Growth response of barley and tomato to nitrogen stress and its control by abscisic acid,water relations and photosynthesis

Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO ₃ ^- pools and in NO ₃ ^- flux to the xylem, particularly in tomato which had smaller tissue NO ₃ ^- reserves. Even in barley, tissue NO ₃ ^- reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon NO ₃ ^- flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf ABA content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.Abbreviation and symbols ABA abscisic acid - c_i leaf internal CO₂ concentration - L_p root hydraulic conductance     

Nitrogen retranslocation in plants of maize,lupin and cocklebur

Summary In a split root experiment translocation of N from shoot to root was studied using¹⁵NO ₃ ^– . The three plant species selected for this experiment differed significantly with respect to root NRA. For lupin, maize and cocklebur about 80, 50 and 6% of all absorbed NO ₃ ^– was assmilated in the roots, respectively.Although NO ₃ ^– was reduced in the roots of lupin and maize plants to a greater extent than required for the roots' demand for organic N, a significant phloem flow of N from shoot to roots was found in these plants. Unexpectedly, for cocklebur, the plant with the very low root NRA, the fraction of total N present in the root that has been imported from the shoot was only half that as found for lupin and maize.     

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance     

Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) and Corn (Zea mays L.) Seedlings: I. N Study

Nitrate reduction in roots and shoots of 7-day-old barley seedlings, and 9-day-old corn seedlings was investigated. The N-depleted seedlings were transferred for 24 h or 48 h of continuous light to a mixed nitrogen medium containing both nitrate and ammonium. Total nitrate reduction was determined by ¹⁵N incorporation from ¹⁵NO₃⁻, translocation of reduced ¹⁵N from the roots to the shoots was estimated with reduced ¹⁵N from ¹⁵NH₄⁺ assimilation as tracer, and the translocation from the shoots to the roots was measured on plants grown with a split root system. A model was proposed to calculate the nitrate reduction by roots from these data. For both species, the induction phase was characterized by a high contribution of the roots which accounted for 65% of the whole plant nitrate reduction in barley, and for 70% in corn. However, during the second period of the experiment, once this induction process was finished, roots only accounted for 20% of the whole plant nitrate reduction in barley seedlings, and for 27% in corn. This reversal in nitrate reduction localization was due to both increased shoot reduction and decreased root reduction. The pattern of N exchanges between the organs showed that the cycling of reduced N through the plant was important for both species. In particular, the downward transport of reduced N increased while nitrate assimilation in roots decreased. As a result, when induction was achieved, the N feeding of the roots appeared to be highly dependent on translocation from the leaves.     

Sulphate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L.) roots

During the first 4 d after the removal of SO ₄ ^2- from cultures of young barley plants, the net uptake of ¹⁵N-nitrate and the transport of labelled N to the shoot both decline. This occurred during a period in which there was no measurable change in plant growth rate and where the incorporation of [³H]leucine into membrane and soluble proteins was unaffected. Reduced N translocation was associated with six- to eightfold increases in the level of asparagine and two- to fourfold increases in glutamine in root tissue; during the first 4 d of SO ₄ ^2- deprivation there were no corresponding increases in amides in leaf tissue. The provision of 1 mol · m^–3 methionine halted, and to some extent reversed the decline in NO ₃ ^- uptake and N translocation which occurred during continued SO ₄ ^2- deprivation. This treatment had relatively little effect in lowering amide levels in roots. Experiments with excised root systems indicated that SO ₄ ^2- deprivation progressively lowered the hydraulic conductivity, Lp, of roots; after 4 d the Lp of SO ₄ ^2- -deprived excised roots was only 20% of that of +S controls. In the expanding leaves of intact plants, SO ₄ ^2- deprivation for 5 d was found to lower stomatal conductance, transpiration and photosynthesis, in the order given, to 33%, 37% and 18% of control values. The accumulation of amides in roots is probably explained by a failure to export either the products of root nitrate assimilation or phloem-delivered amino-N. This may be correlated with the lowered hydraulic conductivity. Enhanced glutamine and-or asparagine levels probably repressed net uptake of NO ₃ ^- and ¹³NO ₃ ^- influx reported earlier (Clarkson et al. 1989, J. Exp. Bot. 40, 953–963). Attention is drawn to the similar hydraulic signals occurring in the early stages of several different types of mineral-nutrient stresses.Abbreviations Asn asparagine - Gln glutamine - Lp hydraulic conductivity J.L.K. is extremely grateful to the British Council for supporting his working visit to Long Ashton. We thank John Radin for helpful discussion and encouragement.     

Daily changes in nitrate influx,efflux and metabolism in maize and pearl millet

Maize (Zea mays L.) and pearl millet (Pennisetum americanum (L.) Leeke) seedlings were exposed to [¹⁵N]nitrate for 1-h periods at eight times during a 24-h period (16–8 h light-dark for maize; 14–10 h for millet). Influx of [¹⁵N]nitrate as well as its reduction and translocation were determined during each period. The efflux of previously absorbed [¹⁴N]nitrate to the uptake solution was also estimated. No marked diurnal changes in [¹⁴N]nitrate efflux or [¹⁵N]nitrate influx were evident in maize. In contrast, [¹⁴N]nitrate efflux from millet increased and eventually exceeded [¹⁵N]nitrate influx during the late dark and early light periods, resulting in net nitrate efflux from the roots. The dissimilarity of their diurnal patterns indicates that influx and efflux are independently regulated. In both species, [¹⁵N]nitrate reduction and ¹⁵N translocation to shoots were curtailed more by darkness than was [¹⁵N]nitrate influx. In the light, maize reduced 15% and millet 24% of the incoming [¹⁵N]nitrate. In darkness, reduction dropped to 11 and 17%, respectively. Since the accumulation of reduced-¹⁵N in shoots declined abruptly in darkness, whereas that in roots was little affected, it is suggested that in darkness [¹⁵N]nitrate reduction occurred primarily in roots. The decrease in nitrate uptake and reduction in darkness was not related to efflux, which remained constant in maize and did not respond immediately to darkness in pearl millet.Paper No. 6722 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh     

Fate of nitrate during initial nitrate utilization by nitrogen-depleted dwarf bean

The fate of nitrate and nitrogen-15 was followed during the apparent induction phase (6h) for nitrate uptake by N-depleted dwarf bean (Phaseolus vulgaris L. ev. Witte Krombek). Experiments were done with intact plants and with detached root systems. Qualitatively and quantitatively, xylem exudation from detached roots was a bad estimate of the export of NO^?₃ or NO^?₃-¹⁵N from roots of intact plants. In vivo nitrate reductase activity (NRA) agreed well with in situ reduction, calculated as the difference between uptake and accumulation in whole plants, provided NRA was assayed with merely endogenous nitrate as substrate (‘actual’ NRA). The majority (75%) of the entering nitrate remained unmetabolized. Both nitrate reduction and nitrate accumulation occurred predominantly in the root system. Some (< 25%) of the root-reduced nitrate-N was translocated to the shoot. Nitrate uptake occurred against the concentration gradient between medium and root cells, and probably against the gradient of the electro-chemical potential of nitrate. Part of the energy expended for NO^?₃ absorption came from the tops, since decapitation and ringing at the stem base restricted nitrate uptake.     

Distribution of reducing power between photosynthetic carbon and nitrogen assimilation in Scenedesmus

Fixation of CO₂ and N assimilation were studied in synchronous cultures of Scenedesmus obtusiusculus Chod. under saturating and limiting light. Within the photon-flux range studied, the cells maintained C to N assimilation ratios of 7–10 with either NO ₃ ^- , NO ₂ ⁺ or NH ₄ ⁺ as the N source. Competitive interactions between C and N assimilation were pronounced under light limitation and were proportional to the oxidation status of the N source. Fixation of CO₂ at saturating light was also slightly reduced by NO ₂ ^- and NH ₄ ⁺ . In the absence of CO₂, NO ₃ ^- uptake and reduction was light-saturated at a comparatively low photon flux, whereas NO ₂ ^- uptake and reduction was considerably faster in the absence of CO₂ than in its presence. The pools of reduced pyridine nucleotides (NADPH and NADH) were largely unaffected by the presence or absence of the different N sources. The regulatory influences of CO₂ fixation on N assimilation are discussed in terms of coupling between the rates of CO₂ fixation and NH ₄ ⁺ assimilation, as well as the existance of control mechanisms for NO ₃ ^- uptake and reduction.Abbreviations Chl chlorophyll - PF photon flux     

The effect of endogenous and externally supplied nitrate on nitrate uptake and reduction in sugarbeet seedlings

The pericarp of the dormant sugarbeet fruit acts as a storage reservoir for nitrate, ammonium and -amino-N. These N-reserves enable an autonomous development of the seedling for 8–10 d after imbibition. The nitrate content of the seed (1% of the whole fruit) probably induces nitrate-reductase activity in the embryo enclosed in the pericarp. Nitrate that leaks out of the pericarp is reabsorbed by the emerging radicle. Seedlings germinated from seeds (pericarp was removed) without external N-supply are able to take up nitrate immediately upon exposure via a low-capacity uptake system (v_max = 0.8 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.12 mM). We assume that this uptake system is induced by the seed nitrate (10 nmol/seed) during germination. Induction of a high-capacity nitrate-uptake system (v_max = 3.4 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.08 mM) by externally supplied nitrate occurs after a 20-min lag and requires protein synthesis. Seedlings germinated from whole fruits absorb nitrate via a highcapacity uptake mechanism induced by the pericarp nitrate (748 nmol/pericarp) during germination. The uptake rates of the high-capacity system depend only on the actual nitrate concentration of the uptake medium and not on prior nitrate pretreatments. Nitrate deprivation results in a decline of the nitrate-uptake capacity (t_1/2 of v_max = 5 d) probably caused by the decay of carrier molecules. Small differences in K_s but significant differences in v_max indicate that the low- and high-capacity nitrate-uptake systems differ only in the number of identical carrier molecules.Abbreviations NR nitrate reductase - pFPA para-fluorophenylalanine This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Utilization of nitrite and nitrate by dwarf bean

The uptake and conversion of NO ₂ ^- and the effect of NO ₂ ^- on the uptake and reduction of NO ₃ ^- were examined in N-depleted Phaseolus vulgaris L. Nitrite uptake at 0.1 mmol dm^-3 was against an electrochemical gradient and became constant after one or two initial phases. Steadystate uptake declined with increasing ambient NO ₂ ^- concentration (0–0.7 mmol dm^-3). In this concentration range root oxygen consumption was unaffected by NO ₂ ^- , indicating that the decrease of NO ₂ ^- uptake was not related to respiration. After 6 h NO ₂ ^- supply, about one-third of the absorbed NO ₂ ^- had accumulated, mainly in the root system. Oxidation of NO ₂ ^- to NO ₃ ^- was not observed. The apparent induction period for NO ₃ ^- uptake was about 6 h in control plants and 3.5 h in plants that were pretreated for 18 h with NO ₂ ^- . In contrast, the time course of NO ₂ ^- uptake was unaffected by pretreatment with NO ₃ ^- . Steadystate NO ₃ ^- uptake was less affected by NO ₂ ^- than was steady-state NO ₂ ^- uptake by NO ₃ ^- . Nitrate reductase activity (NRA) in leaves and roots was induced by both NO ₃ ^- and NO ₂ ^- . In roots, induction with NO ₂ ^- was faster than with NO ₃ ^- , but there was no difference in NRA after 5 h. Nitrite inhibited NRA in the roots of NO ₃ ^- -induced plants and thus seems to stimulate the induction, but not the activity of induced nitrate reductase. In view of the observed differences in time course and mutual competition, a common uptake mechanism for NO ₂ ^- and NO ₃ ^- seems unlikely. Expression of the NO ₂ ^- effect on the induction of NO ₃ ^- uptake required more time than the induction itself. We therefore conclude that NO ₂ ^- is not the physiological inducer of NO ₃ ^- uptake.Abbreviations NR(A) nitrate reductase (activity) - BM basal medium     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

Alteration in enrichment of NO 3 − and reduced-N in xylem exudate during and after extended plant exposure to15NO 3 −

Summary Distinctly different patterns of¹⁵N enrichment were observed in the nitrate and reduced-N fractions of xylem exudate from soybean plants during and after 5 to 6 days of exposure to¹⁵NO ₃ ^– . Within 1 d after changes in solution NO ₃ ^– label, more than 90% of the exudate NO ₃ ^– originated from the exogenous supply. Alterations in the enrichment of exudate reduced-N were much slower, however, and the enrichment reached only 40% even after 5 d of continuous exposure to¹⁵NO ₃ ^– . Taking into account possible reduction of endogenous NO ₃ ^– and delayed translocation of NO ₃ ^– reduction products, it was concluded that root reduction could have contributed only 30 to 42% of the reduced-N found in the exudate.     

The influence of NO3 - and NH4 + nutrition on the carbon and nitrogen partitioning characteristics of wheat (Triticum aestivum L.) and maize (Zea mays L.) plants

The carbon and nitrogen partitioning characteristics of wheat (Triticum aestivum L.) and maize (Zea mays L.) grown hydroponically at a constant pH on either 4 mM or 12 mM NO₃ ^- or NH₄ ⁺ nutrition were investigated using either ¹⁴C or ¹⁵N techniques. Greater allocation of ¹⁴C to amino-N fractions occurred at the expense of allocation of ¹⁴C to carbohydrate fractions in NH₄ ⁺-compared to NO₃ ^--fed plants. The [¹⁴C]carbohydrate:[¹⁴C]amino-N ratios were 1.5-fold and 2.0-fold greater in shoots and roots respectively of 12 mM NO₃ ^--compared to 12 mM NH₄ ⁺-fed wheat. In both 4 mM and 12 mM N-fed maize the [¹⁴C]carbohydrate:[¹⁴C]amino-N ratios were approximately 1.7-fold and 2.0-fold greater in shoots and roots respectively of NO₃ ^--compared to NH₄ ⁺-fed plants. Similar results were observed in roots of wheat and maize grown in split-root culture with one root-half in NO₃ ^--and the other in NH₄ ⁺-containing nutrient media. Thus the allocation of carbon to the amino-N fractions occurred at the expense of carbohydrate fractions, particularly within the root. Allocation of ¹⁴N and ¹⁵N within separate sets of plants confirmed that NH₄ ^--fed plants accumulated more amino-N compounds than NO₃ ^--fed plants. Wheat roots supplied with ¹⁵NH₄ ⁺ for 8 h were found to accumulate ¹⁵NH₄ ⁺ (8.5 g ¹⁵N g^-1 h^-1) whereas in maize roots very little ¹⁵NH₄ ⁺ accumulated (1.5 g ¹⁵N g^-1 h^-1)It is proposed that the observed accumulation of ¹⁵NH₄ ⁺ in wheat roots in these experiments is the result of limited availability of carbon within the roots of the wheat plants for the detoxification of NH₄ ⁺, in contrast to the situation in maize. Higher photosynthetic capacity and lower shoot: root ratios of the C₄ maize plants ensure greater carbon availability to the root than in the C₃ wheat plants. These differences in carbon and nitrogen partitioning between NO₃ ^--and NH₄ ⁺-fed wheat and maize could be responsible for different responses of wheat and maize root growth to NO₃ ^- and NH₄ ⁺ nutrition.     

Nitrate Uptake by Roots as Regulated by Nitrate Reduction Products of the Shoot

A mechanism is proposed by which secondary products of nitrate reduction in the shoot control the uptake of nitrate by the roots. KNO₃ enters the roots and is translocated to the shoot where nitrate is reduced and, at the same time, malate is produced. The reduction of nitrate is stoichiometric to the synthesis of malate (1). Part of the K-malate moves down to the root system in which malate is oxidized, yielding KHCO₃ which exchanges for KNO₃. Nitrate reduction in the shoot promotes the synthesis of malate which, after its translocation to the root, allows the preferential uptake of nitrate. Thus, plants reducing large amounts of nitrate may take up the anion without a superfluous accumulation of the cation. Furthermore, the utilization of nitrate by the shoot regulates its uptake by the root.     

Nitrate Uptake and Assimilation by Wheat Seedlings during Initial Exposure to Nitrate

Nitrate uptake, reduction, and translocation were examined in intact, 14-day-old, nitrogen-depleted wheat (Triticum vulgare var. Knox) seedlings during a 9-hour exposure to 0.2 mm Ca (NO(3))(2). The nitrate uptake rate was low during the initial 3-hour period, increased during the 3- to 6-hour period, and then declined. By the 3rd hour, 14% of the absorbed nitrate had been reduced, and this increased to 36% by the 9th hour. Shoots accumulated reduced (15)N more rapidly than roots and the ratio of reduced (15)N to (15)N-nitrate was higher in the shoots. A significant proportion of the total reduction occurred in the root system under these experimental conditions. Accumulation of (15)N in ethanol-insoluble forms was evident in both roots and shoots by the 3rd hour and, after 4.5 hours, increased more rapidly in shoots than in roots.An experiment in which a 3-hour exposure to 0.2 mm Ca ((15)NO(3))(2) was followed by a 12-hour exposure to 0.2 mm Ca ((14)NO(3))(2) revealed a half-time of depletion of root nitrate of about 2.5 hours. A large proportion of this depletion, however, was due to loss of (15)N-nitrate to the ambient (14)N-nitrate solution. The remaining pool of (15)N-nitrate was only slowly available for reduction. Total (15)N translocation to the shoot was relatively efficient during the first 3 hours after transfer to Ca ((14)NO(3))(2) but it essentially ceased after that time in spite of significant pools of (15)N-nitrate and alpha-amino-(15)N remaining in the root tissue.     

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     

Morphogenesis of lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen

Combined light and transmission electron microscopy were used to examine the effect of nitrate on the development of root nodules in lucerne (alfalfa, Medicago sativa L.) following induction by the nitrogen-fixing symbiont, Rhizobium meliloti. The timing of NO ₃ ^- addition was varied in order to study its effect on all of the recognized morphogenetic steps of nodule formation. Roots of plants inoculated in the presence of 18 mM NO ₃ ^- had straight root hairs which were devoid of adherent rhizobia and infection threads, and developed no nodules. However, nodules were formed on roots if 18 mM NO ₃ ^- was added 5 d after inoculation. At this time, the initiation of nodule primordia had already commenced in the root cortex. The histology and ultrastructure of young nodules which had developed for 5 d in the absence of NO ₃ ^- and another 5 d in the presence of 18 mM NO ₃ ^- resembled nodules developing under N-free conditions, except that in the infection threads within the infection zone of the nodule 1) some bacteria tended to loose their normal shape and gain more electron density, indicating premature degradation, and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO ₃ ^- levels in the medium, lysis and degeneration of the bacteria released from the infection threads were observed in the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other hand, the nodule meristems continued to proliferate even after 12 d of exposure of 18 mM NO ₃ ^- . This was the only morphogenetic step of root nodulation which was insensitive to levels of combined nitrogen that completely prevented infection if present at the time of inoculation. These data indicate that all of the recognized steps of root nodule morphogenesis in which the bacteria play a key role are sensitive to the inhibitory effect of combined nitrogen.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Translocation of nitrogen in osmotically stressed wheat seedlings

Wheat (Triticum aestivum L., cv. Drabant) seedlings were grown in a ‘split root’ system where either the whole root system or one root half was subjected to osmotic stress for 24 h, using 200 g polyethylene glycol (PEG, molecular weight 4000) dm^?3 nutrient solution. ¹⁵N-Labelled nitrate was fed to one of the root compartments and total N and ¹⁵N-labelling were measured in plant material and xylem sap. Untreated plants translocated 87% of the N taken up to the shoot, and 10% of this was then retranslocated back to the root. Recalculated on a root nitrogen basis, 36% of the label recovered in the root after 24 h had passed through the shoot. Significant labelling of xylem sap collected from non-labelled roots indicated cycling of organic N through the roots. PEG-treatment of the whole root system caused significant water loss in both roots and shoots. Uptake of nitrate and retranslocation of N to roots were inhibited, whereas cycling of organic nitrogen through the root was still measurable. Treatment of half the root system with PEG had minor effects on shoot water content, but reduced the water content of the treated root part. The total uptake of nitrate by the root system was unaffected, and the effect on the treated root half was comparatively small. Nitrate reductase activity (NRA) declined in PEG-treated roots even if high nitrate uptake rates were maintained. Shoot NRA was unaffected by osmotic stress. The data indicate that the reduction in water content of the root per se has only small effects on nitrate uptake. Major inhibition of nitrate uptake was observed only after treatment of a sufficiently large portion of the root system to given an effect on shoot water content.