Nitrate transport and signalling

Physiological measurements of nitrate (NO(3)(-)) uptake by roots have defined two systems of high and low affinity uptake. In Arabidopsis, genes encoding both of these two uptake systems have been identified. Most is known about the high affinity transport system (HATS) and its regulation and yet measurements of soil NO(3)(-) show that it is more often available in the low affinity range above 1 mM concentration. Several different regulatory mechanisms have been identified for AtNRT2.1, one of the membrane transporters encoding HATS; these include feedback regulation of expression, a second component protein requirement for membrane targeting and phosphorylation, possibly leading to degradation of the protein. These various changes in the protein may be important for a second function in sensing NO(3)(-) availability at the surface of the root. Another transporter protein, AtNRT1.1 also has a role in NO(3)(-) sensing that, like AtNRT2.1, is independent of their transport function. From the range of concentrations present in the soil it is proposed that the NO(3)(-)-inducible part of HATS functions chiefly as a sensor for root NO(3)(-) availability. Two other key NO(3)(-) transport steps for efficient nitrogen use by crops, efflux across membranes and vacuolar storage and remobilization, are discussed. Genes encoding vacuolar transporters have been isolated and these are important for manipulating storage pools in crops, but the efflux system is yet to be identified. Consideration is given to how well our molecular and physiological knowledge can be integrated as well to some key questions and opportunities for the future.     

Transpiration, potassium uptake and flow in tobacco as affected by nitrogen forms and nutrient levels

BACKGROUND AND AIMS: Ammonium can result in toxicity symptoms in many plants when it is supplied as the sole source of N. In this work, influences of different nitrogen forms at two levels (2 and 15 mm N) on growth, water relations and uptake and flow of potassium were studied in plants of Nicotiana tabacum 'K 326'. METHODS: Xylem sap from different leaves was collected from 106-d-old tobacco plants cultured in quartz sand by application of pressure to the root system. Whole-shoot transpiration for each of the treatments was measured on a daily basis by weight determination. KEY RESULTS: Total replacement of NO(3)(-)N by NH(4)(+)-N caused a substantial decrease in dry weight gain, even when plants grew under nutrient deficiency. Increasing nutrient concentration resulted in a greater net dry weight gain when nitrogen was supplied as NO(3)(-) or NH(4)NO(3), but resulted in little change when nitrogen was supplied as NH(4)(+). NH(4)(+)-N as the sole N-source also caused reduction in transpiration rate, changes in plant WUE (which depended on the nutrient levels) and a decrease in potassium uptake. However, the amount of xylem-transported potassium in the plants fed with NH(4)(+) was not reduced: it was 457 % or 596 % of the potassium currently taken up at low or high nutrient level, respectively, indicating a massive export from leaves and cycling of potassium in the phloem. CONCLUSIONS: Ammonium reduces leaf stomatal conductance of tobacco plants. The flow and partitioning of potassium in tobacco plants can be changed, depending on the nitrogen forms and nutrient levels.     

Oxidative pentose phosphate pathway-dependent sugar sensing as a mechanism for regulation of root ion transporters by photosynthesis

Root ion transport systems are regulated by light and/or sugars, but the signaling mechanisms are unknown. We showed previously that induction of the NRT2.1 NO(3)(-) transporter gene by sugars was dependent on carbon metabolism downstream hexokinase (HXK) in glycolysis. To gain further insights on this signaling pathway and to explore more systematically the mechanisms coordinating root nutrient uptake with photosynthesis, we studied the regulation of 19 light-/sugar-induced ion transporter genes. A combination of sugar, sugar analogs, light, and CO(2) treatments provided evidence that these genes are not regulated by a common mechanism and unraveled at least four different signaling pathways involved: regulation by light per se, by HXK-dependent sugar sensing, and by sugar sensing upstream or downstream HXK, respectively. More specific investigation of sugar-sensing downstream HXK, using NRT2.1 and NRT1.1 NO(3)(-) transporter genes as models, highlighted a correlation between expression of these genes and the concentration of glucose-6-P in the roots. Furthermore, the phosphogluconate dehydrogenase inhibitor 6-aminonicotinamide almost completely prevented induction of NRT2.1 and NRT1.1 by sucrose, indicating that glucose-6-P metabolization within the oxidative pentose phosphate pathway is required for generating the sugar signal. Out of the 19 genes investigated, most of those belonging to the NO(3)(-), NH(4)(+), and SO(4)(2-) transporter families were regulated like NRT2.1 and NRT1.1. These data suggest that a yet-unidentified oxidative pentose phosphate pathway-dependent sugar-sensing pathway governs the regulation of root nitrogen and sulfur acquisition by the carbon status of the plant to coordinate the availability of these three elements for amino acid synthesis.     

In vivo gas exchange measurement of the site and dynamics of nitrate reduction in soybean

A gas analysis system was built to study the relationship between the reductant cost of NO(3)(-) assimilation and the measured rate of CO(2) and O(2) exchange in roots, leaves, and stems+ petioles of soybean (Glycine max L. Merr. cv Maple glen) plants. The measurements were used to calculate the diverted reductant utilization rate (DRUR = 4*[measured rate of CO(2) + measured rate of O(2)], in moles of high-energy electron [e(-)] per gram per hour) in plants in the presence (N+) and absence (N-) of NO(3)(-). The differences in DRUR between the N+ and N- treatments provided a measure of the NO(3)(-)-coupled DRUR of 25-d-old plants, whereas a (15)NO(3)(-)-enriched nutrient solution was used to obtain an independent measure of the rate of NO(3)(-) assimilation. The measured reductant cost for the whole plant was 9.6 e(-) per N assimilated, a value within the theoretical range of four to 10 e(-) per N assimilated. The results predicted that shoots accounted for about 55% of the whole-plant NO(3)(-) assimilation over the entire day, with shoots dominating in the light, and roots in the dark. The gas analysis approach described here holds promise as a powerful, noninvasive tool to study the regulation of NO(3)(-) assimilation in plant tissue.     

A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis

Up-regulation of the high-affinity transport system (HATS) for NO(3)(-) and stimulation of lateral root (LR) growth are two important adaptive responses of the root system to nitrogen limitation. Up-regulation of the NO(3)(-) HATS by nitrogen starvation is suppressed in the atnrt2.1-1 mutant of Arabidopsis (Arabidopsis thaliana), deleted for both NRT2.1 and NRT2.2 nitrate transporter genes. We then used this mutant to determine whether lack of HATS stimulation affected the response of the root system architecture (RSA) to low NO(3)(-) availability. In Wassilewskija (Ws) wild-type plants, transfer from high to low NO(3)(-) medium resulted in contrasting responses of RSA, depending on the level of nitrogen limitation. Moderate nitrogen limitation (transfer from 10 mm to 1 or 0.5 mm NO(3)(-)) mostly led to an increase in the number of visible laterals, while severe nitrogen stress (transfer from 10 mm to 0.1 or 0.05 mm NO(3)(-)) promoted mean LR length. The RSA response of the atnrt2.1-1 mutant to low NO(3)(-) was markedly different. After transfer from 10 to 0.5 mm NO(3)(-), the stimulated appearance of LRs was abolished in atnrt2.1-1 plants, whereas the increase in mean LR length was much more pronounced than in Ws. These modifications of RSA mimicked those of Ws plants subjected to severe nitrogen stress and could be fully explained by the lowered NO(3)(-) uptake measured in the mutant. This suggests that the uptake rate of NO(3)(-), rather than its external concentration, is the key factor triggering the observed changes in RSA. However, the mutation of NRT2.1 was also found to inhibit initiation of LR primordia in plants subjected to nitrogen limitation independently of the rate of NO(3)(-) uptake by the whole root system and even of the presence of added NO(3)(-) in the external medium. This indicates a direct stimulatory role for NRT2.1 in this particular step of LR development. Thus, it is concluded that NRT2.1 has a key dual function in coordinating root development with external NO(3)(-) availability, both indirectly through its role as a major NO(3)(-) uptake system that determines the nitrogen uptake-dependent RSA responses, and directly through a specific action on LR initiation under nitrogen-limited conditions.     

Major alterations of the regulation of root NO(3)(-) uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis

The role of AtNrt2.1 and AtNrt2.2 genes, encoding putative NO(3)(-) transporters in Arabidopsis, in the regulation of high-affinity NO(3)(-) uptake has been investigated in the atnrt2 mutant, where these two genes are deleted. Our initial analysis of the atnrt2 mutant (S. Filleur, M.F. Dorbe, M. Cerezo, M. Orsel, F. Granier, A. Gojon, F. Daniel-Vedele [2001] FEBS Lett 489: 220-224) demonstrated that root NO(3)(-) uptake is affected in this mutant due to the alteration of the high-affinity transport system (HATS), but not of the low-affinity transport system. In the present work, we show that the residual HATS activity in atnrt2 plants is not inducible by NO(3)(-), indicating that the mutant is more specifically impaired in the inducible component of the HATS. Thus, high-affinity NO(3)(-) uptake in this genotype is likely to be due to the constitutive HATS. Root (15)NO(3)(-) influx in the atnrt2 mutant is no more derepressed by nitrogen starvation or decrease in the external NO(3)(-) availability. Moreover, the mutant also lacks the usual compensatory up-regulation of NO(3)(-) uptake in NO(3)(-)-fed roots, in response to nitrogen deprivation of another portion of the root system. Finally, exogenous supply of NH(4)(+) in the nutrient solution fails to inhibit (15)NO(3)(-) influx in the mutant, whereas it strongly decreases that in the wild type. This is not explained by a reduced activity of NH(4)(+) uptake systems in the mutant. These results collectively indicate that AtNrt2.1 and/or AtNrt2.2 genes play a key role in the regulation of the high-affinity NO(3)(-) uptake, and in the adaptative responses of the plant to both spatial and temporal changes in nitrogen availability in the environment.     

Cytokinins and auxin communicate nitrogen availability as long-distance signal molecules in pineapple (Ananas comosus)

This work aimed at identifying a possible role of phytohormones in long-distance (root-shoot) signaling under nitrogen deficiency. Three-months old pineapple plants were transferred from Murashige and Skoog (MS) medium to nitrogen-free MS (-N). During the first 24h on -N, 20 plants were harvested every 4h. After 30 days in -N, the remaining plants were transferred back to regular MS (+N) and 20 plants harvested every 4h for the first 24h. Following the harvests, endogenous levels of nitrate (NO(3)(-)), indole-3-acetic acid (IAA), isopentenyladenine (iP), isopentenyladenine riboside (iPR), zeatin (Z) and zeatin riboside (ZR) were analyzed in roots and leaves. In N-starved plants, the NO(3)(-) level dropped by 20% in roots between the first (4h) and the second harvest (8h). In leaves a reduction of 20% was found 4h later. Accumulation of IAA peaked in leaves at 16h. In roots, the accumulation of IAA only started at 16h while the leaf content was already in decline, which suggests that the hormone might have traveled from the leaves to the roots, communicating N-shortage. The contents of the four cytokinins were generally low in both, shoot and roots, and remained almost unchanged during the 24h of analysis. After N re-supply, roots showed a NO(3)(-) peak at 8h whereas the foliar concentration increased 4h later. Hormone levels in roots climaxed at 8h, this coinciding with the highest NO(3)(-) concentration. In leaf tissue, a dramatic accumulation was only observed for Z and ZR, and the peak was seen 4h later than in roots, suggesting that Z-type cytokinins might have traveled from the roots to the leaves. These findings provide evidence that there is a signaling pathway for N availability in pineapple plants, communicated upwards through cytokinins (N-supplemented plants) and downwards through auxin (N-starved plants).     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.     

Leaf-atmosphere NH(3) exchange of white clover (Trifolium repens L.) in relation to mineral N nutrition and symbiotic N(2) fixation.

Plant-atmosphere NH(3) exchange was studied in white clover (Trifolium repens L. cv. Seminole) growing in nutrient solution containing 0 (N(2) based), 0.5 (low N) or 4.5 (high N) mM NO(3)(-). The aim was to show whether the NH(3) exchange potential is influenced by the proportion of N(2) fixation relative to NO(3)(-) supply. During the treatment, inhibition of N(2) fixation by NO(3)(-) was followed by in situ determination of total nitrogenase activity (TNA), and stomatal NH(3) compensation points (chi(NH(3))) were calculated on the basis of apoplastic NH4(+) concentration ([NH4(+)]) and pH. Whole-plant NH(3) exchange, transpiration and net CO(2) exchange were continuously recorded with a controlled cuvette system. Although shoot total N concentration increased with the level of mineral N application, tissue and apoplastic [NH4(+)] as well as chi(NH(3)) were equal in the three treatments. In NH(3)-free air, net NH(3) emission rates of <1 nmol m(-2) s(-1) were observed in both high-N and N(2)-based plants. When plants were supplied with air containing 40 nmol mol(-1) NH(3), the resulting net NH(3) uptake was higher in plants which acquired N exclusively from symbiotic N(2) fixation, compared to NO(3)(-) grown plants. The results indicate that symbiotic N(2) fixation and mineral N acquisition in white clover are balanced with respect to the NH4(+) pool leading to equal chi(NH(3)) in plants growing with or without NO(3)(-). At atmospheric NH(3) concentrations exceeding chi(NH(3)), the NH(3) uptake rate is controlled by the N demand of the plants.     

Inoculation of cranberry (Vaccinium macrocarpon) with the ericoid mycorrhizal fungus Rhizoscyphus ericae increases nitrate influx

Despite the ubiquitous presence of ericoid mycorrhizal (ERM) fungi in cranberry (Vaccinium macrocarpon), no prior studies have examined the effect of ERM colonization on NO(3)(-) influx kinetics. Here, (15)NO(3)(-) influx was measured in nonmycorrhizal and mycorrhizal cranberry in hydroponics. Mycorrhizal cranberry were inoculated with the ERM fungus Rhizoscyphus (syn. Hymenoscyphus) ericae. (15)NO(3)(-) influx by R. ericae in solution culture was also measured. Rhizoscyphus ericae NO(3)(-) influx kinetics were linear when mycelium was exposed for 24 h to 3.8 mm NH(4)(+), and saturable when pretreated with 3.8 mm NO(3)(-), 50 microm NO(3)(-), or 50 microm NH(4)(+). Both low-N pretreatments induced greater NO(3)(-) influx than either of the high-N pretreatments. Nonmycorrhizal cranberry exhibited linear NO(3)(-) influx kinetics. By contrast, mycorrhizal cranberry had saturable NO(3)(-) influx kinetics, with c. eightfold greater NO(3)(-) influx than nonmycorrhizal cranberry at NO(3)(-) concentrations from 20 microm to 2 mm. There was no influence of pretreatments on cranberry NO(3)(-) influx kinetics, regardless of mycorrhizal status. Inoculation with R. ericae increased the capacity of cranberry to utilize NO(3)(-)-N. This finding is significant both for understanding the potential nutrient niche breadth of cranberry and for management of cultivated cranberry when irrigation water sources contain nitrate.     

Ecophysiological adjustment of two Sphagnum species in response to anthropogenic nitrogen deposition

Here, it was investigated whether Sphagnum species have adjusted their nitrogen (N) uptake in response to the anthropogenic N deposition that has drastically altered N-limited ecosystems, including peatlands, worldwide. A lawn species, Sphagnum balticum, and a hummock species, Sphagnum fuscum, were collected from three peatlands along a gradient of N deposition (2, 8 and 12 kg N ha(-1) yr(-1)). The mosses were subjected to solutions containing a mixture of four N forms. In each solution one of these N forms was labeled with (15)N (namely (15)NH(+)(4), (15)NO(-)(3) and the amino acids [(15)N]alanine (Ala) and [(15)N]glutamic acid (Glu)). It was found that for both species most of the N taken up was from , followed by Ala, Glu, and very small amounts from NO(-)(3). At the highest N deposition site N uptake was reduced, but this did not prevent N accumulation as free amino acids in the Sphagnum tissues. The reduced N uptake may have been genetically selected for under the relatively short period with elevated N exposure from anthropogenic sources, or may have been the result of plasticity in the Sphagnum physiological response. The negligible Sphagnum NO(-)(3) uptake may make any NO(-)(3) deposited readily available to co-occurring vascular plants.     

Deposition of ammonium and nitrate in the roots of maize seedlings supplied with different nitrogen salts

This study measured total osmolarity and concentrations of NH(4)(+), NO(3)(-), K(+), soluble carbohydrates, and organic acids in maize seminal roots as a function of distance from the apex, and NH(4)(+) and NO(3)(-) in xylem sap for plants receiving NH(4)(+) or NO(3)(-) as a sole N-source, NH(4)(+) plus NO(3)(-), or no nitrogen at all. The disparity between net deposition rates and net exogenous influx of NH(4)(+) indicated that growing cells imported NH(4)(+) from more mature tissue, whereas more mature root tissues assimilated or translocated a portion of the NH(4)(+) absorbed. Net root NO(3)(-) influx under Ca(NO(3))(2) nutrition was adequate to account for pools found in the growth zone and provided twice as much as was deposited locally throughout the non-growing tissue. In contrast, net root NO(3)(-) influx under NH(4)NO(3) was less than the local deposition rate in the growth zone, indicating that additional NO(3)(-) was imported or metabolically produced. The profile of NO(3)(-) deposition rate in the growth zone, however, was similar for the plants receiving Ca(NO(3))(2) or NH(4)NO(3). These results suggest that NO(3)(-) may serve a major role as an osmoticant for supporting root elongation in the basal part of the growth zone and maintaining root function in the young mature tissues.     

硝酸盐供应对玉米侧根生长的影响

以两个玉米(Zea mays L.)自交系478和Wu312为研究材料,采用琼脂培养方法,研究不同浓度NO-3对侧根生长的影响.结果表明,在外部浓度0.01～1.0mmol/L范围内,NO-3供应能显著增加侧根的长度及根生物量.但当NO-3供应超过1.0 mmo1/L后,侧根长度开始下降.当NO-3供应分别在超过5.0(Wu312)与10(478)mmol/L后,侧根密度显著下降.在10 mmol/LNO-3下,Wu312的侧根生长几乎完全被抑制.而478在20 mmol/L时,侧根密度仍可达到其最大值的30%(主根)～50%(胚根).植株地上部全氮及硝酸盐含量随NO-3供应的增加而升高,二者与侧根长度、侧根密度及冠根比的数学函数关系相同.     

Nitrate transporters in plants: structure, function and regulation

Physiological studies have established that plants acquire their NO(-3) from the soil through the combined activities of a set of high- and low-affinity NO(-3) transport systems, with the influx of NO(-3) being driven by the H(+) gradient across the plasma membrane. Some of these NO(-3) transport systems are constitutively expressed, while others are NO(-3)-inducible and subject to negative feedback regulation by the products of NO(-3) assimilation. Here we review recent progress in the characterisation of the two families of NO(-3) transporters that have so far been identified in plants, their structure and their regulation, and consider the evidence for their roles in NO(-3) acquisition. We also discuss what is currently known about the genetic basis of NO(-3) induction and feedback repression of the NO(-3) transport and assimilatory pathway in higher plants.     

Root-zone acidity and nitrogen source affects Typha latifolia L. growth and uptake kinetics of ammonium and nitrate

The NH(4)(+) and NO(3)(-) uptake kinetics by Typha latifolia L. were studied after prolonged hydroponics growth at constant pH 3.5, 5.0, 6.5 or 7.0 and with NH(4)(+) or NO(3)(-) as the sole N-source. In addition, the effects of pH and N source on H(+) extrusion and adenine nucleotide content were examined. Typha latifolia was able to grow with both N sources at near neutral pH levels, but the plants had higher relative growth rates, higher tissue concentrations of the major nutrients, higher contents of adenine nucleotides, and higher affinity for uptake of inorganic nitrogen when grown on NH(4)(+). Growth almost completely stopped at pH 3.5, irrespective of N source, probably as a consequence of pH effects on plasma membrane integrity and H(+) influx into the root cells. Tissue concentrations of the major nutrients and adenine nucleotides were severely reduced at low pH, and the uptake capacity for inorganic nitrogen was low, and more so for NO(3)(-)-fed than for NH(4)(+)-fed plants. The maximum uptake rate, V(max), was highest for NH(4)(+) at pH 6.5 (30.9 micro mol h(-1) g(-1) root dry weight) and for NO(3)(-) at pH 5.0 (31.7 micro mol h(-1) g(-1) root dry weight), and less than 10% of these values at pH 3.5. The affinity for uptake as estimated by the half saturation constant, K((1/2)), was lowest at low pH for NH(4)(+) and at high pH for NO(3)(-). The changes in V(max) and K((1/2)) were thus consistent with the theory of increasing competition between cations and H(+) at low pH and between anions and OH(-) at high pH. C(min) was independent of pH, but slightly higher for NO(3)(-) than for NH(4)(+) (C(min)(NH(4)(+)) approximately 0.8 mmol m(-3); C(min)(NO(3)(-)) approximately 2.8 mmol m(-3)). The growth inhibition at low pH was probably due to a reduced nutrient uptake and a consequential limitation of growth by nutrient stress. Typha latifolia seems to be well adapted to growth in wetland soils where NH(4)(+) is the prevailing nitrogen compound, but very low pH levels around the roots are very stressful for the plant. The common occurrence of T. latifolia in very acidic areas is probably only possible because of the plant's ability to modify pH-conditions in the rhizosphere.