Efficiency of nitrate uptake in spinach: impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with 13N- and 15N-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration. Plants were grown at different nitrate concentrations (0.8 and 4 mM), with mineral composition of growth and uptake solutions identical. Nitrate influx, efflux and net nitrate uptake rate (NNUR) were independent of the external nitrate concentration, despite differences in internal nitrate concentration. At both N regimes, NNUR was adequate to meet the N demand for growth. RGR-related signals predominantly determined the nitrate fluxes. At high RGR (0.25 g g-1 day-1), nitrate influx was 20 to 40% lower and nitrate efflux was 50 to 70% lower than at lower RGR (0.17 g g-1 day-1); efflux:influx ratio (E:I) declined from 0.5 at low RGR to 0.2 at higher RGR. Thus, the efficiency of NNUR substantially increased with increasing RGR. Differences in nitrate translocation between morning and afternoon coincided with differences in nitrate efflux, which is in accordance with the suggested regulation of nitrate efflux by the root cytoplasmic nitrate concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influx,efflux and net uptake of nitrate in Quercus suber seedlings

Nitrate influx, efflux and net nitrate uptake were measured for the slow-growing Quercus suber L. (cork-oak) to estimate the N-uptake efficiency of its seedlings when grown with free access to nitrate. We hypothesise that nitrate influx, an energetically costly process, is not very efficiently controlled so as to avoid losses through efflux, because Q. suber has relatively high respiratory costs for ion uptake. Q. suber seedlings were grown in a growth room in hydroponics with 1 mM NO₃ ^-. Seedlings were labelled with ¹⁵NO₃ ^- in nutrient solution for 5 min to measure influx and for 2 h for net uptake. Efflux was calculated as the difference between influx and net uptake. Measurements were made in the morning, afternoon and night. The site of nitrate reduction was estimated from the ratio of NO₃ ^- to amino acids in the xylem sap; the observed ratio indicated that nitrate reduction occurred predominantly in the roots. Nitrate influx was always much higher than net acquisition and both tended to be lower at night. High efflux occurred both during the day and at night, although the proportion of ¹⁵NO₃ ^- taken up that was loss through efflux was proportionally higher during the night. Efflux was a significant fraction of influx. We concluded that the acquisition system is energetically inefficient under the conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

On the discrepancies between a colorimetric and isotopic method for measuring nitrate utilization in nutrient-depleted waters: implications for the design of experimental protocols in new production studies

Colorimetric analyses of nitrate disappearance from seawater have been compared with isotopic analyses of ¹⁵N-labelled nitrate incorporation into particulate matter. The slope (1.41) of a regression line calculated from 19 sample pairs gathered during 6 time-series experiments and 2 single end-point incubations showed that nitrate incorporation is positively related to changes in nitrate concentration but that it accounts only for 71% of nitrate disappearance. ¹⁵N-isotope dilution as a consequence of nitrification, if any, would not fully explain discrepancies between the two analytical procedures. A further possible mechanism responsible for the imbalance between nitrate-incorporation and -disappearance rates suggests losses of ¹⁵N label from plankton biomass to an unanalyzed pool (dissolved organic nitrogen?) which increase (up to 65%) with incubation time. The lack of ¹⁵N-mass balance calls for the need to consider additional nitrogen pools in ¹⁵N budgets of isotope experiments and not only substrate and biomass pools as has been done so far.     

Effects of NaCl salinity on 15N-nitrate fluxes and specific root length in the halophyte Plantago maritima L.

The effect of salinity on nitrate influx, efflux, nitrate net uptake rate and net nitrogen translocation to the shoot was assessed in a ¹⁵N steady state labelling experiment in the halophyte Plantago maritima L. raised for 14 days on solution supplied with 50, 100 and 200 mol m^–3 sodium chloride or without sodium chloride. Additionally, salinity induced changes in root morphology were determined. Specific root length increased upon exposure to elevated sodium chloride concentrations due to variations in biomass allocation and length growth of the tap root. Changes in root morphology, however, had a minor effect on nitrate fluxes when expressed on a root fresh weight basis. The decreased rate of nitrate net uptake in plants grown on elevated levels of sodium chloride was almost entirely due to a decrease in nitrate influx. Expressed as a proportion of influx, nitrate efflux remained unchanged and was even lower at the highest salinity level. At all sodium chloride concentrations applied the initial rate of nitrogen net translocation to the shoot decreased relative to the rate of nitrate net uptake. It is concluded that under steady state conditions the negative effect of sodium chloride on the rate of nitrate net uptake at non growth-limiting salinity levels was due to the interaction between sodium chloride and nitrate transporters in the root plasma membrane and/or processes mediating the translocation of nitrogen compounds, possibly nitrate, to the shoot.     

Growth requirement for N as a criterion to assess the effects of physical manipulation on nitrate uptake fluxes in spinach

The effects of physical manipulation of hydroponically grown plants of spinach (Spinacia oleracea L., cvs Subito and Glares) on nitrate uptake fluxes were studied in a long-term experiment (3 days), and in short-term label experiments (2 h) with ¹³N-nitrate and ¹⁵N-nitrate. In the long-term experiment, net nitrate uptake rate (NNUR) was measured by following the nitrate depletion in the uptake solution, which was replaced at regular intervals. In the short-term experiments, NNUR and nitrate influx were measured by simultaneous application of ¹³N-nitrate and ¹⁵N-nitrate. Plants were gently transferred into the labelled uptake solution, as is usually done in nutrient uptake studies. In addition, a more severe physical manipulation was carried out, including blotting of the roots, to mimic pretreatments which involve more handling of the plants prior to uptake measurements. Nitrate influx was measured immediately after physical manipulation and after 2 h of recovery. To assess the impact of the physical manipulation the experimentally determined nitrate uptake fluxes were compared with the N demand for growth, defined as relative growth rate (RGR) times plant nitrogen concentration (PNC) of parallel plants, which were left undisturbed. Nitrate influx and efflux were both subject to changes after physical manipulation of the plants. Physical handling, however, did not always result in an alteration of NNUR, which complicates the determination of the length of the recovery period. The impact of the handling and the time course of the recovery depended on the severity of the disturbance and were independent of the light conditions during the experiments. Even after a gentle transfer of the plants, recovery, in most cases, was not complete within 2 h. The data emphasise the need for minimal disturbance of plants during the last hours prior to nutrient uptake measurements.     

The effect of root temperature on the induction of nitrate reductase activities and nitrogen uptake rates in arctic plant species

We investigated whether six arctic plant species have the potential to induce nitrate reductase (NR) activity when exposed to NO₃ ^--nitrogen under controlled environment conditions, using an in vivo assay that uses the rate of NO₂ ^--accumulation to estimate potential NR activity. We also assessed the effect of low root temperatures on NR activity, growth and nitrogen uptake (using ¹⁵N applications) in two of the selected species. Five of the six species (Cerastium alpinum, Dryas intergrifolia, Oxyria digyna, Saxifraga cernua and Salix arctica) were capable of inducing NR activity when exposed to solutions containing 0.5 mM NO₃ ^- at 20°C for 10 days. Although in vivo NR activity was not induced in Saxifraga oppositifolia under controlled conditions, we conclude that it was capable of growing successfully on NO₃ ^-, due to the presence of moderate rates of NR activity observed in both NH₄ ⁺-grown and NO₃ ^--treated plants. Exposure of O. digyna and D. integrifolia to 3°C root temperatures for two weeks, with the shoots kept at 20°C, resulted in root and leaf NR activity rates of NO₃ ^--treated plants being reduced to rates exhibited by NH₄ ⁺-grown plants. Although these decreases in NR in both species appeared to be due to limitations in NO₃ ^--uptake and growth rate (rather than direct low-temperature inhibition of NR synthesis per se), direct low-temperature inhibition of root NR synthesis could not be ruled out. In contrast to the temperature insensitivity of NH₄ ⁺ uptake in D. integrifolia, NO₃ ^--uptake in D. integrifolia was inhibited by low root temperatures. We conclude that the selected arctic species have the genetic potential to utilize NO₃ ^--nitrogen, and that low root temperatures, in conjunction with other environmental limitations, may be responsible for the lack of induction of NR in D. integrifolia and Salix arctica under field conditions.     

The course of 15N-ammonium nitrate in a spring barley cropping system

¹⁵N-labelled ammonium nitrate was applied to spring barley growing on a Cambisol soil in western Switzerland. Immobilization, plant uptake and disappearance of inorganic nitrogen were followed at frequent intervals. Fertilizer nitrogen disappeared shortly after its application, mainly through immobilization by soil microorganisms and absorption by the crop. Some of the added nitrogen was probably denitrified as a result of humid conditions during the first days after fertilizer application. At the end of the growing season, 31% of the added nitrogen was recovered from the aerial barley plants, and 56% was immobilized by microorganisms. Most of the fertilizer nitrogen not used by the crop was immobilized in the upper 0–30 cm soil layer. This prevented downward movement of nitrate and limited nitrogen losses. Fertilizer efficiency was mainly determined by the competition between crop uptake and microbial immobilization. Careful consideration of the time of fertilization, taking into account plant growth and weather conditions, can result in an increase in fertilizer efficiency and minimal pollution.     

Differences in uptake kinetics of ammonium and nitrate in legumes and cereals

Influx isotherms were obtained for nitrate and ammonium from three legumes, Cajanus cajan (L.) Millsp., Cicer arietinum L. and Arachis hypogaea L. and three cereals, Sorghum bicolor (L.) Moench., Pennisetum glaucum L. and Zea mays L. The transition in influx isotherms for both nitrogen sources was found to be within the concentration range (0.05–2.5 mM) tested. There were significant differences in Km and Vmax for ammonium between legumes and cereals. The difference in the kinetic properties for nitrate uptake between the two groups of plants only became apparent at the higher concentration tested. Legumes translocated absorbed nitrate and ammonium to shoots more rapidly than cereals. Results show that there are significant differences in uptake and translocation of ammonium and nitrate between legumes and cereals.     

Nitrate transport processes in Fagus- Laccaria-Mycorrhizae

The contribution of influx and efflux of NO₃ ^- on NO₃ ^- net uptake has been studied in excised mycorrhizae of 18–20 week old beech (Fagus sylvatica L.) trees. Net uptake rates of NO₃ ^- followed uniphasic Michaelis-Menten kinetics in the concentration range between 10 μM and 1.0 mM external NO₃ ^-, with an apparent K_m of 88±7 μM, and a V_max of 110±7 nmol g^-1 root f.wt. h^-1. The relative xylem loading of N, i.e. the portion of NO₃ ^- taken up that was loaded into the xylem vessels as NO₃ ^- plus reduced N, was constant over the concentration range tested (4.6–7.7%). NO₃ ^- influx proceeded linearly with increasing external NO₃ ^- supply. When the assumed regulators of net NO₃ ^- uptake, i.e. NH₄ ⁺ or L-glutamate, were applied together with NO₃ ^-, net uptake rates of NO₃ ^- decreased. This inhibitory effect was caused by a reduction of NO₃ ^- influx rather than an enhanced efflux. The comparison of the present data with a recent study with non-mycorrhizal beech roots (Kreuzwieser et al., 1997; J. Exp. Bot. 48, 1431–1438) revealed that mycorrhization leads to reduced rates of NO₃ ^- net uptake. This effect is caused by reduced influx plus enhanced efflux of NO₃ ^- as compared with non-mycorrhizal beech roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relationships between functional traits and inorganic nitrogen acquisition among eight contrasting European grass species

Backgrounds and Aims Leaf functional traits have been used as a basis to categoize plants across a range of resource-use specialization, from those that conserve available resources to those that exploit them. However, the extent to which the leaf functional traits used to define the resource-use strategies are related to root traits and are good indicators of the ability of the roots to take up nitrogen (N) are poorly known. This is an important question because interspecific differences in N uptake have been proposed as one mechanism by which species’ coexistence may be determined. This study therefore investigated the relationships between functional traits and N uptake ability for grass species across a range of conservative to exploitative resource-use strategies.Methods Root uptake of NH4+ and NO3–, and leaf and root functional traits were measured for eight grass species sampled at three grassland sites across Europe, in France, Austria and the UK. Species were grown in hydroponics to determine functional traits and kinetic uptake parameters (I_max and K_m) under standardized conditions.Key Results Species with high specific leaf area (SLA) and shoot N content, and low leaf and root dry matter content (LDMC and RDMC, respectively), which are traits associated with the exploitative syndrome, had higher uptake and affinity for both N forms. No trade-off was observed in uptake between the two forms of N, and all species expressed a higher preference for NH4+.Conclusions The results support the use of leaf traits, and especially SLA and LDMC, as indicators of the N uptake ability across a broad range of grass species. The difficulties associated with assessing root properties are also highlighted, as root traits were only weakly correlated with leaf traits, and only RDMC and, to a lesser extent, root N content were related to leaf traits.     

Significance of organic nitrogen acquisition for dominant plant species in an alpine meadow on the Tibet plateau, China

Though the potential of plants to take up organic N (e.g., amino acids) is well established, the true significance of organic N acquisition to plant N nutrition has not yet been quantified under field conditions. Here we demonstrate that organic N contributes significantly to the annual N uptake of three dominant plant species (Kobresia humilis, Saussurea superba and Stipa aliena) of alpine meadows on the Tibet Plateau, China. This was achieved by using double-labelled (¹⁴C and ¹⁵N) algae as a source for slow and continuous release of amino acids, and tracing both labels in the above- and below-ground plant biomass. Four months after addition of algae, between 0.5% and 2.6% of ¹⁴C and 5% and 14% of ¹⁵N from added algae were recovered in the plants, which translate into an uptake of organic N between 0.3 mg N m⁻² and 1.5 mg N m⁻². The calculated contribution of organic N to total N uptake was estimated to range between 21% and 35% for K. humilis, and between 13% and 21% for S. aliena and S. superba, respectively, implying that organic N uptake by grassland plants is quantitatively significant under field conditions in the studied alpine meadows. This finding has important ecological implications with regard to competition for organic N between microorganisms and plant roots.     

北京典型树种木质组织碳释放速率温度敏感性的时间变化规律和铅锤分异特征

采用红外气体分析法(IRGA)于2014年1—12月原位测定了北京市4个典型树种(国槐Sophora japonica,旱柳Salix matsudana,华北落叶松Larix principis-rupprechtii和侧柏Platycladus orientalis)在不同高度上的木质组织CO_2通量速率(E_(CO_2)),旨在比较不同树种间E_(CO_2)及其温度敏感性(Q_(10))的时间变化规律和铅锤分异特征。研究结果显示:(1)4个树种的E_(CO_2)均表现为单峰型季节变化规律,生长月份内的E_(CO_2)显著高于非生长月份,温度和枝干的径向生长是影响E_(CO_2)季节变化的主要因素;(2)E_(CO_2)对温度的敏感性在夏季月份明显降低,且出现明显的垂直分异:Q_(10)随测量高度的增加而增加,呈现出非连续的阶梯分布;(3)在日间尺度上,阔叶树种E_(CO_2)对温度的感性系数Q_(10)出现昼夜不对称现象,晚上Q_(10)明显升高。准确量化E_(CO_2)的时间变化规律和铅锤分异特征,细化不同时间尺度下E_(CO_2)对温度的响应特征,成为准确估算木质组织碳排放的前提条件。     

The interaction of defoliation and nutrient uptake in Sporobolus kentrophyllus,a short-grass species from the serengeti plains

Summary Sporobolus kentrophyllus, a grazing-tolerant C₄ grass from the southeastern Serengeti Plains, was grown in solution culture to examine the effects of clipping on the uptake, preference and subsequent transport of varying nitrogen forms. Clipping reduced offtake mass, crown mass ane root mass, resulting in a 58% decline in plant mass. Proportional biomass allocation to roots decreased with clipping, while tillering rates increased. Clipping also increased the nitrogen concentrations of all tissues, and plant nitrogen uptake (nitrogen accumulated throughout the experiment per gram root). The ¹⁵N concentrations (% atom excess) of all tissues were higher in clipped compared with unclipped plants, and the average ¹⁵N uptake rate of clipped plants was twice that of unclipped plants. The relative ¹⁵N allocation to aboveground mass, a measure of canopy sink strength, was higher in clipped plants. Plants fed ¹⁵N-ammonium or ¹⁵N-nitrate during the ¹⁵N pulse experiment had greater ¹⁵N tissue concentrations compared with urea-fed plants, and ¹⁵N uptake rates were higher in ammonium-fed and nitrate-fed plants, compared with urea-fed plants. The relative magnitudes of these differences were higher when plants were clipped. Clipped plants had higher uptake rates for potassium, phosphorus and sodium, while differences between clipping treatments for calcium, iron, and magnesium were indistinguishable. Rapid uptake rates for species on the southeastern Serengeti plains, particularly during grazing periods, have important implications for nutrient cycling in this system.     

The expression of genes coding for auxin carriers in different tissues and along the organ can explain variations in auxin transport and the growth pattern in etiolated lupin hypocotyls

Novel cDNA clones encoding putative auxin influx and efflux carriers have been isolated and characterized from etiolated lupin (Lupinus albus L) hypocotyls. The full length of LaAUX1 and LaPIN1 and the partial length of LaPIN3 were obtained and the deduced amino acid sequence revealed a high degree of identity with the corresponding auxin carrier proteins from several species. The expression of these genes depended on the tissue, the hypocotyl zone and seedling age. LaAUX1 and LaPIN3 were expressed in stele and outer tissues, while LaPIN1 was restricted to the stele. From the above-mentioned results and taking into account the role proposed for the efflux carrier PIN1, it is suggested that LaPIN1 could mediate the basipetal auxin transport already described in this organ. LaAUX1 might facilitate auxin influx in the transport cells. The expression of the three genes decreased down the hypocotyl. The basipetally decreasing gradient in the expression of LaPIN1 coincides with previous results showing a similar gradient in the intensity and polarity of auxin transport. The decisive role ascribed to PIN1 in polar auxin transport due to its localization in the basal end of transporting cells and the existence of such a gradient in the expression of LaPIN1 support the hypothesis of a barrier effect (generated by decreasing auxin transport) previously proposed by our research group as being responsible for the auxin gradient, which controls the growth pattern in etiolated lupin hypocotyls. José Sánchez-Bravo, Manuel Acosta and Carlos Nicolás contributed equally to the paper. Nucleotide sequence database accession numbers: LaAUX1: AM235387; LaPIN1: AM235388; LaPIN3: AM407405.     

Complementarity in the use of nitrogen forms in a temperate broad-leaved mixed forest

Background: The complementary use of different forms of soil nitrogen (N) might lead to a higher productivity of mixed forests than monocultures, but convincing evidence for temperate mixed forests is scarce.

Aims: We searched for species differences in N uptake rates and the preference for NH₄⁺, NO₃^? or glycine among five temperate broad?leaved tree species (Acer pseudoplatanus, Carpinus betulus, Fagus sylvatica, Fraxinus excelsior, Tilia cordata) in a mature mixed stand.

Methods: ¹⁵N tracer was added to the soil and its accumulation in fine root biomass was analysed after 10 min, 1 h and 1 d.

Results: The estimated root uptake rates of the species were in the range of 5–46 µg N g^?1 root h^?1 for NH₄⁺, 6–86 µg N g^?1 h^?1 for NO₃^? and 4–29 µg N g^?1 h^?1 for glycine during the first hour after tracer application. Carpinus, Tilia and Acer tended to prefer NH₄⁺ over NO₃^?, while Fraxinus showed equal preference for both N forms and Fagus seemed to prefer NO₃^?.

Conclusions: The five co-existing tree species differed in uptake rates and partly in their N form preference, but complementarity in the use of different N forms seems to be of minor importance in this forest because tree species appear to be rather flexible in their N form use.     

Distribution of15N in the soil-plant system during a four-year field lysimeter study with barley (Hordeum distichum L.) and perennial meadow fescue (Festuca pratensis Huds.)

An annual cereal, barley, and a perennial grass ley, meadow fescue, were grown in field lysimeters in Sweden and fertilized with 12 and 20g Ca(NO₃)₂-N m⁻² yr⁻¹, respectively. Isotope-labeled (¹⁵N) fertilizer was added during year 1 of the study, whereafter similar amounts of unlabeled N were added during years 2 and 3. The grass ley lysimeters were ploughed after the growing season of year 3 and sown with barley during year 4. The barley harvest in year 1 removed 59% of the added fertilizer N, while the fertilizer N export by two meadow fescue harvests in year 1 was 65%. The labeled N export decreased rapidly after year 1, especially in the barley, but increased slightly after ploughing of the grass ley. The microbial biomass, measured with the chloroform fumigation method, incorporated a maximum of 1.4–1.7% of the labeled N during the first seven weeks after application. Later on, the incorporation stabilized at less than 1% in both cropping systems. The susceptibility of the residual labeled N to mineralization was evaluated three years after application by means of long-term laboratory incubations. The curves of cumulative mineralized N were described by a two-component first-order regression model that differentiated between an available and a more recalcitrant fraction of potentially mineralizable N. There was no difference in the amounts of potentially mineralizable N between the cropping systems. The labeled N comprised 5 and 2% of the amounts of potentially mineralizable N in the available and more recalcitrant fraction, respectively. The mineralization rate constants for the labeled N were almost twice as high as for the total potentially mineralizable N. The available fraction of the total potentially mineralizable N was 12%, while twice that proportion of the labeled N was available. It was concluded that the short-term ley did not differ from the annual crop with respect to the early disposition of the fertilizer N and the behaviour of the residual organic N.     

Importance of soil cover box area for the determination of N2O emissions from arable soils

To obtain nutrients mineralised from organic matter in the soil, plants have to respond to its heterogeneous distribution. We measured the timing of nitrogen uptake by wheat from a localised, ¹⁵N labelled organic residue in soil, as well as the timing of changes in root length density. We calculated the rates of N uptake per unit root length (inflows) for roots growing through the residue and for the whole root system. A stimulated local inflow appeared to be the main mechanism of exploitation of the residue N during the first five days of exploitation. 8% of the N that the plants would ultimately obtain from the residue was captured in this period. Roots then proliferated in the residue. This, together with a rapidly declining N inflow, contributed to the capture, over the next seven days, of 63% of the N that the plants derived from the residue. After that time, massive root proliferation occurred in the residue, but relatively little further N was captured.     

Spatial dynamics of rotifers in a large lowland river,the Elbe,Germany: How important are retentive shoreline habitats for the plankton community?

A stable nitrogen isotope analysis was used to clarify the relative importance of denitrification and nitrate uptake by plants in the nitrate reduction in a reed belt of L. Kamisagata (N 37°49′, E 138°53′, alt. 4.5 m, depth 30–80 cm, area 0.025 km²), one of about 20 sand dune lakes in Japan. A very high concentration of NO ₃ ⁻ -N with 19.0 ± 5.9 mg N l⁻¹ in spring sources decreased during passage through the reed belt along two set transect lines about 120 m long in any season, whereas progressive enrichment in ¹⁵N-NO ₃ ⁻ in flowing water was detected. Loss rate of nitrate ranged from 38.4 to 73.1% with an average of 56.7 ± 11.6%. Enrichment factors calculated using a Rayleigh curve method ranged from −1.03 to −5.12‰. The contribution of denitrification to nitrate loss ranged from 6 to 28%, with a mean of 19.5% (±7.0), whereas that of plant uptake was from 72 to 94%, with a mean of 80.5% (±7.0), indicating the importance of vegetation in a sand dune riparian zone. A technique using the variation of natural abundance of ¹⁵N may provide useful information on the nitrate dynamics in artificial or natural wetlands under a non-destructive condition.