hour period in ¹⁵NO₃⁻ resulted in efflux of the latter. Presence of Mg²⁺, rather than Ca²⁺, in the ambient ¹⁵NO₃⁻ solution resulted in a decidedly increased rate of ¹⁴NO₃⁻ efflux and a slight but significant increase in ¹⁵NO₃⁻ influx. Accordingly, net NO₃⁻ influx was slightly depressed. A model in accordance with these observations is presented; its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous NO₃⁻ within unstirred layers from the passive pathway to the active uptake site.     

The Uptake of NO3?, NO2?, and NH4+ by Intact Wheat (Triticum aestivum) Seedlings : I. Induction and Kinetics of Transport Systems

The inducibility and kinetics of the NO₃⁻, NO₂⁻, and NH₄⁺ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for and the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO₃⁻ and NO₂⁻ transporters. Evidence for substrate inducibility of the NH₄⁺ transporters requiring 5 hours is presented. The transport of NO₃⁻ was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO₂⁻ and NH₄⁺. The K_m values for NO₃⁻, NO₂⁻, and NH₄⁺ were, respectively, 0.027, 0.054, and 0.05 millimolar. The K_m for mechanism II of NO₃⁻ transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.     

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.     

Role of Nitrate and Nitrite in the Induction of Nitrite Reductase in Leaves of Barley Seedlings

The role of NO₃⁻ and NO₂⁻ in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO₂⁻/gram fresh weight × hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO₂⁻ and NO₃⁻ induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO₂⁻ and NO₃⁻ over a 24 hour period). In NO₃⁻-supplied leaves, NiR induction occurred at an ambient NO₃⁻ concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 millimolar. Nitrate accumulated in NO₂⁻-fed leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NiR as did equivalent amounts of NO₃⁻ accumulating in NO₃⁻-fed leaves. Induction of NiR in NO₂⁻-fed leaves was not seen until NO₃⁻ was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO₃⁻, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO₃⁻ to NO₂⁻ was inhibited by WO₄²⁻, the induction of NiR was inhibited only partially. The results indicate that in barley leaves NiR is induced by NO₃⁻ directly, i.e. without being reduced to NO₂⁻, and that absorbed NO₂⁻ induces the enzyme activity indirectly after being oxidized to NO₃⁻ within the leaf.     

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.     

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.     

Biosensor Determination of the Microscale Distribution of Nitrate, Nitrate Assimilation, Nitrification, and Denitrification in a Diatom-Inhabited Freshwater Sediment

High-resolution NO₃⁻ profiles in freshwater sediment covered with benthic diatoms were obtained with a new microscale NO₃⁻ biosensor characterized by absence of interference from chemical species other than NO₂⁻ and N₂O. Analysis of the microprofiles obtained indicated no nitrification during darkness, high rates of nitrification and a tight coupling between nitrification and denitrification during illumination, and substantial rates of NO₃⁻ assimilation during illumination. Nitrification during darkness could be induced by purging the bulk water with O₂ gas, indicating that the stimulatory effect on nitrification by illumination was caused by algal production of O₂. NH₄⁺ addition did not stimulate nitrification during darkness when O₂ was restricted to the upper 1-mm layer, and there was thus a low nitrification potential in the permanently oxic top 1 mm of the sediment.