Novel Proteins of the High-Affinity Nitrate Transporter Family NRT2, SaNRT2.1 and SaNRT2.5, from the Euhalophyte Suaeda altissima: Molecular Cloning and Expression Analysis

Two genes of nitrate transporters SaNRT2.1 and SaNRT2.5, putative orthologs of high-affinity nitrate transporter genes AtNRT2.1 and AtNRT2.5 from Arabidopsis thaliana, were cloned from the euhalophyte Suaeda altissima. Phylogenetic bioinformatic analysis demonstrated that the proteins SaNRT2.1 and SaNRT2.5 exhibited higher levels of homology to the corresponding proteins from the plants of family Amaranthaceae; the similarity of amino acid sequences between proteins SaNRT2.1 and SaNRT2.5 was lower (54%). Both SaNRT2.1 and SaNRT2.5 are integral membrane proteins forming 12 transmembrane helices as predicted by topological modeling. An attempt to demonstrate nitrate transporting activity of SaNRT2.1 or SaNRT2.5 by heterologous expression of the genes in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the only yeast nitrate transporter was not successful. The expression patterns of SaNRT2.1 and SaNRT2.5 were studied in S. altissima plants that were grown in hydroponics under either low (0.5 mM) or high (15 mM) nitrate and salinity from 0 to 750 mM NaCl. The growth of the plants was strongly inhibited by low nitrogen supply while stimulated by NaCl; it peaked at 250 mM NaCl for high nitrate and at 500 mM NaCl for low nitrate. Under low nitrate supply, nitrate contents in S. altissima roots, leaves and stems were reduced but increased in leaves and stems as salinity in the medium increased. Potassium contents remained stable under salinity treatment from 250 to 750 mM NaCl. Quantitative real-time PCR demonstrated that without salinity, SaNRT2.1 was expressed in all organs, its expression was not influenced by nitrate supply, while SaNRT2.5 was expressed exclusively in roots—its expression rose about 10-fold under low nitrate. Salinity increased expression of both SaNRT2.1 and SaNRT2.5 under low nitrate. SaNRT2.1 peaked in roots at 500 mM NaCl with 15-fold increase; SaNRT2.5 peaked in roots at 500 mM NaCl with 150-fold increase. It is suggested that SaNRT2.5 ensures effective nitrate uptake by roots and functions as an essential high-affinity nitrate transporter to support growth of adult S. altissima plants under nitrogen deficiency.


Introduction
Nitrogen is an important biogenic element.For terrestrial plants, the main source of nitrogen is nitrate, which is a dominating nitrogen form in aerated soils [1,2].Plants absorb nitrate from the soil solution using specific transport mechanisms and molecular systems that function in their plasma membranes of root epidermal and cortical cells.
The availability of soil nitrate varies a lot due to its high turnover rate.It is explained by nitrate leaching, denitrification and different season-related edaphic factors [3].Driven by the necessity to adapt to the wide range of changing nitrate concentrations, plants evolved transport systems with different affinities to nitrate [4,5].According to the various concentrations of soil nitrate, the nitrate-transporting systems of plants could be divided to two groups, namely, low-affinity transport systems (LATS) and high-affinity transport systems (HATS).The LATS act at concentrations higher than 0.5 mM NO 3 − ; the HATS act at low NO 3 − concentrations (saturation in the range of 0.2 to 0.5 mM) [6].Concentrations of nitrate present in soils are often in the micromolar range, which limits plant growth [5].Low nitrate concentrations induce high-affinity transport systems (HATS) of plants; HATS allow for absorbing nitrate from the soil solution with concentrations below 0.5 mM [6,7].Experiments with model salt-sensitive glycophyte Arabidopsis thaliana demonstrated that the important role in nitrogen supply under low nitrate concentration in the ambient medium is exhibited by the high-affinity nitrate transporters of the NRT2 family.Genes for seven members of the NRT2 family, AtNRT2.1-AtNRT2.7,were identified in the Arabidopsis genome [8].The genes were found based on their similarity to AtNRT2.1 [8,9].The transporters of the NRT2 family of Arabidopsis are the best studied so far.It was demonstrated that the activity of high-affinity nitrate transporters AtNRT2.1,AtNRT2.2,AtNRT2.4 and AtNRT2.5 is localized in roots and linked to nitrate uptake [6,[10][11][12].Transporters AtNRT2.1 and AtNRT2.2play an essential role under low nitrate concentrations [10,11].Transporters AtNRT2.4 and AtNRT2.5 are also involved in high-affinity nitrate uptake but their activity is expressed under conditions of nitrate starvation only while AtNRT2.4 is important for nitrate uptake at very low external NO 3 − concentrations [6,12].Long-term nitrate starvation strongly induces the expression of AtNRT2.5;AtNRT2.5 brings the main contribution into high-affinity nitrate uptake under these conditions [6].AtNRT2.7 is specifically expressed in seeds; it is the only NRT2 transporter located in tonoplast for loading NO 3 − into vacuoles [13].Complete genome sequencing of plant species is rapidly progressing; 3517 genomes were sequenced from 1575 plant species from 2000 to 2024 [14].This helped to discover many sequences encoding nitrate transporters of different families including NRT2.Five NRT2 genes have been identified in the rice genome [15].Four ZmNRT2 genes have been identified in the maize genome (ZmNRT2.1,ZmNRT2.2,ZmNRT2.3 and ZmNRT2.5)[16].Five nitrate transporter genes of the NRT2 family have been found in wheat (Triticum urartu) [17,18].High-affinity nitrate transporters belonging to the NRT2 family were also found in a wide range of other higher plant species, including barley (Hordeum vulgare) [19], wild soybean (Glycine soja) [20], rapeseed (Brassica napus) [21], spinach (Spinacea oleraecae) [22] and Poncirus trifoliata [23].
Salinization reduces nitrate availability to plants.One of the main reasons is the competition between NO 3 − and Cl − for anion transporters [24][25][26].For plants growing in soils with low (micromolar) nitrate concentrations, this could be especially crucial.The uptake of NO 3 -and assimilation of nitrogen are suppressed in glycophytes under salinity [27].
It is hypothesized that halophytes, the plants naturally inhabiting saline soils, absorb nitrate under salinity more efficiently than glycophytes [28,29].Anion-transporting proteins in the plasma membrane of halophyte root cells function when exposed to much more concentrated solutions of Na + and Cl − ions than the orthologous proteins of glycophytes, suggesting structural peculiarities for these proteins of halophytes and, correspondingly, their distinct physicochemical properties compared to the glycophytic orthologs.The characteristics ensure that the anion transport systems of halophytes bind nitrate and transport it via plasmalemma when nitrate concentration is orders of magnitude lower than the concentration of chloride.So, understanding the effects of salinity on nitrogen uptake in halophytes may be beneficial for creating salinity-tolerant agricultural plants.Nevertheless, available information about anion-transporting proteins of halophytes is scarce.
It is reasonable to assume that under the conditions of nitrate deficiency and at the same time of salinity with high (over 200 mM) NaCl in nutrient or soil solution, the key role in nitrate uptake and transport in plants is played by specific high-affinity nitrate transporters.Here, we describe the cloning of coding sequences for two high-affinity nitrate transporter genes, SaNRT2.1 and SaNRT2.5, from the euhalophyte Suaeda altissima (Amaranthaceae, Suaedoideae).Many members of the genus Suaeda inhabit highly saline soils and are characterized by extreme salt tolerance, above 750 mM NaCl [30][31][32].S. altissima Pall. is a herbaceous halophytic plant with very high salinity tolerance, sharing the same halophyte features as other representatives of the genus Suaeda.It is one of the most salt tolerant plants, able to complete its life cycle at 1 M NaCl concentrations [33].Under natural conditions, S. altissima inhabits the shores of the largest salt lake in Europe, Elton.
Newly identified nitrate transporters from S. altissima, SaNRT2.1 and SaNRT2.5, are putative orthologs of high-affinity nitrate transporters AtNRT2.1 and AtNRT2.5 from A. thaliana.The relative abundance of SaNRT2.1 and SaNRT2.5 transcripts in S. altissima organs was measured for plants grown at various nitrate and chloride concentrations in nutrient solutions.The ability of SaNRT2.1 and SaNRT2.5 to transport nitrate was examined in a heterologous system, by functional complementation analysis in the mutant strain ∆ynt1 of yeast Hansenula (Ogatae) polymorpha.H. polymorpha is a suitable model organism to study heterologous plant nitrate-transporting mechanisms since it is able to take up and metabolize nitrate as the only nitrogen source [34,35].Gene YNT1 (yeast nitrate transporter 1) encodes the only high-affinity nitrate transporter in H. polymorpha [36].In the mutant strain ∆ynt1, used in this work, the gene YNT1 is deleted.

Growth Parameters and Anion Accumulation in S. altissima Organs
Quantitative analyses were completed to determine the effects of low nitrate (0.5 mM NO 3 − ) and high nitrate (15 mM NO 3 − ) conditions and NaCl treatments on the overall growth of S. altissima plants after 6 weeks in hydroponics.
According to expectations, the growth of plants under low nitrate conditions was essentially reduced compared to the conditions when plants grew under high nitrate conditions (Figure 1).Addition of NaCl to the nutrient solution stimulated growth of the euhalophyte under both low nitrate (0.5 mM NO 3 − ) and high nitrate (15 mM NO 3 − ) conditions; the stimulation was strikingly significant when 250 mM NaCl was added to the ambient medium with 15 mM NO 3 − .Under low nitrate conditions, the growth of plants was maximal under 500 mM NaCl added (Figure 1).
Measurements of nitrate contents in organs of Suaeda plants that grew under different concentrations of nitrate and chloride (NaCl) in the medium demonstrated that nitrate accumulated in high amounts in leaves and stems of plants growing in the medium with high nitrate and without NaCl (Figure 2a).Addition of 250 mM NaCl to the nutrient solution led to considerable drop in nitrate contents in organs of the euhalophyte.The further increase in NaCl in the nutrient solution resulted in the gradual decrease in nitrate in roots and leaves of Suaeda but nitrate contents remained about the same in stems of the plants.High nitrate contents in organs of euhalophyte grown in the nutrient solution without NaCl could be explained by the strategy of S. altissima to accumulate ions in vacuoles of cells in leaves and stems to maintain their low osmotic potential [33].Sodium and chloride ions are accumulated in vacuoles of leaves and stems when NaCl is present in the nutrient solution while K + and nitrate presumably substitute them in the absence of NaCl (Figures 2 and 3).
The nitrate contents in the organs of S. altissima plants were low for low nitrate concentrations (0.5 mM) in the medium (Figure 2b), it corresponded to the reduced growth of the euhalophyte (Figure 1b,d).However, under the conditions of low nitrate in the nutrient solution, the increase in NaCl in the medium did not decrease but slightly increased total nitrate contents in the organs of the plants (Figure 2b), suggesting the functioning of specialized high-affinity nitrate transporting systems in roots of S. altissima that are able to bind and transport nitrate against the background of high external chloride concentrations.Measurements of nitrate contents in organs of Suaeda plants that grew under different concentrations of nitrate and chloride (NaCl) in the medium demonstrated that nitrate accumulated in high amounts in leaves and stems of plants growing in the medium with high nitrate and without NaCl (Figure 2a).Addition of 250 mM NaCl to the nutrient solution led to considerable drop in nitrate contents in organs of the euhalophyte.The further increase in NaCl in the nutrient solution resulted in the gradual decrease in nitrate in roots and leaves of Suaeda but nitrate contents remained about the same in stems of the plants.High nitrate contents in organs of euhalophyte grown in the nutrient solution without NaCl could be explained by the strategy of S. altissima to accumulate ions in vacuoles of cells in leaves and stems to maintain their low osmotic potential [33].Sodium and chloride ions are accumulated in vacuoles of leaves and stems when NaCl is present in the nutrient solution while K + and nitrate presumably substitute them in the absence of NaCl (Figures 2 and 3).The nitrate contents in the organs of S. altissima plants were low for low nitrate concentrations (0.5 mM) in the medium (Figure 2b), it corresponded to the reduced growth of the euhalophyte (Figure 1b,d).However, under the conditions of low nitrate in the nutrient solution, the increase in NaCl in the medium did not decrease but slightly increased total nitrate contents in the organs of the plants (Figure 2b), suggesting the functioning of specialized high-affinity nitrate transporting systems in roots of S. altissima that are able to bind and transport nitrate against the background of high external chloride concentrations.

Identification of the Full-Length Coding Sequences SaNRT2.1 and SaNRT2.5 for High-Affinity Nitrate Transporters and In Silico Analysis of the Protein Structures
Earlier, we identified the partial coding sequences (CDSs) of SaNRT2.1 and SaNRT2.5 (GenBank IDs: MK580128.1 and MK580129.1,accordingly), genes of high-affinity nitrate transporters from S. altissima [37].Identification of the partial CDS of S. altissima NRT2 genes was carried out assuming similarity of putative S. altissima genes with homologous genes from the halophytes S. fruticosa and S. glauca, which are closely related to S. altissima.Coding nucleotide sequences of S. fruticosa and S. glauca homologous genes were obtained by in silico analysis of the de novo assembled transcriptomes of these halophytes.The  Earlier, we identified the partial coding sequences (CDSs) of SaNRT2.1 and SaNRT2.5 (GenBank IDs: MK580128.1 and MK580129.1,accordingly), genes of high-affinity nitrate transporters from S. altissima [37].Identification of the partial CDS of S. altissima NRT2 genes was carried out assuming similarity of putative S. altissima genes with homologous genes from the halophytes S. fruticosa and S. glauca, which are closely related to S. altissima.Coding nucleotide sequences of S. fruticosa and S. glauca homologous genes were obtained by in silico analysis of the de novo assembled transcriptomes of these halophytes.The short-read RNA arrays for the transcriptome assembling were taken by us from the BioProject database, portal NCBI (Acc.No. #PRJNA279962, https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA279962, accessed on 22 March 2024, and #PRJNA295637, https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA295637, accessed on 22 March 2024).
Here, based on the partial SaNRT2.1 and SaNRT2.5 CDSs, we obtained 3 ′ -and 5 ′ -end sequences of their cDNAs (Figure 4a,b).Then, using the experimental approach described in "Materials and Methods", the complete cDNA sequences for the genes SaNRT2.1 and SaNRT2.5 were obtained from S. altissima (Figure 4c,d   In silico analysis carried out using the online resource InterPro (version 98.0, http:// www.ebi.ac.uk/interpro/, accessed on 22 March 2024) as well as Protein BLAST at the NCBI portal (https://blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on 22 March 2024) confirmed that the two identified transporters belong to the family of high-affinity nitrate transporters NRT2.In a phylogenetic tree based on amino acid sequences, SaNRT2.1 is located within a clade with NRT2.1 proteins from other Amaranthaceae-Amaranthus tricolor, Beta vulgaris, Chenopodium quinoa, Spinacea oleracea (Figure 5a).Accordingly, SaNRT2.5 lies in a clade with NRT2.5 proteins from the same plant species (Figure 5a).
The online service WoLF PSORT II predicted that both SaNRT2.1 and SaNRT2.5 resided in the plasma membrane.According to the topology model predicted using DeepTMHMM software (version 1.0.24),both SaNRT2.1 and SaNRT2.5 are integral membrane proteins forming 12 hydrophobic transmembrane helices (TMH); both N-and C-ends are in the cytoplasmic compartment (Figure 5b).Every six transmembrane helixes forming a group are connected by a central cytoplasmic loop, which is relatively short in Suaeda NRT2s.Such topology is generally typical for proteins of the MFS (major facilitator superfamily) [38].MFS transporters are secondary carriers consisting of a single polypeptide capable of transporting small molecules of solutes in response to chemiosmotic ion gradients.They function as uniporters, symporters or antiporters.MFS proteins contain 12 transmembrane regions.Proteins of this superfamily are involved in the absorption of nitrate from the soil by plant roots.The SaNRT2.1 and SaNRT2.5 sequences were aligned with NRT2 sequences from some other plants (Figure 6).The alignment reveals a number of conservative motifs in the polypeptide chains and among them the MFS-conserved motif G-xxx-D-xx-G-x-R and nitrate-nitrite transporter family motif G-W/L-G-N-M/L-G-G-G [39].Table 1 shows the The SaNRT2.1 and SaNRT2.5 sequences were aligned with NRT2 sequences from some other plants (Figure 6).The alignment reveals a number of conservative motifs in the polypeptide chains and among them the MFS-conserved motif G-xxx-D-xx-G-x-R and nitrate-nitrite transporter family motif G-W/L-G-N-M/L-G-G-G [39].Table 1 shows the similarity of proteins SaNRT2.1 and SaNRT2.5 to each other and to proteins of the NRT2 family from A. thaliana.Similarity of SaNRT2.1 to SaNRT2.5 is not high: about 50%.Protein SaNRT2.1 demonstrates higher similarity to Arabidopsis NRT2 proteins with the exception of the AtNRT2.7 than to halophytic protein SaNRT2.5.The similarity of SaNRT2.5 to Arabidopsis NRT2 proteins is about the same as to SaNRT2.1.It is worth noting that AtNRT2.5 is also the most divergent protein of the whole AtNRT2 protein family where the genes are expressed in roots (Table 1).We investigated expression of SaNRT2.1 and SaNRT2.5 in organs of S. altissima under different nitrate concentrations in the NS and different salinities.Observed patterns of SaNRT2.1 and SaNRT2.5 expressions were different (Figures 7 and 8).
SaNRT2.5 was expressed mainly in roots of S. altissima; the expression of the gene in the other organs of S. altissima was negligibly low (Figure 7b).The level of SaNRT2.5 expression in the roots was essentially influenced by concentrations of nitrate and chloride in the nutrient solution.In the absence of NaCl in the nutrient solution, the expression of SaNRT2.5 was significantly reduced at high nitrate concentrations (Figure 7b) and was nearly completely suppressed with rising NaCl concentrations (Figure 8b).On the contrary, the expression of SaNRT2.5 in the roots of plants that were grown under nitrate deficiency dramatically increased with the rise in NaCl; the maximum expression was achieved at 500 mM NaCl, similar to the expression of SaNRT2.1 (Figure 8b).Expression of SaNRT2.5 in other S. altissima organs remained negligibly low as the concentration of NaCl in the medium increased (Figure 8d,f) The levels of SaNRT2.1 expression were comparable in all the studied organs; in roots, leaves and stems of the halophyte; independently of nitrate supply (low or high supply) when NaCl was absent in the nutrient solution (Figure 7a).An increase in NaCl concentrations in the nutrient solution changed the pattern.The expression of SaNRT2.1 in the roots of plants under low nitrate essentially increased; it reached maximum at 500 mM NaCl (Figure 8a).The expression of SaNRT2.1 changed insignificantly in leaves and stems under the conditions (Figure 8c,e).Under high nitrate concentrations in the nutrient solution, SaNRT2.1 expression in roots increased but remained at a low level compared to the expression observed at low nitrate concentration; the expression of SaNRT2.1 in leaves and stems was nearly not influenced by the treatment (Figure 8a,c,e).
SaNRT2.5 was expressed mainly in roots of S. altissima; the expression of the gene in the other organs of S. altissima was negligibly low (Figure 7b).The level of SaNRT2.5 expression in the roots was essentially influenced by concentrations of nitrate and chloride in the nutrient solution.In the absence of NaCl in the nutrient solution, the expression of SaNRT2.5 was significantly reduced at high nitrate concentrations (Figure 7b) and was nearly completely suppressed with rising NaCl concentrations (Figure 8b).On the contrary, the expression of SaNRT2.5 in the roots of plants that were grown under nitrate deficiency dramatically increased with the rise in NaCl; the maximum expression was achieved at 500 mM NaCl, similar to the expression of SaNRT2.1 (Figure 8b).Expression of SaNRT2.5 in other S. altissima organs remained negligibly low as the concentration of NaCl in the medium increased (Figure 8d,f) It should be noted that the level of SaNRT2.5 expression was 10 times higher than the level of SaNRT2.1 expression when comparing expression of SaNRT2.1 and SaNRT2.5 genes in roots of S. altissima plants that were grown in the medium with NaCl under nitrate deficiency (Figure 8a,b).It should be noted that the level of SaNRT2.5 expression was 10 times higher than the level of SaNRT2.1 expression when comparing expression of SaNRT2.1 and SaNRT2.5 genes in roots of S. altissima plants that were grown in the medium with NaCl under nitrate deficiency (Figure 8a,b).

Experiments on Functional Complementation of Yeast Mutant ∆ynt1 by SaNRT2.1 and SaNRT2.5 Expression in H. polymorpha Cells
In order to demonstrate the nitrate transporting function of the SaNRT2.1 and SaNRT2.5 proteins, knockout mutant strain ∆ynt1 of the methylotrophic yeast H. polymorpha was transformed with yeast integrative vector constructs pCHLXSaNRT2.1 or pCHLXSaNRT2.5,carrying the coding sequences SaNRT2.1 or SaNRT2.5, respectively.Unlike the yeast wildtype (WT) strain, the growth of the mutant ∆ynt1 strain lacking the only high-affinity nitrate transporter YNT1 was suppressed on a minimal SD medium containing NO 3 − at concentrations ranging from 0.2 to 5 mM (Figure 9).Unfortunately, there was no noticeable recovery in the growth of the mutant ∆ynt1 strain transformed by the vectors with coding sequences of heterologous nitrate carriers SaNRT2.1 or SaNRT2.5 (Figure 9).The reasons for this will be discussed in the Discussion Section.

SaNRT2.5 Expression in H. polymorpha Cells
In order to demonstrate the nitrate transporting function of the SaNRT2.1 and SaNRT2.5 proteins, knockout mutant strain Δynt1 of the methylotrophic yeast H. polymorpha was transformed with yeast integrative vector constructs pCHLXSaNRT2.1 or pCHLXSaNRT2.5,carrying the coding sequences SaNRT2.1 or SaNRT2.5, respectively.Unlike the yeast wild-type (WT) strain, the growth of the mutant Δynt1 strain lacking the only high-affinity nitrate transporter YNT1 was suppressed on a minimal SD medium containing NO3 − at concentrations ranging from 0.2 to 5 mM (Figure 9).Unfortunately, there was no noticeable recovery in the growth of the mutant Δynt1 strain transformed by the vectors with coding sequences of heterologous nitrate carriers SaNRT2.1 or SaNRT2.5 (Figure 9).The reasons for this will be discussed in the Discussion Section.

Discussion
Nitrogen is an essential and one of the most important mineral nutrients for plants.The growth of S. altissima under the controlled experimental conditions of hydroponics demonstrated that low concentrations of nitrate in the nutrient solution (0.5 mM) expectedly reduced plant growth, by about five times in fresh and dry weights of shoots and stems, but less for roots, compared to plants growing at high nitrate concentrations (15

Discussion
Nitrogen is an essential and one of the most important mineral nutrients for plants.The growth of S. altissima under the controlled experimental conditions of hydroponics demonstrated that low concentrations of nitrate in the nutrient solution (0.5 mM) expectedly reduced plant growth, by about five times in fresh and dry weights of shoots and stems, but less for roots, compared to plants growing at high nitrate concentrations (15 mM) (Figure 1).Concentrations of nitrate in S. altissima plant organs were also markedly reduced under low nitrate, by 5 times for roots and by over 20 times for leaves and stems (Figure 2a,b).However, contrary to glycophytes, the growth of euhalophyte S. altissima was significantly stimulated by increasing NaCl concentrations in the nutrient solution, peaking at 250 mM NaCl for high nitrate supply and even at 500 mM NaCl for low nitrate (Figure 1).
Experiments with species of the Suaeda genus, S. physophora and S. salsa, demonstrated that NaCl application significantly increased leaf NO 3 − concentrations under N-sufficient conditions thus indicating that NaCl may have a promoting effect on nitrate uptake in some halophytes [29,40].Different results obtained in our experiments.The concentration of nitrate in S. altissima organs dropped 2-4 times under salinity for high nitrate supply though still remained higher than under low nitrate treatment and did not essentially decrease with increasing salinity up to 750 mM NaCl (Figure 2a).This is the typical effect for most plants under salinity (e.g., [27] for rice and tomato and references therein).However, although increasing salinity treatment did not reduce nitrate concentrations (apart from a slight initial decrease in the roots) in S. altissima organs under low nitrate, an increase was observed in the leaves and stems (Figure 2b), similar to findings with halophytes S. physophora and S. salsa [29,40].This indicates that high-affinity nitrate transporters in the roots of the halophyte are able to take up nitrate under high salinity; when potential competition with Cl − ions is expected, the concentrations of Cl − ions are nearly three orders of magnitude higher than concentrations of nitrate.
The salinity treatment from 0 to 750 mM NaCl influenced the concentrations of other measured ions in S. altissima plants.For high nitrate supply, chloride contents increased nearly linearly in all plant organs with increasing NaCl concentrations in the nutrient solution, while for low nitrate, the concentrations of chloride increased more slowly with signs of saturation at 750 mM NaCl (Figure 2c,d).Lower concentrations of chloride in S. altissima roots and higher in leaves under high nitrate supply, up to twofold at 750 mM NaCl, indicate that the halophyte accumulates chloride ions in leaf vacuoles, confirming the earlier results [33].The deficit of nitrate limited growth and distorted the strategy.Chloride contents were similar for all the organs (apart at 500 mM NaCl) (Figure 2d).Sodium contents in S. altissima organs also increased nearly linearly as the concentration of sodium in the nutrient solution increased up to 750 mM, for both high and low nitrogen supply with two-and-more-fold lower Na + in roots than in leaves (Figure 3a,b).It supported again the preferences of the halophyte to accumulation of ions in leaf vacuoles [33].Concentrations of potassium and K + -to-Na + ratios are more indicative for characterizing the development of salinity stress ( [41,42]; proved for Arabidopsis-Thellungiella pair in [43]; for varieties of pepper in [44]; etc.).The loss of potassium from plant roots under salinity is assumed as one of determinants for salinity tolerance (e.g., reviewed in [42,45]).Potassium concentrations were higher under high nitrate without salinity in all the studied organs of S. altissima (Figure 3c,d).Rising salinity to 250 mM NaCl dropped the K + concentrations under high nitrate in all organs, with a higher decrease in leaves and stems (Figure 3c).The further increase in salinity did not change the K + concentrations, suggesting that the plants are adapted to the new high salinity environment.At low nitrate, the potassium concentration dropped in leaves only and remained about the same as under high nitrate under all the salinity treatments (0-750 mM) (Figure 3d).The K + concentrations under salinity were highest in roots for high nitrate and in roots and stems under low nitrate, suggesting the active role of the organs in ion uptake and transport (Figure 3c,d).
In the present study, the coding sequences of two genes, SaNRT2.1 and SaNRT2.5, for high-affinity nitrate transporters of the NRT2 family were cloned from euhalophyte S. altissima.Newly identified nitrate transporters from S. altissima, SaNRT2.1 and SaNRT2.5, are the putative orthologs of high-affinity nitrate transporters AtNRT2.1 and AtNRT2.5 from A. thaliana.The phylogenetic bioinformatic analysis demonstrated that the proteins SaNRT2.1 and SaNRT2.5 exhibit higher levels of homology to the corresponding proteins from the plants of family Amaranthaceae-Amaranthus tricolor, Beta vulgaris, Chenopodium quinoa, and Spinacea oleracea (Figure 5a).At the same time, the similarity of amino acid sequences between proteins SaNRT2.1 and SaNRT2.5 is not that high (54%).SaNRT2.1 demonstrated higher similarity in amino acid sequences to orthologs from A. thaliana (75%) than to SaNRT2.5 (Table 1).SaNRT2.5 in turn demonstrated a modest similarity to all corresponding orthologs from A. thaliana.
The topological models of SaNRT2.1 and SaNRT2.5 proteins (Figure 5b) predict 12 transmembrane domains in each of the proteins, corresponding to topological of proteins belonging to the MFS (major facilitator superfamily) with 12 transmembrane domains.Proteins of this superfamily are involved in the uptake of nitrate from soil by plant roots.All NRT2s are high-affinity transporters.the available nitrate is low, the highaffinity transport system is activated and plays a leading role in nitrate uptake by a plant [39,46].The observed changes in expression of SaNRT2.1 and SaNRT2.5 under salinity and different (high and low) nitrate concentrations in the nutrient solution (Figure 8) point to potential and highly probable participation of the high-affinity transporters SaNRT2.1 and SaNRT2.5 in nitrate uptake by S. altissima plants under salinity.At the same time, the expression patterns of the genes for the two transporters significantly differ, which could be linked to the functional differences between SaNRT2.1 and SaNRT2.5 in intact S. altissima plants.When NaCl is absent in the medium, the gene SaNRT2.1 is expressed at comparable levels in all studied organs of S. altissima; concentration of nitrate in the nutrient solution (low or high) did not essentially influence the level of expression (Figure 7a).On the other hand, the gene SaNRT2.5 is expressed almost only in the roots; its level of expression is sharply increased under low nitrate (Figure 7b).An increase in NaCl in the nutrient solution from 0 to 750 mM under low nitrate increased the expression of both SaNRT2.1 and SaNRT2.5, especially for roots, from 15 to 150 times at 500 mM NaCl, correspondingly.The expression level of SaNRT2.5 exceeds the expression level of SaNRT2.1 in roots by an order of magnitude (Figure 8a,b).Based on the analysis of the gene expression, it is highly likely that transporter SaNRT2.5 is a main player in ensuring effective nitrate uptake by roots and functions as an essential nitrate transporter to support the growth of adult halophyte plants under nitrogen deficiency.The two-threefold increase in expression of the nitrate transporter gene SsNRT2.1 under salinity of 200 mM NaCl and 500 mM NaCl at the background of low (0.5 mM) nitrate was also demonstrated for euhalophyte Suaeda salsa [31].The discovered regular patterns of SaNRT2.1 and SaNRT2.5 expression under NaCl conditions were similar to the patterns of expression for homologous genes in A. thaliana.In particular, four Arabidopsis genes, AtNRT2.1,AtNRT2.4,AtNRT2.5 and AtNRT2.6 showed strong preferential expression in the roots while conditions of low nitrate treatment significantly upregulated expression of AtNRT2.1,AtNRT2.4 and AtNRT2.5 [6,[10][11][12].Nevertheless, under low nitrate conditions, AtNRT2.1 is the main transporter for nitrate uptake and transport in roots [7,11].The expression of AtNRT2.5 is highly induced after a long period of starvation.Under these conditions, AtNRT2.5 becomes the most abundantly expressed gene of the AtNRT2 family in roots and leaves, although AtNRT2.5 transcripts were mainly present in roots and at much lower levels in shoots [6,11,47].AtNRT2.5 expression decreased under conditions of sufficient nitrate supply [7,12].The same pattern was observed in our experiments for SaNRT2.5 (and SaNRT2.1)expressed in roots (Figure 8a,b).
A direct attempt to prove the nitrate transporting function of SaNRT2.1 and SaNRT2.5 proteins from S. altissima was chosen using the heterologous expression system.The mutant strain ∆ynt1 of yeast H. polymorpha is lacking the only nitrate transporter of the organism.Hence, the growth of the ∆ynt1 mutant is suppressed at medium with nitrate as a single nitrogen source while the heterologously expressed nitrate transporters AtNPF6.3 from A. thaliana and SaNPF6.3 from S. altissima was effective in the growth rescue of the mutant [35,48].Unfortunately, the heterologous expression of SaNRT2.1 or SaNRT2.5 genes in the yeast mutant did not rescue its growth at medium with nitrate.The same result was obtained when barley HvNRT2.1 and HvNRT2.2cDNAs were used to complement a ∆ynt1 mutant of H. polymorpha [39].This complementation was only partial because, for unknown reasons, the nitrate uptake activity of the transformants was very low compared to the wild type [39].The reasons for this absence of complementation could be multiple.The simplest ones are that the level of synthesized proteins SaNRT2.1 or SaNRT2.5 is too low in the heterologous systems, such that they are not delivered properly to the cellular yeast membrane, misfolded or not translated at all.A more elegant explanation comes from the fact that some proteins of NRT2 family require partner protein from the family NAR2/NRT3 for their proper functionality as nitrate transporters [47,49,50].The interaction of NRT2 and NAR2 was demonstrated to enhance the nitrate transport activity of a high-affinity transport system [51,52] There are, however, some NRT2 that apparently function without these accessory proteins [50].So, this unsuccessful complementation of yeast ∆ynt1 mutant by heterologous expression of SaNRT2.1 or SaNRT2.5 genes could be explained by the omitted protein of family NAR2/NRT3 from S. altissima in the yeast heterologous expression system.So far, none of the genes for NAR2/NRT3 proteins have been cloned and studied in S. altissima.Further research is needed to elucidate the reasons for lack of activity of SaNRT2.1 and SaNRT2.5 in the yeast heterologous expression system.The identification and characterization of NAR proteins from S. altissima and understanding of their potential interactions with SaNRT2.1 and SaNRT2.5 is one of the ways in this direction.

Plant Material
Seeds of Suaeda altissima (L.) Pall.were harvested from the plants growing on the shores of the salt lake Elton (Volgograd region, Russia).Seeds were germinated in wet sand at 24 • C. Fourteen days after germination, the seedlings were transferred to aerated Robinson-Downton nutrient solution (NS) [53] in 3 L vessels, 4 plants per vessel and 3 vessels per experimental point.NS was supplied with nitrate and chloride as KNO 3 and NaCl, respectively.NS contained 0.5 mM NO 3 − (low-nitrate medium) or 15 mM NO 3 − (high-nitrate medium) and four increasing Cl − concentrations (0, 250, 500, 750 mM) for each NO 3 − condition.Plants were grown in hydroponic conditions at 24 • C, 60-70% relative humidity and under 16/8 h light/dark cycle for 4 weeks (young plants) or 6 weeks (adult plants), depending on the type of experiment.The plants were illuminated with high-pressure sodium lamps DNaZ_400 (Reflux, Novocherkassk, Russia) at a light flux of 300 µmol photons m −2 s −1 .To study the long-term salinity effects on the growth characteristics of Suaeda plants and expression of the nitrate transporter genes in Suaeda organs, NaCl was added to the nutrient solution on the 7th day after the transfer of the seedlings from the wet sand to the vessels.To avoid salt shock, NaCl was added gradually in increments of 50 or 100 mM per day, up to the final concentrations of 250, 500 or 750 mM; no NaCl was added to the NS for control plants.
All chemicals used in this study for preparing Robinson-Downton nutrient solution [53] were of PTC (plant culture-tested) grade or molecular biology grade and manufactured by Central Drug House (P) Ltd. (New Delhi, India).Sodium chloride (extra-pure grade) was from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India).

Yeast Strain and Vectors Used in This Study
Methylotrophic yeast Hansenula polymorpha double-auxotrophic strains DL-1 (leu2 ura3 genotype) (wild-type strain, WT strain) and yeast integrative vectors pCCUR2 and pCHLX were used in this study.The strain DL-1 (leu2 ura3) was transformed with plasmids pCCUR2 and pCHLX carrying the URA and LEU genes, respectively, to ensure the growth of the yeast strains without additional nitrogen sources, leucine and uracil, when performing complementation tests.Plasmids pCCUR2 and pCHLX were kindly provided by Michael Agafonov (Federal Research Center "Fundamentals of Biotechnology", Russian Academy of Sciences, Moscow, Russia).Yeast cells were transformed by the lithium method [54] or by electroporation [55] using an Eppendorf device (Eppendorf, Framingham, MA, USA).H. polymorpha mutant strain ∆ynt1 with a deleted YNT1 gene encoding the only high-affinity nitrate transporter in H. polymorpha was produced from the wild-type H. polymorpha strain DL-1 (leu2 ura3) by us earlier [48].The mutant strain ∆ynt1 (ynt1: BleoR/ZeoR, leu2, ura3) was also transformed with pCCUR2 and pCHLX integrative plasmids carrying the URA and LEU genes, respectively, to ensure the growth of the yeast strains without additional nitrogen sources in the selective media, namely, leucine and uracil, when performing complementation tests.

Plant Organ Fresh and Dry Weight Analysis
Quantitative analyses of the overall growth parameters of S. altissima plants were made after 6 weeks of growing plants in hydroponics.Root, stem and leaf fresh weights (FWs) were measured after removal of the entire plant from the hydroponics system.The respective dry weights (DWs) were measured after drying plant material in drying oven Binder FD115 (Tuttlingen, Germany) at 90 • C for 48 h.Water extracts from the samples of dried S. altissima organs (roots, stems and leaves) were prepared by incubating 100 mg of homogenized samples in 10 mL aliquots of boiling deionized water for 10 min.Concentrations of Cl − and NO 3 − in the extracts were determined using ion-selective electrodes (respectively, Elite-261 and Elite-021, Niko-Analit, Moscow, Russia).

Extraction of Total RNA from Plant Material and First-Strand cDNA Synthesis
For total RNA extraction S. altissima plants after 4 weeks of growing in hydroponics were used.Plant organs (roots, leaves, stems) were sampled (approximately 1 g fresh weight of each sample), frozen in liquid nitrogen and stored at −80 • C for the further use.
Total RNA from S. altissima plant organs was isolated by the hot phenolic method [56] and used as a template for the total first-strand cDNA synthesis.For amplification of the 3 ′ -and 5 ′ -ends of the SaNRT2.1 and SaNRT2.5 transcripts by the Step-Out RACE method, the first strand of cDNA was synthesized on the total RNA template isolated from Suaeda roots using MINT revertase (Evrogen, Moscow, Russia).Full-length cDNAs of the SaNRT2.1 and SaNRT2.5 genes were also amplified on the total RNA template isolated from Suaeda roots.To obtain full-length cDNAs of the SaNRT2.1 and SaNRT2.5 and quantify the representation of the gene transcripts in S. altissima organs, first-strand cDNA synthesis was performed on the total RNA templates using (dT)15 primer and MMLV revertase (Evrogen, Moscow, Russia).

Quantitative Analysis of SaNRT2.1 and SaNRT2.5 Transcripts in S. altissima Organs
Quantitative analysis of SaNRT2.1 and SaNRT2.5 transcripts was performed by qRT-PCR using a LightCycler ® 96 System (Roche Diagnostics Corporation, Indianapolis, IN, USA).The cDNA templates for the amplification of SaNRT2.1 and SaNRT2.5 fragments were synthesized on the total RNAs templates, isolated from the organs of S. altissima plants grown in the NS supplied with various nitrate and NaCl concentrations.A ready-to-use reaction mixture with intercalating dye SYBR Green I for real-time PCR (Evrogen, Moscow, Russia) was used.The S. altissima gene of elongation factor 1 alpha SaeEF1alpha (GenBank ID: MN076325.1)was used as an internal control.The expression of this housekeeping gene has been shown to be constitutive within the different plant organs under changing experimental conditions [58].To amplify the SaeEF1alpha fragment, SaeEF1alfa_F1 and SaeEF1alfa_R1 primers were used.The results obtained were processed using LightCycler 96SW 1.1 software.The relative expression profile was calculated by the 2 −∆∆CT method.For the amplification of SaNRT2.1 and SaNRT2.5 fragments, the primer pairs SaNRT2.1_F1 and SaNRT2.1_R1,and SaNRT2.5_F2and SaNRT2.5_R2were used.The results are based on three biological and three analytical replicates.

Cultivation of H. polymorpha WT Strain and ∆ynt1 Transformants
Cells of H. polymorpha WT strain and mutant ∆ynt1 strain were grown in a rich YPD medium (1% yeast extract, 2% peptone, 2% glucose).After co-transformation with pCHLX and pCCUR2 vectors, yeast cells were grown on a minimal synthetic SD medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 2% glucose) with the addition of 0.5% (NH 4 ) 2 SO 4 as a nitrogen source.All solid media contained 2% agar.
For complementation assay, at first, H. polymorpha WT and ∆ynt1 strains co-transformed with pCCUR2 and pCHLX vectors (with or without SaNRT2.1/SaNRT2.5insert) were grown in 10 mL of minimal SD medium containing 0.5% ammonium sulfate overnight at 37 • C.Then, the obtained cultures were centrifuged for 5 min at 2500 g, the precipitates were washed with sterile water, resuspended in the water and 2 µL suspension samples were plated on agarized SD medium containing KNO 3 instead of ammonium sulfate at concentrations ranging from 0.2 to 5 mM.The resulting samples were incubated at 37 • C for 2-3 days until colonies appeared.
All manipulations with H. polymorpha were performed according to the protocols generally accepted for the yeast [55].Yeast transformants were selected on minimal selective media in the absence of leucine and/or uracil.Transformants that contained the insertion in the genome were validated by PCR with Hp_DL-1_Chr1_R primers for genomic DNA and standard M13_F primers for pCHLX vectors.

Statistical Analysis
The results presented in Figures 1-3, 7 and 8 were deduced from three biological replicates and each of them was performed in three analytical replicates.The significant difference was analyzed by using Student's t-test.A p-value < 0.05 was considered to be statistically significant.Standard deviations are given in the figures.

Conclusions
Concluding, two genes of high-affinity nitrate transporters, SaNRT2.1 and SaNRT2.5, were cloned from the euhalophyte Suaeda altissima, which is able to grow and proliferate at 1 M NaCl.The expression patterns of SaNRT2.1 and SaNRT2.5 were studied for the plants grown under low (0.5 mM) or high (15 mM) nitrate with salinity ranging from 0 to 750 mM NaCl.SaNRT2.1 was expressed in all organs when SaNRT2.5 was expressed exclusively in roots.Under low nitrate in medium, salinity increased the expression of both genes: at 500 mM NaCl, SaNRT2.1 peaked in the roots with a 15-fold rise; SaNRT2.5 peaked in the roots, rising 150-fold.An attempt to demonstrate nitrate transporting activity of SaNRT2.1 or SaNRT2.5 in the yeast heterologous expression system was not successful; hence, future research is aimed at finding the partner protein of the NAR2/NRT3 family from S. altissima and functional characterization of the nitrate transporters SaNRT2.1 and SaNRT2.5.

Figure 1 .
Figure 1.Fresh weights (FWs) (a,b) and dry weights (DWs) (c,d) of organs (roots, leaves, stems) of S. altissima plants grown at high nitrate (15 mM) (a,c) or low nitrate (0.5 mM) (b,d) and at various NaCl concentrations in the nutrient solution.A p-value < 0.05 was considered statistically significant.* p < 0.05.Standard deviations are given.

Figure 2 .
Figure 2. Nitrate (NO3 − ) (a,b) and chloride (Cl − ) (c,d) contents in the organs (roots, leaves, stems) of S. altissima plants grown at high nitrate supply of 15 mM (a,c) or low nitrate supply of 0.5 mM (b,d) and at various NaCl concentrations in the nutrient solution.A p-value < 0.05 was considered statistically significant.* p < 0.05, ** p < 0.01.Standard deviations are given.

Figure 2 .
Figure 2. Nitrate (NO 3 − ) (a,b) and chloride (Cl − ) (c,d) contents in the organs (roots, leaves, stems) of S. altissima plants grown at high nitrate supply of 15 mM (a,c) or low nitrate supply of 0.5 mM (b,d) and at various NaCl concentrations in the nutrient solution.A p-value < 0.05 was considered statistically significant.* p < 0.05, ** p < 0.01.Standard deviations are given.

Figure 3 .
Figure 3. Sodium (Na + ) (a,b) and potassium (K + ) (c,d) contents in the organs (roots, leaves, stems) of S. altissima plants grown at high nitrate supply of 15 mM (a,c) or low nitrate supply of 0.5 mM (b,d) and at various NaCl concentrations in the nutrient solution.A p-value < 0.05 was considered statistically significant.* p < 0.05, ** p < 0.01.Standard deviations are given.

Figure 3 .
Figure 3. Sodium (Na + ) (a,b) and potassium (K + ) (c,d) contents in the organs (roots, leaves, stems) of S. altissima plants grown at high nitrate supply of 15 mM (a,c) or low nitrate supply of 0.5 mM (b,d) and at various NaCl concentrations in the nutrient solution.A p-value < 0.05 was considered statistically significant.* p < 0.05, ** p < 0.01.Standard deviations are given.

Figure 4 .
Figure 4. (a,b): Analysis of 3′-end and 5′-end fragments (indicated by arrows with asterisks) of SaNRT2.1 and SaNRT2.5 coding sequences synthesized on the total cDNA template from S. altissima roots using Step-Out RACE technology.(c,d): Analysis of the full-length SaNRT2.1 (c) and SaNRT2.5(d) coding sequences that were used for further cloning in vector pCHLX.DNA fragments were separated by electrophoresis in 1% agarose gel.M-DNA molecular weight markers.

Figure 4 .
Figure 4. (a,b): Analysis of 3 ′ -end and 5 ′ -end fragments (indicated by arrows with asterisks) of SaNRT2.1 and SaNRT2.5 coding sequences synthesized on the total cDNA template from S. altissima roots using Step-Out RACE technology.(c,d): Analysis of the full-length SaNRT2.1 (c) and SaNRT2.5(d) coding sequences that were used for further cloning in vector pCHLX.DNA fragments were separated by electrophoresis in 1% agarose gel.M-DNA molecular weight markers.

Figure 5 .
Figure 5. (a) An unrooted phylogenetic tree of the SaNRT2.1 and SaNRT2.5 transporters of S. altissima and other plant NRT2 homologs.The phylogenetic tree was built in MEGA 11 using the maximum likelihood method based on the Jones-Taylor-Thornton model.The number of bootstrap replicates was 1000; the values of bootstrap support are indicated near the nodes.The NRT2 protein sequences were extracted from the NCBI portal ((https://www.ncbi.nlm.nih.gov/protein,accessed on 14 March 2024).Names of plant species and protein IDs are given in Table S2.(b) Membrane topology of proteins SaNRT2.1 and SaNRT2.5 predicted using DeepTMHMM software version 1.0.24.Both SaNRT2.1 and SaNRT2.5 form 12 transmembrane helices; N-and C-ends are located in cytoplasm.

Figure 5 .
Figure 5. (a) An unrooted phylogenetic tree of the SaNRT2.1 and SaNRT2.5 transporters of S. altissima and other plant NRT2 homologs.The phylogenetic tree was built in MEGA 11 using the maximum likelihood method based on the Jones-Taylor-Thornton model.The number of bootstrap replicates was 1000; the values of bootstrap support are indicated near the nodes.The NRT2 protein sequences were extracted from the NCBI portal (https://www.ncbi.nlm.nih.gov/protein,accessed on 14 March 2024).Names of plant species and protein IDs are given in Table S2.(b) Membrane topology of proteins SaNRT2.1 and SaNRT2.5 predicted using DeepTMHMM software version 1.0.24.Both SaNRT2.1 and SaNRT2.5 form 12 transmembrane helices; N-and C-ends are located in cytoplasm.

Figure 7 .Figure 7 .
Figure 7. Relative abundance of SaNRT2.1 (a) and SaNRT2.5(b) transcripts in the organs of S. altissima plants grown in the nutrient solution containing low (0.5 mM) or high (15 mM) NO3 − concentrations.There was no NaCl in the medium.The relative abundance of SaNRT2.1 transcripts in the roots of the plants grown in the nutrient medium containing 0.5 mM NO3 − was taken as 100 percent.A p-value < 0.05 was considered to be statistically significant.* p < 0.05.Standard deviations are given.

Figure 8 .
Figure 8. Relative abundance of SaNRT2.1 (a,c,e) and SaNRT2.5 (b,d,f) transcripts in the organs of S. altissima plants (roots (a,b), leaves (c,d), stems (e,f)) grown in the nutrient medium containing low (0.5 mM) or high (15 mM) NO3 − concentrations under increasing NaCl concentrations.The relative abundance of SaNRT2.1 transcripts in the roots of the plants grown in the nutrient medium without NaCl and containing 0.5 mM NO3 − was taken as 100 percent.A p-value < 0.05 was considered to be statistically significant.* p < 0.05.Standard deviations are given.

Figure 8 .
Figure 8. Relative abundance of SaNRT2.1 (a,c,e) and SaNRT2.5 (b,d,f) transcripts in the organs of S. altissima plants (roots (a,b), leaves (c,d), stems (e,f)) grown in the nutrient medium containing low (0.5 mM) or high (15 mM) NO 3 − concentrations under increasing NaCl concentrations.The relative abundance of SaNRT2.1 transcripts in the roots of the plants grown in the nutrient medium without NaCl and containing 0.5 mM NO 3 − was taken as 100 percent.A p-value < 0.05 was considered to be statistically significant.* p < 0.05.Standard deviations are given.

Figure 9 .
Figure 9. Complementation assay of the H. polymorpha mutant strain Δynt1 transformed by pCHLX-SaNRT2.1 or pCHLXSaNRT2.5 constructs during growth on minimal agarized SD medium supplied with different concentrations of nitrate (from 0.2 to 5 mM).Wild-type yeast strain DL-1 and the Δynt1 mutant transformed with the empty vector pCHLX were taken as controls.Approximately 10 5 cells of each strain were plated on Petri dishes and incubated at 37 °C for 3 days.

Figure 9 .
Figure 9. Complementation assay of the H. polymorpha mutant strain ∆ynt1 transformed by pCH-LXSaNRT2.1 or pCHLXSaNRT2.5 constructs during growth on minimal agarized SD medium supplied with different concentrations of nitrate (from 0.2 to 5 mM).Wild-type yeast strain DL-1 and the ∆ynt1 mutant transformed with the empty vector pCHLX were taken as controls.Approximately 10 5 cells of each strain were plated on Petri dishes and incubated at 37 • C for 3 days.