Nitrate Fluxes and Nitrate Reductase Activity of Suspension-Cultured Tobacco Cells (Effects of Internal and External Nitrate Concentrations).

Cell suspensions of tobacco (Nicotiana tabacum L., cv KY14) were used to determine the responses of NO3- uptake and NO3- reductase activity (NRA) to exogenous NO3- levels in the absence of long-distance NO3- transport. Tobacco cells grown with complete Murashige and Skoog medium for 7 d were subcultured for 3 d with NH4+-free media containing 0, 5, 10, 20, 30, and 40 mM NO3-. Cell NO3-, in vitro NRA, NO3- influx, and efflux of cell NO3- were determined. The NRA increased as cell NO3- increased. Cell NO3- efflux values increased as cell NO3- level increased. Cells with low intracellular NO3- had greater NO3- influx than cells with high intracellular NO3-. Woolf-Augustinsson-Hofstee transformations of the NO3- influx kinetic data revealed patterns characteristic of a high- and low-affinity two-component NO3- uptake system. Apparent Vmax values generally decreased and Km values increased as cell NO3- concentration increased. The NRA of cells supplied with 10 and 20 mM NO3- after 3-d growth in N- free medium increased about 5-fold within 2 h and then remained constant for the next 2 h, whereas NRA of cells supplied with 5 mM NO3- increased only 2-fold during the 4-h period. Intracellular NO3- and other N metabolites associated with cell NO3- levels exerted differential effects on the NO3- influx activity and NRA of tobacco cells cultured in suspension. Expression of high NRA was correlated with both high external and intracellular NO3-, whereas maximum NO3- influx activity required a low (depleted) level of cell NO3-.

Wheat vegetative nitrogen compositional changes in response to reduced reproductive sink strength.

N redistribution patterns and the N composition of vegetative tissues above the peduncle node of wheat (Triticum aestivum L.) plants with altered reproductive sink strength were evaluated to determine the role of vegetative storage proteins in the temporary storage of excess N destined for export. The degree of leaf senescence symptoms (loss of chlorophyll, total N, and ribulose-1,5-bisphosphate carboxylase/oxygenase) were initially reduced, but the complete senescence of vegetative tissues proceeded even for plants completely lacking reproductive sinks. Plants with 50% less sink strength than control plants with intact spikes redistributed vegetative N to the spike almost as effectively as the control plants. Plants without reproductive sinks exported less N from the flag leaf and had flag leaf blades and peduncle tissues with higher soluble protein and alpha-NH(2) amino acid levels than control plants. An abundant accumulation of polypeptides in the soluble protein profiles of vegetative tissues was not evident in plants with reduced sink strength. Storage of amino acids apparently accommodates any excess N accumulated by vegetative tissues during tissue reproductive growth. Any significant role of vegetative storage proteins in the N economy of wheat is unlikely.

Nitrate use by tobacco cells in response to N-stress and ammonium nutrition.

Characterization of NO 3 (-) use by suspension cultured tobacco cells during a culture cycle is needed to take advantage of cell cultures for further study of the biochemical regulation of NO 3 (-) uptake induction and decay processes. Tobacco (Nicotiana tabacum L., cv. Ky14) cells were cultured with media containing different N sources. Cells cultured with a mixture of NO 3 (-) and NH 4 (+) (40 mM NO 3 (-) plus 20 mM NH 4 (+) , in Murashige and Skoog media) initially grew slightly faster but attained the same maximum cell culture density as those cultured with 40 mM NO 3 (-) only. Cells subcultured with N-free media grew at a similar rate for the first 3 d as those cells grown with N, then ceased further growth. The cessation of growth of cells subcultured with N-free media coincided with depletion of cell NO 3 (-) . The NO 3 (-) influx of cells subcultured with N-free media increased eleven-fold and those grown with N increased four- to five-fold before declining. Maximal NO 3 (-) influx rates occurred at the onset of the stationary growth phase for N-stressed cells, while cells grown with N reached maximums prior to the stationary phase of cell growth. Cells grown with a mixture of NO 3 (-) and NH 4 (+) had lower NO 3 (-) reductase (NR) activity and higher cell NO 3 (-) levels than those of cells grown with NO 3 (-) only. The NR activity of cells subcultured with N-free media peaked within 1 d after subculture before declining to a constitutive level when cell NO 3 (-) was depleted. The level of cell NO 3 (-) plays a critical role in the expression of the NO 3 (-) uptake and reduction processes. The transitions in the expression of NO 3 (-) uptake and reduction activities of tobacco cell suspension cultures should prove valuable for further study of the biochemical and molecular basis for the regulation of these processes.

Phosphorus stress effects on assimilation of nitrate.

An experiment was conducted to investigate alterations in uptake and assimilation of NO(3) (-) 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 (15)NO(3) (-) during the 12 hour light period to determine changes in NO(3) (-) 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 (15)NO(3) (-) per gram root dry weight and translocation of absorbed (15)NO(3) (-) 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 (15)NO(3) (-) and (15)N 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-(15)N in the shoot during the 12 hour (15)NO(3) (-) exposure periods. The results indicate that alterations in NO(3) (-) 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.

Effect of dietary protein level on nitrogen metabolism in the growing bovine: I. Nitrogen recycling and intestinal protein supply in calves.

Eight Angus heifer calves (234 kg) were assigned to either a high (HP; 126 g N/d) or low (LP; 66.5 g N/d) protein intake. Calves received 4.8 kg DM/d consisting of 30% cottonseed hulls and 70% corn-soybean meal in equal portions at 4-h intervals. Single doses of 14C- and 15N-urea and 15N-ammonium sulfate were injected into the blood urea-N (BUN) and ruminal NH3-N (RAN) pools, respectively, to measure rate of flux through, and transfer of N between, these and bacterial N. Nitrogen balance was greater (P less than .05) for HP than for LP (56.9 vs 25.1 g N/d), but abomasal N flow as a percentage of N intake was greater (P less than .05) for LP than for HP (124 vs 71.1%). Pool size and net synthesis rate for both RAN and BUN pools were greater (P less than .05) for HP than for LP. Calves fed HP degraded more (P less than .05) BUN in the gastrointestinal tract than calves fed LP (37.4 vs 14.0 g N/d). Quantities of RAN absorbed from the rumen also were greater (P less than .05) for HP than for LP (14.2 vs 2.8 g N/d). The proportion of total gastrointestinal BUN degradation occurring in the rumen averaged 53 and 26% for LP and HP. Data are interpreted to suggest that net incorporation of BUN into bacterial protein (urea recycling) is inversely related to level of protein intake.

Effect of dietary protein level on nitrogen metabolism in the growing bovine: II. Diffusion into and utilization of endogenous urea nitrogen in the rumen.

Six Angus heifer calves (234 kg) were assigned to either a high (HP; 126.1 g N/d) or low (LP; 66.5 g N/d) protein intake to evaluate ruminal criteria associated with movement of blood urea-N (BUN)-derived NH3-N from the rumen wall into interior ruminal digesta. Calves received 4.8 kg DM/d of diets containing 30% cottonseed hulls and 70% cornsoybean meal in equal portions at 4-h intervals. Following single i.v. injections of 15N-urea, ruminal fluid was collected serially for 4 h postinjection from digesta located adjacent to the rumen wall (wall-proximate digesta; WPD) and from the center of the rumen digesta mass after manual agitation (center mixed digesta; CMD). Mean ruminal NH3-N (RAN) concentrations were higher (P less than .05) for HP than for LP, but were not affected (P greater than .05) by digesta sampling site. Ruminal urease activity was higher (P less than .05) for LP than for HP and tended (P = .14) to be higher for WPD than for CMD. Area under the 15N enrichment curve (AUC) ratios between sampling sites (WPD/CMD x 100) for RAN were greater (P less than .05) for LP than for HP. However, AUC ratios for bacterial N were not affected (P greater than .05) by protein level. Whereas BUN-derived 15NH3 appeared to thoroughly equilibrate with RAN in interior ruminal digesta with HP, there appeared to be a declining enrichment gradient for RAN from the rumen wall to the interior ruminal digesta with LP. Data are interpreted to suggest that bacteria at or near the rumen wall may preferentially utilize some BUN-derived NH3-N entering through the rumen wall in calves fed LP diets.

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

An experiment was conducted to investigate the relative changes in NO(3) (-) 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 (15)NO(3) (-) for 6 hour intervals during a normal 12 hour light period and a subsequent period of darkness lasting 42 hours. Uptake of (15)NO(3) (-) 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 (15)NO(3) (-) assimilation processes, which were distinctly different than those in (15)NO(3) (-) uptake. During the extended dark period, translocation of absorbed (15)N out of the root to the shoot varied rhythmically. The adjustments were independent of (15)NO(3) (-) 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 (15)NO(3) (-) always was limited more than uptake. The assimilation of (15)N 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 (15)NO(3) (-) reduction and synthesis of insoluble reduced-(15)N even when (15)NO(3) (-) uptake was severely restricted and minimal carbohydrate reserves remained in the tissue.

Main stem sink manipulation in wheat : effects on nitrogen allocation to tillers.

The role of main stem (MS) sink size on N use by field-grown soft red winter wheat (Triticum aestivum L. cv Hart) was determined. At Feeke's growth stage 8 (last leaf just visible), 100 micromoles of 99 atom% (15)N-ammonium was injected into the lower MS. At anthesis, MS sink size was adjusted by removal of 0, 33, 66, and 100% of the MS spikelets; tiller spikes were left intact. The MS and tiller average kernel size was unaffected by MS sink manipulations. The MS kernel N concentration increased when MS spikelets were removed. Tiller kernel N concentrations were unaffected except when the entire MS reproductive sink was removed, which caused an increase in tiller kernel N concentration. Net losses of MS vegetative N during grain fill were similar for all treatments except for plants lacking MS spikelets, which mobilized 30% less N from the MS. Labeled N was predominately (>90%) associated with the insoluble reduced N fraction of plant tissues at anthesis. Allocation of labeled N to tillers was not proportional to reduction in MS sink size. These results indicate that the reproductive sink on an individual culm has first priority for vegetative N mobilized during grain fill even when sink demand is reduced substantially.

Development of accelerated net nitrate uptake : effects of nitrate concentration and exposure time.

Upon initial nitrate exposure, net nitrate uptake rates in roots of a wide variety of plants accelerate within 6 to 8 hours to substantially greater rates. Effects of solution nitrate concentrations and short pulses of nitrate (

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 NO(3) (-), 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 (15)NO(3) (-) for 12 hours in darkness and then returned to a solution containing 1.0 millimolar (14)NO(3) (-) for the 12 hours ;chase' period in the light. Another set of plants was exposed to (15)NO(3) (-) during the light period to allow a direct comparison of contributions of substrate from the endogenous and exogenous sources. At the end of the (15)NO(3) (-) exposure in the dark, 70% of the absorbed (15)NO(3) (-) remained unreduced, and 83% of this unreduced NO(3) (-) was retained in roots. The pool of endogenous (15)NO(3) (-) 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 NO(3) (-), which had accumulated in the root prior to the previous dark period, also was depleted in the light. When exogenous (15)NO(3) (-) was supplied during the light period, the rate of assimilation progressively increased, reflecting an increased rate of uptake and decreased accumulation of NO(3) (-) in the root tissue. The dark-absorbed endogenous NO(3) (-) in the root was the primary source of substrate for whole-plant NO(3) (-) reduction in the first 6 hours of the light period, and exogenous NO(3) (-) was the primary source of substrate thereafter. It is concluded that retention of NO(3) (-) 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 NO(3) (-) assimilation in photosynthetic tissues.

Effect of dietary protein level on nitrogen metabolism in lambs: studies using 15N-nitrogen.

Six wether lambs (31 kg) were randomly assigned to two treatments (three lambs/treatment): a high protein intake (HP; 21 g N/d) or a low protein intake (LP; 12 g N/d). Each lamb received 860 g/d dry matter (DM) of a pelleted diet (75% corn-soybean meal, 25% cottonseed hulls) offered hourly in 24 equal portions. Single injections of 15N-labelled compounds were made into the ruminal NH3-N and blood urea-N pools to measure the rate of flux through, and transfer of N between, these and the bacterial N pool. Total tract digestibilities of DM and N were lower (P less than .05) for the LP than the HP treatment. Abomasal flows of total, feed or bacterial N tended to be greater (P greater than .05) in lambs fed HP than LP. Lambs fed HP excreted more (P less than .01) urinary N, yet retained a greater (P less than .01) amount of N than lambs fed LP (6.2 vs 1.8 and 9.7 vs 4.1 g N/d, respectively). Pool size and production rate for both ruminal NH3-N and blood urea-N were greater (P less than .05) for the HP than LP treatment. Lambs consuming HP degraded more (P less than .05) blood urea-N in the gastro-intestinal tract (13.4 vs 6.9 g N/d); however, lambs fed LP degraded a greater (P less than .05) percentage of synthesized body urea-N (88.7 vs 71.8%). Ruminal NH3-N absorption was greater (P less than .01) for the HP than LP treatment (3.1 vs .5 g N/d). Although the percentage of bacterial N derived from ruminal NH3-N was similar (P greater than .05) between diets (51.1 vs 63.9), a greater (P less than .05) percentage of bacterial N was derived from blood urea-N in lambs fed LP than HP (77.1 vs 30.2%). Lambs fed LP incorporated a greater (P less than .10) amount of blood urea-N into bacterial N than lambs fed HP (5.5 vs 2.6 g N/d). These data are interpreted to suggest that blood urea-N may provide a substantial quantity of N for bacterial protein synthesis and, thus, may be an important source of protein in the deficient animal. In addition, urea recycling may play an important role in the recovery of ruminal NH3-N lost through absorption in animals fed a high level of protein.

Postanthesis nitrate assimilation in winter wheat : in situ flag leaf reduction.

When adequate levels of soil NO(3) (-) are available, concurrent NO(3) (-) absorption and assimilation, and mobilization of vegetative N reserves accumulated prior to anthesis, may be used to supply N to developing wheat (Triticum aestivum L.) kernels. Vegetative wheat components (stems, leaves, spike) are known to possess NO(3) (-) reductase activity, but the in situ utilization of NO(3) (-) translocated to the shoot has not been studied. Assimilation and partitioning of (15)N was determined in winter wheat ;Doublecrop.' At 7 days after anthesis, the stem immediately above the peduncle node was heat girdled to block phloem export from the flag leaf. Control plants were not girdled. One day later, 50 micromoles of (15)NO(3) (-) (98 atom percent (15)N) was injected into the penultimate internodal lacuna, after which (15)NO(3) (-) utilization was determined sequentially over a 5 day period. Based on differences in spike accumulation of reduced (15)N excess between treatments and the amount of reduced (15)N excess remaining in the flag leaf, it was estimated that the flag leaf contributed 37% of the total reduced (15)N excess in the injected shoot. The lower shoot contribution was 18% and that of the peduncle plus spike was 45%.

Variation in nitrogen use efficiency among soft red winter wheat genotypes.

Nitrogen use efficiency (NUE), defined as grain dry weight or grain nitrogen as a function of N supply, was evaluated in 25 soft red winter wheat genotypes for two years at one location. Significant genotypic variation was observed for NUE, nitrogen harvest index, and grain yield. Genotype x environment interaction for these traits was not significant. Several variables including N uptake efficiency (total plant N as a function of N supply), grain harvest index, and N concentration at maturity were evaluated for their role in determining differences in NUE. Nitrogen uptake efficiency accounted for 54% of the genotypic variation in NUE for yield and 72% of the genotypic variation in NUE for protein. A path coefficient analysis revealed that the direct effect of uptake efficiency on NUE was high relative to indirect effects.

Partitioning of previously-accumulated nitrate to translocation, reduction, and efflux in corn roots.

The effect of nitrate uptake, or its absence, on the utilization of nitrate previously accumulated by dark-grown, decpitated maize (Zea mays L., cv. DeKalb XL-45) seedlings was examined. Five-d-old plants that had been pretreated with 50 mM (14)NO 3 (-) for 20 h were exposed for 8 h to nutrient solutions containing either no nitrate or 50 mM (15)NO 3 (-) , 98.7 atom % (15)N. The ambient solution, xylem exudate, and plant tissue were analyzed to determine the quantities of previously-accumulated (endogenous) (14)NO 3 (-) that were translocated to the xylem, lost to the solution, or reduced within the tissue during the 8-h period. Energy was continuously available to the roots from the attached endosperm. In the absence of incoming nitrate, appreciable reduction and translocation of the endogenous (14)NO 3 (-) occurred, but efflux of (14)NO 3 (-) to the external solution was minimal. In contrast, during (15)NO 3 (-) uptake, there was considerable efflux of (14)NO 3 (-) as well as translocation of (14)NO 3 (-) to the xylem, but little (14)NO 3 (-) was reduced. Thus there appeared to be an inverse relationship between (14)NO 3 (-) efflux and reduction. The data are tentatively interpreted on the basis of a model which envisages (a) two storage locations within roots, one of which primarily supplies nitrate for translocation and the other of which primarily supplies nitrate for outward passage through plasmalemma, and (b) the majority of nitrate reduction as occurring during or immediately following influx across the plasmalemma, with endogenous (14)NO 3 (-) initially moving outward being recycled inward and thereby being reduced.

Restricted nitrate influx and reduction in corn seedlings exposed to ammonium.

The effect of ambient ammonium (0.5 millimolar [(14)NH(4)](2)SO(4)) added to a nutrient solution containing 1.0 millimolar K(15)NO(3), 99 atom per cent (15)N, upon [(15)N]nitrate assimilation and utilization of previously accumulated [(14)N]nitrate was investigated. Corn seedlings, 5-day-old dark-grown decapitated (experiment I) and 10-day-old light-grown intact (experiment II), which had previously been grown on K(14)NO(3) nutrient solution, were used. In both experiments, the presence of ambient ammonium decreased [(15)N]nitrate influx (20% after 6 hours) without significantly affecting the efflux of previously accumulated [(14)N]nitrate. In experiment I, relative reduction of [(15)N]nitrate (reduction as a percentage of influx) was inhibited more than was [(15)N]nitrate influx. Nevertheless, in experiment I, where all reduction could be assigned to the root system, the absolute inhibition of reduction during the 12 hours (13 micromoles/root) was less than the absolute inhibition in influx (24 micromoles/root). The data suggest that the influence of ammonium on [(15)N]nitrate influx could not be totally accounted for by the decrease in the potential driving force which resulted from restricted reduction; an additional impact on the influx process is indicated. Reduction of [(15)N]nitrate in experiment II after 6 hours accounted for 30 and 18% of the tissue excess (15)N in the control and ammonium treatments, respectively. Relative distribution of (15)N between roots and exudate (experiment I), or between roots and shoots (experiment II) was not affected by ammonium. On the other hand, the accumulation of [(15)N]nitrate in roots, shoots, and xylem exudate was enhanced by ammonium treatment compared to the control, whereas the accumulation of reduced (15)N was inhibited.

Minimizing Nitrate Reduction during Kjeldahl Digestion of Plant Tissue Extracts and Stem Exudates : APPLICATION TO N STUDIES.

From 10 to 60% of the nitrate present in plant tissue extracts and stem exudates of corn (Zea mays L.) was found to be reduced during Kjeldahl digestion, even in the absence of added reducing agents. This reduction is of particular concern in [(15)N]nitrate assimilation studies, because it results in an overestimate of nitrate reduction. To overcome this problem, a method was developed for removing nitrate prior to Kjeldahl digestion, thereby preventing nitrate reduction. The procedure utilizes hydrogen peroxide for partial oxidation of organic matter in order to minimize the nitration of organic compounds. The free nitrates are then volatilized as nitric acid from concentrated sulfuric acid at 95 degrees C. When the proposed method was used as a pretreatment to Kjeldahl digestion, less than 0.5% of the applied nitrate was recovered in the reduced nitrogen fraction of plant tissue extracts and stem exudates.

Nitrate Accumulation, Assimilation, and Transport by Decapitated Corn Roots : EFFECTS OF PRIOR NITRATE NUTRITION.

The effects of accumulated [(14)N]nitrate and its utilization in decapitated, 5-day-old dark-grown corn roots on influx, accumulation, xylem deposition, and reduction of concurrently absorbed nitrate during an 18-hour exposure to 0.5 millimolar K(15)NO(3) nutrient solution were examined. A 20-hour pretreatment in 15.0 millimolar K(14)NO(3) high nitrate (HN) resulted in a 2-fold greater tissue nitrate level than pretreatment in 0.5 millimolar K(14)NO(3) low nitrate (LN). Upon transfer to the 0.5 millimolar K(15)NO(3) solution, the net nitrate uptake rate in HN roots after 2 hours was 52% of the LN rate, but increased to 93% at the end of the uptake period. Despite an enhanced [(14)N]nitrate efflux from HN roots to the uptake solution, the efflux differences between the two pretreatments did not compensate for the decrease in net nitrate uptake. The [(15)N]nitrate influx rate was initially restricted by 33% in the HN roots compared to LN roots, but it had decreased to 7% by the end of the 18-hour uptake period. At this time, the total tissue nitrate levels were similar for both pretreatments. The rate of accumulation of [(15)N]nitrate in the tissue was relatively constant for both pretreatments, but was 25% less in HN roots. Of the previously accumulated [(14)N]nitrate, 52 and 46% remained after 18 hours in the LN and HN roots, respectively. The [(14)N]nitrate decline for HN roots was initially more rapid than in the LN roots which was linear over time. Xylem transport and efflux more than accounted for the decline in [(14)N]nitrate of LN roots and all but 4% in the HN roots which was attributed to reduction. Compartmentation of the previously accumulated nitrate was evident from the higher atom per cent (15)N of xylem nitrate compared to that of the tissue nitrate of both LN and HN roots. During the first 2 hours, xylem transport of [(14)N]nitrate by the HN roots was 49% greater than for LN roots, while [(15)N]nitrate transport was 9% less in HN roots compared to LN roots. Even though the reduction of [(15)N]nitrate in HN roots was 31% less than LN roots during the first 2 hours, [(15)N]nitrate was reduced more rapidly than the previously accumulated [(14)N]nitrate. After the first 4 hours, the relative partitioning of absorbed [(15)N]nitrate between accumulation, reduction, and translocation was similar regardless of pretreatment.