Regulation of the High-Affinity Nitrate Transport System in Wheat Roots by Exogenous Abscisic Acid and Glutamine

Nitrate is a major nitrogen （N） source for most crops. Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels. Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency. The present study investigated the regulation of the high-affinity nitrate transport system （HATS） by exogenous abscisic acid （ABA） and glutamine （Gin） in wheat （Triticum aestivum L.） roots. Wheat seedlings grown in nutrient solution containing 2 mmol/L nitrate as the only nitrogen source for 2weeks were deprived of N for 4d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gin in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h. Treated wheat plants were then divided into two groups. One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L ^15N-labeled nitrate. The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate. Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction. When Gin was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced. These results imply that the inhibition by Gin of NRT2 expression occurs only when nitrate is present in the growth media. Although exogenous ABA and Gin induced HATS genes in the roots of wheat, they did not induce nitrate influx.     

Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mm nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 x atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mm nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.     

Nitrate Signaling and the Two Component High Affinity Uptake System in Arabidopsis

Nitrate transporters are important for nitrogen acquisition by plants and in algae some require two gene products, NRT2 and NAR2, for function. The NRT2 family was already described and the recent identification of a family of the NAR2-type genes in higher plants showed that there was a homologue in Arabidopsis, AtNAR2.1. Using heterologous expression in yeast and oocytes we showed that the two Arabidopsis AtNRT2.1 and AtNAR2.1 proteins interacted to give a functional high affinity nitrate transport system (HATS). The gene knock out mutant atnar2.1-1 is deficient specifically for HATS activity and the resulting growth phenotype on low nitrate concentration is more severe than for the atnrt2.1-1 knock out mutant. Physiological characterisation of the plant N status and gene expression revealed a pattern that was characteristic of severe nitrogen deficiency. Consistent with the down regulation of AtNRT2.1 expression, the atnar2.1-1 plants also displayed the same phenotype as atnrt2.1 mutants in lateral root (LR) response to low nitrate supply. Using atnar2.1-1 plants constitutively expressing the NpNRT2.1 gene, we now show a specific role for AtNAR2.1 in LR response to low nitrate supply. AtNAR2.1 is also involved in the repression of LR initiation in response to high ratios of sucrose to nitrogen in the medium. Therefore the two component system itself is likely to be involved in the signaling pathway integrating nutritional cues for LR architecture regulation. Using a green fluorescent protein-NRT2.1 protein fusion we show the essential role of AtNAR2.1 for the presence of AtNRT2.1 to the plasma membrane.Key Words: high affinity nitrate transport, nitrate transporter, nitrate signalling, root growth     

A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi

In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X₂-CX₁₇-C-X₂-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X₂-C-X₁₈-C-X₂-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light.     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

The nitrate reductase promoter of birch directs differential reporter gene expression in tissues of transgenic tobacco

A 1535 bp promoter of the nitrate reductase gene (nia) from birch (Betula pendula) and a series of 5′ deletions were fused to the β-glucuronidase (GUS) gene and introduced into Nicotiana plumbaginifolia. In transgenic plants the NR promoter sequences directed strong GUS expression in the root epidermal hair cells, and in phloem cells of leaf and stem vascular tissue. The NR promoter confers also a significant stimulation of the GUS gene expression by nitrate. These findings might indicate that nitrate flow is one of the signals involved into tissue and cell specific expression of the NR promoter GUS fusions. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of N-remobilisation and the uptake of NH4+ and NO3- by Lolium perenne L. in laminae growth following defoliation under field conditions

Several studies have previously shown that shoot removal of forage species, either by cutting or herbivore grazing, results in a large decline in N uptake (60%) and/or N₂ fixation (80%). The source of N used for initial shoot growth following defoliation relies mainly on mobilisation of N reserves from tissues remaining after defoliation. To date, most studies investigating N-mobilisation have been conducted, with isolated plants grown in controlled conditions. The objectives of this study were for Lolium perenne L., grown in a dense canopy in field conditions, to determine: 1) the contribution of N-mobilisation, NH₄ ⁺ uptake and NO₃ ^- uptake to growing shoots after defoliation, and 2) the contribution of the high (HATS) and low (LATS) affinity transport systems to the total plant uptake of NH₄ ⁺ and NO₃ ^-. During the first seven days following defoliation, decreases in biomass and N-content of roots (34% and 47%, respectively) and to a lesser extent stubble (18% and 43%, respectively) were observed, concomitant with mobilisation of N to shoots. The proportion and origin of N used by shoots (derived from reserves or uptake) was similar to data reported for isolated plants. Both HATS and LATS contributed to the total root uptake of NH₄ ⁺ and NO₃ ^-. The V_max of both the NH₄ ⁺ and NO₃ ^- HATS increased as a function of time after defoliation, and both HATS systems were saturated by substrate concentrations in the soil at all times. The capacity of the LATS was reduced as soil NO₃ ^- and NH₄ ⁺ concentrations decreased following defoliation. Data from ¹⁵N uptake by field-grown plants, and uptake rates of NH₄ ⁺ and NO₃ ^- estimated by excised root bioassays, were significantly correlated, though uptake was over-estimated by the later method. The results are discussed in terms of putative mechanisms for regulating N uptake following severe defoliation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The characterization of inhibition of net nitrate uptake by salt in salt-tolerant barley (Hordeum vulgare L. cv. California Mariout)

Barley seedlings (Hordeum vulgare L. cv. California Mariout) grown hydroponically for 14-19 d without addition of NaCl were used for describing the effects of salt application on net nitrate uptake and for the calculation of kinetic parameters. The addition of NaCl, KCl, CaCl2, and Na2SO4 to the uptake solution in the experiments led to similar inhibition of nitrate uptake, only at low and very high salt concentrations were ion-specific effects found. The same decrease in nitrate uptake can also be achieved by sorbitol or betaine at corresponding osmolalities. Thus it was concluded that the inhibition of uptake was caused mainly by the osmotic effects of salts. Differences in the mechanisms of inhibition were detected between the two systems of nitrate uptake (high affinity system: HATS, and low affinity system: LATS). The HATS was inhibited non-competitively by NaCl, an apparent Ki of 60 mol m^-3 was calculated using a Dixon-plot. Fitting an equation assuming a non-competitively inhibited HATS by computer program to the raw data resulted in an apparent Ki of about 37 mol m^-3. In contrast, the LATS was affected in a complex way: up to 60 mol m^-3 NaCl the affinity was increased, which led to a stimulation of nitrate uptake at low nitrate concentrations (<2 mol m^-3). An inhibition of the LATS became obvious at concentrations above 3 mol m^-3 nitrate (for all applied salt concentrations) or with 100 mol m^-3 NaCl (throughout the whole nitrate range). Related plots of the data pointed to a competitive effect.Key words: Hordeum vulgare L., net nitrate uptake, high affinity transport system (HATS), low affinity transport system (LATS), salt, inhibition, apparent kinetic parameters.      

Defensin gene family in <Emphasis Type="Italic">Medicago truncatula:</Emphasis> structure,expression and induction by signal molecules

A large gene family encoding the putative cysteine-rich defensins was discovered in Medicago truncatula. Sixteen members of the family were identified by screening a cloned seed defensin from M. sativa (Gao et al. 2000) against the Institute for Genomic Research’s (TIGR) M. truncatula gene index (MtGI version 7). Based on the comparison of their amino acid sequences, M. truncatula defensins fell arbitrarily into three classes displaying extensive sequence divergence outside of the eight canonical cysteine residues. The presence of Class II defensins is reported for the first time in a legume plant. In silico as well as Northern blot and RT-PCR analyses indicated these genes were expressed in a variety of tissues including leaves, flowers, developing pods, mature seed and roots. The expression of these genes was differentially induced in response to a variety of biotic and abiotic stimuli. For the first time, a defensin gene (TC77480) was shown to be induced in roots in response to infection by the mycorrhizal fungus, Glomus versiforme. Northern blot analysis indicated that the tissue-specific expression patterns of the cloned Def1 and Def2 genes differed substantially between M. truncatula and M. sativa. Furthermore, the induction profiles of the Def1 and Def2 genes in response to the signaling molecules methyl jasmonate, ethylene and salicylic acid differed markedly between these two legumes.     

Gene structure and expression of the high-affinity nitrate transport system in rice roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.     

Characterization of the Ferredoxin-Gogat gene (OsGog2 clone) expression in rice

Ferredoxin-dependent glutamate synthase (Fd-Gogat; EC 1.4.7.1) in leaf and root plastids is the last enzyme involved in the pathway of nitrate assimilation in higher plants. Arabidopsis thaliana expresses two different genes: the first, light regulated, specific of green tissues and the second expressed in other tissues. In this work, we investigated whether in our clone, OsGog2 AC Y12595, this gene is up-regulated by light or it is expressed under darkness. Fd-Gogat specific activity, protein and mRNA increased after light treatment in rice shoots. In roots, the activity and the protein content remained constant, whereas the mRNA is repressed by light treatment. The results obtained using a specific probe, situated in the 3′ untranslated region of the OsGog2 cDNA, indicated that OsGog2 gene is up-regulated by light and that its expression is tissue specific and suggested that a dark expressed Fd-Gogat gene could be present in rice similarly as in Arabidopsis.     

T-DNA-insert-independent mutations induced in transformed plant cells duringAgrobacterium co-cultivation

Transformation frequencies were determined for 1n, 2n, and 4n Nicotiana plumbaginifolia protoplast cultures inAgrobacterium-mediated gene transfer experiments. An unexpected large drop (50%) in plating efficiencies was observed in the non-selected (control) 1n populations after transformation treatment with virulent strains. This effect was not observed in the 2n or 4n cultures or in the 1n cultures when treated with avirulent bacteria. The mortality was disproportionally high and could not be explained by the low (0.1–0.5%) transformation efficiency in the 1n population, indicating mutagenesis of the cell populations independently from the T-DNA insertions. Mutagenesis was also indicated in gene tagging experiments where nitrate reductase-deficient (NR^–) mutants were selected from haploidNicotiana plumbaginifolia protoplasts, as well as from leaf disc cultures or protoplasts of diploid plants that were heterozygotic for a mutation either in the NR apoenzyme gene (nia/wt) or one of the molybdenum-containing cofactor genes (cnxA/wt), afterAgrobacterium co-cultivation. The chlorate-resistant isolates were tested for the T-DNA-specific kanamycin resistance trait only after NR-deficiency had been established. Thirty-nine independent NR-deficient mutants were analysed further by Southern blot hybridization. There was no indication of integrated T-DNA sequences in the mutated NR genes, despite the fact that NR-deficient cells were found more frequently in cell populations which became transformed during the treatment than in the populations which did not. These observations suggest that transformation-competent cells undergo mutagenesis during theAgrobacterium gene transfer process not only as a result of stable integration events, but also through accompanying events that do not result in major changes in the mutated loci. The nature of these changes at the molecular level remains to be elucidated.     

The effects of light on induction, time courses, and kinetic patterns of net nitrate uptake in barley

Barley seedlings ( Hordeum vulgare L.) were grown hydroponically with (induced) or without (uninduced) nitrate in a light/dark cycle with high photon flux density to determine the effects of light on time courses, induction and kinetics of net nitrate uptake. Nitrate uptake was induced by external nitrate in both light and dark and was prevented by 1 mol m^–3 p-fluorophenylalanine. In high light, nitrate uptake was about 2-fold higher than in low light. During time course experiments the uptake rates oscillated due to daily light–dark changes. Rates of nitrate uptake also increased at about 2200 h during continuous darkness. This increase coincided approximately with the time at which the dark period started during the previous culture of the plants, indicating that it was due to a mechanism associated with an endogenous diurnal rhythm. When calculating the kinetics of nitrate uptake, a model with two saturable systems, including a high-affinity system (HATS) and a low-affinity system (LATS), gave the best fit to data in all treatments. The apparent affinity of the HATS ranged from 7·7 to 12·2 mmol m^–3 in induced plants in all light conditions. The effect of light on the HATS was mainly an increase of apparent V _max in the step from low to high light. In uninduced plants the HATS operated at a very low activity which was strongly enhanced during induction. Interpretation of the calculated kinetics of the LATS was much more difficult on the basis of net uptake data. The apparent affinity of the LATS increased from 24·3 mol m^–3 in low light up to 0·17 mol m^–3 after acceleration in high light. These extreme changes in apparent affinity of the LATS could not be explained satisfactorily, and the nature of this system is also discussed with respect to the method used.     

Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO₃⁻. The V_max for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks.Additionally, we studied the regulation of root NO₃⁻ uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO₃⁻ and down-regulated by the N status and by NO₃⁻ itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status.The use of various metabolic uncouplers or inhibitors indicated that NO₃⁻ net uptake mediated by iHATS and LATS was an active transport system in both rootstocks.Key Words: Citrus, inducible high affinity transport system (iHATS), constitutive high affinity transport system (cHATS), nitrate uptake, regulation     

Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max)

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 μM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots. Received: 22 November 1997 / Accepted: 26 January 1998     

A comparative kinetic analysis of nitrate and ammonium influx in two early-successional tree species of temperate and boreal forest ecosystems

Root NO₃ ^? and NH₄ ⁺ influx systems of two early‐successional species of temperate (trembling aspen: Populus tremuloides Michx.) and boreal (lodgepole pine: Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) forest ecosystems were characterized. NO₃ ^? and NH₄ ⁺ influxes were biphasic, consisting of saturable high‐affinity (HATS) and constitutive non‐saturable low‐affinity transport systems (LATS) that were evident at low and relatively high N concentrations, respectively. NO₃ ^? influx via HATS was inducible (IHATS); nitrate pre‐treatment resulted in 8–10‐fold increases in the V_max for influx in both species. By contrast, HATS for NH₄ ⁺ were entirely constitutive. In both species, V_max values for NH₄ ⁺ influx were higher than those for NO₃ ^? uptake; the differences were larger in pine (6‐fold) than aspen (1·8‐fold). In aspen, the K_m for NH₄ ⁺ influx by HATS was approximately 3‐fold higher than for IHATS NO₃ ^? influx, while in pine the K_m for IHATS NO₃ ^? influx was approximately 3‐fold higher than for NH₄ ⁺ influx. The aspen IHATS for NO₃ ^? influx appeared to be more efficient than that of pine (V_max values for aspen being approximately 10‐fold higher and K_m values being approximately 13‐fold lower than for pine). By contrast, only small differences in values for the NH₄ ⁺ HATS were evident between the two species. The kinetic parameters observed here probably result from adaptations to the N availabilities in their respective natural habitats; these may contribute to the distribution and niche separation of these species.