Nitrate reductase activity and nitrogen compounds in xylem exudate of Juglans nigra seedlings: relation to nitrogen source and supply

Nitrogen (N) limits plant productivity and its uptake and assimilation may be regulated by N source, N availability, and nitrate reductase activity (NRA). Knowledge of how these factors interact to affect N uptake and assimilation processes in woody angiosperms is limited. We fertilized 1-year-old, half-sib black walnut (Juglans nigra L.) seedlings with ammonium (NH₄ ⁺) [as (NH₄)₂SO₄], nitrate (NO₃ ⁻) (as NaNO₃), or a mixed N source (NH₄NO₃) at 0, 800, or 1,600 mg N plant⁻¹ season⁻¹. Two months following final fertilization, growth, in vivo NRA, plant N status, and xylem exudate N composition were assessed. Specific leaf NRA was higher in NO₃ ⁻-fed and NH₄NO₃-fed plants compared to observed responses in NH₄ ⁺-fed seedlings. Regardless of N source, N addition increased the proportion of amino acids (AA) in xylem exudate, inferring greater NRA in roots, which suggests higher energy cost to plants. Root total NRA was 37% higher in NO₃ ⁻-fed than in NH₄ ⁺-fed plants. Exogenous NO₃ ⁻ was assimilated in roots or stored, so no difference was observed in NO₃ ⁻ levels transported in xylem. Black walnut seedling growth and physiology were generally favored by the mixed N source over NO₃ ⁻ or NH₄ ⁺ alone, suggesting NH₄NO₃ is required to maximize productivity in black walnut. Our findings indicate that black walnut seedling responses to N source and level contrast markedly with results noted for woody gymnosperms or herbaceous angiosperms.     

Use of labeled nitrogen to monitor transition in nitrogen dependence from storage to current-year uptake in mature walnut trees

Summary Nitrogen (N) derived from both the soil during current-year uptake, and the withintree pool of storage N was distinguished in two groups of Serr walnut trees using labeled fertilizer (¹⁵N-depleted ammonium sulfate) applied in different years. Mass spectrometric analysis of N in xylem sap collected periodically in spring allowed quantification of the relative contributions of N from storage and current season uptake and the transition in N usage from previously assimilated (storage) N to the onset of current season uptake of soil N. N derived from storage accounted for > 50% of the xylem sap N during the staminate and pistillate bloom periods and throughout the period of spur leaf expansion.     

Transfer of amino acids and nitrate from the roots into the xylem of Ricinus communis seedlings

The loading of amino acids and nitrate into the xylem was investigated by collection and analysis of root-pressure exudate from the cut hypocotyl stumps of seedlings of Ricinus communis L. Glutamine was found to be the dominant amino acid in the exudate and also to be the amino acid which is transferred to the xylem most rapidly and accumulated to the greatest extent. The comparison between uptake and xylem loading showed significant differences in specificity between these two transport reactions, indicating a different set of transport systems. Nitrate is transferred to the xylem at a higher relative rate than any amino acid despite the great nitrate-storage capacity of the root system. Thus the supply of nitrate to Ricinus plants leads to enhanced nitrogen allocation to the shoots.     

Extraction of tracheal sap from Citrus and analysis of its nitrogenous compounds

Tracheal sap was extracted from sections of stems (0.5 to 1.5 cm in diameter and 7.5 to 15.0 cm in length) of orange trees (Citrus sinensis (L.) Osbeck cv. Washington Navel) by using a combination of the vacuum and liquid displacement methods. The volume of sap obtained and its concentration of nitrogenous compounds were dependent on the volume of displacing liquid used for the extraction. Four ml of water-saturated 1-butanol extracted essentially all of the xylem fluid present in the stem sections without apparent production of artifacts. The time of sampling affected the nitrogen concentration of the tracheal sap, but not the content of xylem nitrogen per volume of stem material. The orientation of the stems in the tree and the diameter of the stems had an effect on their contents of xylem nitrogen, with southeastern orientation and thinner stems showing higher concentrations. We could not detect the presence of ammonium, nitrites or proteins in the tracheal sap of orange trees. Most of the nitrogen was present as amino acids and about 2% of the total in the form of nitrates. The qualitative composition of amino acids, as determined by TLC, was the same both in winter and spring tracheal sap. The main components of the sap were proline and arginine in winter, and these amino acids together with asparagine and aspartic acid in spring.     

Influx of double labelled glutamine into mistletoes (Viscum album) from the xylem sap of its host (Abies alba)

The flux of glutamine into the mistletoe Viscum album from the xylem sap of a coniferous host was analyzed. For this purpose, a perfusion system was used in which the xylem sap of the host was replaced by an artificial perfusion solution. With this system, flux rates into the mistletoe were determined in feeding experiments with the organic nitrogen source U(13)C/(15)N-Gln. At the end of the experiments the delta values of C and N were significantly depleted in the outflow compared to the percolation solution. Since this depletion was higher for C than for N, a combination of Gln uptake and simultaneous uploading of organic compounds in the host xylem can be assumed. Gln was strongly metabolized during its allocation in the mistletoe. As a consequence, the C skeleton of Gln was equally distributed between leaf and stem tissue, whereas N of Gln preferentially accumulated in the stem. Apparently, the C atoms of the Gln taken up are transported faster in the mistletoe to the sink tissues than the N atoms. It is concluded that C liberated from Gln is transported rapidly to different sink tissues, whereas N in the oversupplied mistletoes is transported slowly to sinks in the leaves.     

Nitrate reductase in needles,roots and trunk wood of spruce trees [Picea abies (L.) Karst.]

Summary Nitrate reductase (EC 1.6.6.2) activity (NRA), as measured by an in vivo assay, is present in needle leaves and mycorrhizal fine root tips of adult Norway spruce [Picea abies (L.) Karst.] in at least equal amounts on a fresh weight basis, in both adult and 5-year-old trees. NRA could also be demonstrated in trunk wood of deroted trees after fertilization with 5 mM , exhibiting a longitudinal profile in the trunk. Inducibility in needles can more efficiently be achieved by NO₂ (100 g·m^-3) than by 5 mM nitrate, which is effective only in root-amputated trees. A remarkably high level of needle-NRA in unfertilized trees, which are characterized by a very low level of nitrate in the xylem sap, suggests that NRA in spruce needles may in part be constitutive. Organic-N is a major nitrogen source for the needles even in root-amputated trees, indicating pronounced exchange processes between ray parenchyma and trunk xylem, which in turn are modified by the nitrogen source fed to the trunk stump. Intact trees exhibit a very similar amino acid composition of the xylem sap, regardless of whether or has been fed. The amino acid pattern of the needles is not thrown out of balance by flooding with and , which occurs in fertilized derooted trees. This indicates a distinct potential for homoeostasis of nitrogen entrance-metabolism (i.e. NRA and glutamine synthetase activity) in the needles. In the ectomycorrhiza/fine root-system (EMC), marked differences in NRA were observed depending on root-tip diameter and along the longitudinal profile of the fine roots. EMC-nitrate reductase is strongly enhanced by . Needle-NRA exhibits a circannual rhythm. An early summer maximum is followed by a December minimum. This activity pattern matches well the transitory increase of soluble nitrogen in spring and the total protein maximum in winter. In an indirect way assimilatory NRA may well contribute to nitrogen overfertilization (by consumption of NO_X) as one possible cause of the contemporary decline of spruce populations.     

Seasonal variation of nitrogenous compounds in the xylem sap of Citrus

Samples of tracheal sap of Citrus sinensis (L.) Osbeck cv. Washington Navel were taken from field trees throughout the year and the nitrogen composition of the sap was determined. The nitrogenous fraction of the sap was composed mainly of free amino acids (92–97% of total nitrogen) and nitrates throughout the year. Proline was the most abundant amino acid during almost the entire cycle, and its concentration was especially high during the autumn and winter period. Nevertheless, a significant part (40–60%) of the total organic nitrogen was transported as arginine. Total nitrogen as well as amino acids and nitrates were maximal at spring flush. At spring flush and summer flush there was also a diversification of α-amino nitrogen among different amino acids. During the spring flush, nitrates, asparagine and γ-aminobutyric acid in the xylem sap seemed to have a radicular origin, whereas glutamic acid and arginine were released from the surrounding parenchyma. The results suggest a metabolic transformation in the wood parenchyma of nitrogenous compounds coming from the roots (including reduction of nitrates) and a turnover of different nitrogen metabolites between the xylem and surrounding cells.     

Stem respiration and carbon dioxide efflux of young <Emphasis Type="Italic">Populus deltoides</Emphasis> trees in relation to temperature and xylem carbon dioxide concentration

Oxidative respiration is strongly temperature driven. However, in woody stems, efflux of CO₂ to the atmosphere (E _A), commonly used to estimate the rate of respiration (R _S), and stem temperature (T _st) have often been poorly correlated, which we hypothesized was due to transport of respired CO₂ in xylem sap, especially under high rates of sap flow (f _s). To test this, we measured E _A, T _st, f _s and xylem sap CO₂ concentrations ([CO₂*]) in 3-year-old Populus deltoides trees under different weather conditions (sunny and rainy days) in autumn. We also calculated R _S by mass balance as the sum of both outward and internal CO₂ fluxes and hypothesized that R _S would correlate better with T _st than E _A. We found that E _A sometimes correlated well with T _st, but not on sunny mornings and afternoons or on rainy days. When the temperature effect on E _A was accounted for, a clear positive relationship between E _A and xylem [CO₂*] was found. [CO₂*] varied diurnally and increased substantially at night and during periods of rain. Changes in [CO₂*] were related to changes in f _s but not T _st. We conclude that changes in both respiration and internal CO₂ transport altered E _A. The dominant component flux of R _S was E _A. However, on a 24-h basis, the internal transport flux represented 9–18% and 3–7% of R _S on sunny and rainy days, respectively, indicating that the contribution of stem respiration to forest C balance may be larger than previously estimated based on E _A measurements. Unexpectedly, the relationship between R _S and T _st was sometimes weak in two of the three trees. We conclude that in addition to temperature, other factors such as water deficits or substrate availability exert control on the rate of stem respiration so that simple temperature functions are not sufficient to predict stem respiration.     

Nitrogen relations of natural and disturbed plant communities in tropical Australia

Nitrogen relations of natural and disturbed tropical plant communities in northern Australia (Kakadu National Park) were studied. Plant and soil N characteristics suggested that differences in N source utilisation occur at community and species level. Leaf and xylem sap N concentrations of plants in different communities were correlated with the availability of inorganic soil N (NH⁺ ₄ and NO⁻ ₃). In general, rates of leaf NO⁻ ₃ assimilation were low. Even in communities with a higher N status, including deciduous monsoon forest, disturbed wetland, and a revegetated mine waste rock dump, levels of leaf nitrate reductase, xylem and leaf NO⁻ ₃ levels were considerably lower than those that have been reported for eutrophic communities. Although NO⁻ ₃ assimilation in escarpment and eucalypt woodlands, and wetland, was generally low, within these communities there was a suite of species that exhibited a greater capacity for NO⁻ ₃ assimilation. These “high- NO⁻ ₃ species” were mainly annuals, resprouting herbs or deciduous trees that had leaves with high N contents. Ficus, a high-NO⁻ ₃ species, was associated with soil exhibiting higher rates of net mineralisation and net nitrification. “Low-NO⁻ ₃ species” were evergreen perennials with low leaf N concentrations. A third group of plants, which assimilated NO⁻ ₃ (albeit at lower rates than the high-NO⁻ ₃ species), and had high-N leaves, were leguminous species. Acacia species, common in woodlands, had the highest leaf N contents of all woody species. Acacia species appeared to have the greatest potential to utilise the entire spectrum of available N sources. This versatility in N source utilisation may be important in relation to their high tissue N status and comparatively short life cycle. Differences in N utilisation are discussed in the context of species life strategies and mycorrhizal associations. Received: 5 July 1997 / Accepted: 13 July 1998     

Hydraulic propagation of pressure along immature and mature xylem vessels of roots of Zea mays measured by pressure-probe techniques

Turgor pressure was measured in cortical cells and in xylem elements of excised roots and roots of intact plants of Zea mays L. by means of a cell pressure probe. Turgor of living and hence not fully differentiated late metaxylem (range 0.6–0.8 MPa) was consistently higher than turgor of cortical cells (range 0.4–0.6 MPa) at positions between 40 and 180 mm behind the root tip. Closer to the tip, no turgor difference between the cortex and the stele was measured. The turgor difference indicated that late-metaxylem elements may function as nutrient-storage compartments within the stele. Excised roots were attached to the root pressure probe to precisely manipulate the xylem water potential. Root excision did not affect turgor of cortical cells for at least 8 h. Using the cell pressure probe, the propagation of a hydrostatic pressure change effected by the root pressure probe was recorded in mature and immature xylem elements at various positions along the root. Within seconds, the pressure change propagated along both early and late metaxylems. The half-times of the kinetics, however, were about five times smaller for the early metaxylem, indicating they are likely the major pathway of longitudinal water flow. The hydraulic signal dissipated from the source of the pressure application (cut end of the root) to the tip of the root, presumably because of radial water movement along the root axis. The results demonstrate that the water status of the growth zone and other positions apical to 20 mm is mainly uncoupled from changes of the xylem water potential in the rest of the plant.Abbreviations and Symbols CPP cell pressure probe - EMX early metaxylem - LMX Late metaxylem - P_c cell turgor - P_r root pressure - RPP root pressure probe - t_1/2,c half-time of water exchange across a single cell - t_1/2 half-time of water exchange across multiple cells We thank Antony Matista for his expert assistance in the construction and modification of instruments. The work was supported by grant DCB8802033 from the National Science Foundation and grant 91-37100-6671 from USDA, and by the award of a Feodor Lynen-Fellowship from the Alexander von Humboldt-Foundation (Germany) to J.F.     

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.     

Nitrogen assimilation in citrus trees

Assimilation of ¹⁵N-ammonium and ¹⁵N-nitrate was examined in 3-year-old satsuma mandarin (Citrus unshiu Marcovitch) trees. Experiments were designed to establish the time course of incorporation of nitrogen just taken up into amino compounds. In fine roots, absorbed ¹⁵N-ammonium was actively incorporated into glutamine and then into glutamic acid and asparagine. When feeding ¹⁵N-nitrate, glutamic acid and asparagine were actively synthesized, but glutamine synthesis was comparatively low as compared with that in ammonium feeding. In current leaves and fruits, a clear difference in the labelling patterns of amino acids was found between the ammonium and nitrate feedings. The amino acid most markedly labelled was asparagine in the ammonium feeding and glutamine in the nitrate feeding. Considering the most heavily labelled component in leaves and fruits, the main form of the nitrogen components transported upward in the xylem was discussed.     

Correlation between the seasonal changes in electrogenic activity across root xylem/symplast interface,sap-flow rate and xylem pressure in field trees (<Emphasis Type="Italic">Diospyros kaki</Emphasis>)

To investigate the seasonal changes in the physiological activity of the roots in field trees, Diospyros kaki, we carried out simultaneous and continuous measurements of the trans-root electric potential (TRP), of the rate of sap-flow with transpiration and of the xylem pressure (Px) through several years. The amplitude of the diurnal change and its temperature dependency of both TRP and Px began to increase in late January. These parameters attained their maxima in the late defoliate phase just before unfolding of new leaves at the end of March, thereafter rapidly went down to their minimal levels with increasing transpiration. The low levels continued during the foliate stage until December, and the pattern of the seasonal change in Px was inverse of that in the sap-flow rate. The role of the electrogenic ion pump activity across the interface between root-symplast and xylem vessel as a driving force for acropetal water transport is discussed in relation to those seasonal changes.     

Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets

PII signal transduction plays a pervasive role in microbial nitrogen control. Different phylogenetic lineages have developed various signal transduction schemes around the highly conserved core of the signalling system, which consists of the PII proteins. Among all various bacterial PII signalling systems, the one in cyanobacteria is so far unique: in unicellular strains, the mode of covalent modification is by serine phosphorylation and the interpretation of the cellular nitrogen status occurs by measuring the 2-oxoglutarate levels. Recent advances have been the identification of the phospho-PII phosphatase, the resolution of the crystal structure of PII proteins from Synechococcus and Synechocystis strains and the identification of novel functions of PII regulation in cyanobacteria, which highlight the central role of PII signalling for the acclimation to changing carbon-nitrogen regimes.     

The fate of15N labeled nitrogen applied to mature citrus trees

Summary The efficiency and balance of nitrogen from one year's application was studied in a long-term fertigation experiment. Enriched nitrogen fertilizer, K¹⁵NO₃, was applied to a 22-year-old Shamouti orange tree with a history of high N applications (N₃) and to an N-starved tree (N₁). The distribution of N in the different parts of the trees and in the soil was determined after the experimental trees were excavated. Similar total recovery of the labeled fertilizer N was found in the trees and soil in both treatments (N₁−61.7% N₃−56%). However, the distribution between tree and soil was different. The amount of recovered residual fertilizer in the soil was much larger in the N₃ treatment than in N₁. The highest percentage of fertilizer N was found in the new organs,i.e. fruits, twigs and leaves. The roots and branches took up only 6–14% from the labeled fertilizer. Only 20.9% of the leaf N and 23.4% of the fruit N in the N₃ tree originated in the labeled fertilizer, indicating translocation of N from older parts of the tree to new growth. Evidence was found of storage of N in the wooded branches, while the roots contained a surprisingly small part of labeled fertilizer. Contribution 1599E.     

Nutrient reserves in roots of fruit trees,in particular carbohydrates and nitrogen

Summary In trees, nutrient reserves built up in the previous year are of primary importance for early spring growth. Despite the relatively great importance of roots for nutrient storage, the root system should not be regarded as a special storage organ. Quantitatively, carbohydrates predominate in these reserves, but qualitatively N and other minerals are of more than minor significance. In roots carbohydrates are usually stored in insoluble form, mainly as starch; sorbitol is the predominant soluble compound in apple and peach. For nitrogen reserves, the soluble form predominates in roots, especially arginine in apple and peach, followed by asparagine. The level of reserves usually becomes maximal early in the winter. During leafing-out the reserves are drawn on until, later in the season, the supply of newly produced or absorbed nutrients exceeds the demand and replenishment occurs. The initial carbohydrate reserves do not determine the amount of new growth, whereas reserve nitrogen is of decisive importance for shoot growth vigour. Environmental factors such as light intensity and temperature affect the level of carbohydrates in roots; the concentration can be reduced by defoliation and summer pruning and increased by ample supply of nitrogen fertilizer in the autumn. The main cultural factors that influence nitrogen reserves are the amount and the time of nitrogen fertilization.     

Carbon and nitrogen metabolism in the seagrass, Zostera marina L.: Environmental control of enzymes involved in carbon allocation and nitrogen assimilation

This study experimentally examined influences of environmental variables on the activities of key enzymes involved in carbon and nitrogen metabolism of the submersed marine angiosperm, Zostera marina L. Nitrate reductase activity in leaf tissue was correlated with both water-column nitrate concentrations and leaf sucrose levels. Under elevated nitrate, shoot nitrate reductase activity increased in both light and dark periods if carbohydrate reserves were available. When water-column nitrate was low, glutamine synthetase activity in leaf tissue increased with environmental ammonium. In contrast, glutamine synthetase activity in belowground tissues was statistically related to both nitrate and temperature. At the optimal growth temperature for this species (ca. 25 °C), increased water-column nitrate promoted an increase in glutamine synthetase activity of belowground tissues. As temperatures diverged from the optimum, this nitrate effect on glutamine synthetase was no longer evident. Activities of both sucrose synthase and sucrose-P synthase were directly correlated with temperature. Sucrose-P synthase activity also was correlated with salinity, and sucrose synthase activity was statistically related to tissue ammonium. Overall, the enzymatic responses that were observed indicate a tight coupling between carbon and nitrogen metabolism that is strongly influenced by prevailing environmental conditions, especially temperature, salinity, and environmental nutrient levels.     

A contribution to understanding the internal nitrogen budget of beech (Fagus sylvatica L.)

Summary The seasonal variation in the total nitrogen content of the xylem sap of the lower trunk section was investigated for two middle aged beech tree stands in northern Hessen each containing 130 trees. In addition seasonal changes in the percentage of nitrate in the total nitrogen content are described. The median values of the total nitrogen content of the xylem sap during the spring mobilization period reached 175 and 250 mg/l. During the summer about 35% of the total nitrogen in the xylem sap is in the form of nitrate. Finally, the distribution of NO₃ in the xylem sap along the trunk height was studied for two sample trees for each of the four seasons (n = 8).