Introduction

There are the large areas of saline-alkali land around the world and this problem is continuing to deteriorate. Salt stress is a limiting factor that severely affects plant growth and development, the crop productivity is also significantly inhibited (Munns and Tester 2008). Almost all plants or crops, which are important to humans, are adversely affected by high concentration of salt (Al-Maskri et al. 2010; Yang and Guo 2018). Therefore, it is especially important for the research of salt-tolerant plants.

Puccinellia tenuiflora is a monocotyledonous halophytic species that extensively distributed in the saline land of the Songnen plain in Northeastern China (Yu et al. 2011). The leaves of P. tenuiflora were very tender and more rich in nutrition, which is a good quality forage for livestock (Wei 2016). Therefore, it is used as a typical material for the study of salt tolerance mechanism, because it is the few species that can survive under multiple salt stresses. The molecular mechanisms of P. tenuiflora which can adapt to salt stress deserve further research.

The high affinity nitrate transporter accessory protein (NAR2) plays a crucial role in nitrate absorption and transport. Plant absorption of nitrogen can be classified as high-affinity transport systems (HATS) and low-affinity transport systems (LATS) depending on their absorptive capacity (Kotur and Glass 2015). NRT2 and NAR2 primarily regulate HATS and are responsible for the transport of nitrates under lower nitrogen concentration in plant growing media (Laugier et al. 2012). NRT2.1 is the major contributor to total HATS activity in NRT2 family (Li et al. 2007). CrNRT2.1 did not transport nitrate alone in Chlamydomonas reinhartii, and required CrNAR2 to co-regulate in transport (Quesada et al. 1994; Zhou et al. 2000). A similar result was found in Arabidopsis thaliana, which has NAR2-like genes named as AtNRT3.1. AtNRT3.1; abundantly expressed and proved to be highly sensitive to nitrate induction (Okamoto et al. 2006; Orsel et al. 2006). In Hordeum vulgare, three CrNAR2-like genes were cloned: HvNAR2.1, HvNAR2.2 and HvNAR2.3; while NO^3- is transported only if HvNRT2.1 and HvNAR2.3 are simultaneously present; neither HvNRT2.1 nor HvNAR2.3 show transport activity when present alone (Tong et al. 2005). These findings indicated that NAR2 is widely found in a variety of higher plants and it will specifically interact with NRT2 in the same species to participate the HATS. Recent studies have shown that OsNAR2.1 overexpression manifested better drought tolerance in rice (Chen et al. 2019).

Salt stress can seriously affect the plant uptake of nitrogen. Transcriptional analyses of P. tenuiflora treated with saline-alkali water showed that PutNAR2 was strongly up-regulated. This indicated the significance of PutNAR2 in P. tenuiflora; due to which the adaptation of this species in the saline-alkali lands can never be ignored (Ye et al. 2019).

The expression of PutNAR2.1 may affect the salt tolerance of P. tenuiflora. Through the analysis of changes in salt tolerance of yeast, protein, and plants due to PutNAR2.1 overexpression, we have a preliminary understanding that PutNAR2.1 improving the salt tolerance of organisms. This research gives further insight into the study of salt-tolerant molecular mechanism P. tenuiflora.

Materials and Methods

Bioinformatics analysis of PutNAR2.1

Seeds of P. tenuiflora were obtained from saline-alkali land in Northeast China. The cDNA was obtained from total RNA of P. tenuiflora using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and the reverse transcription PCR kit (Takara, Tokyo, Japan). The specific primers (PutNAR2.1F: 5'-ATGGCTCGGCAAGGAATGGT-3' and PutNAR2.1R: 5'-TTAGTTGTTCTTCTTGCGCTTC-3') were designed by analyzing the P. tenuiflora transcriptome sequence. The PCR product was amplified with cDNA by connecting the template to plasmid pMD18-T Vector (Takara, Tokyo, Japan), and then sequenced.

Conserved domain of PutNAR2.1 in the NCBI database

The sequence of conserved domain of PutNAR2.1 of cloned gene was analyzed by blast search in NCBI, and the amino acid sequence of PutNAR2.1 protein was found highly similar with other species. The homologous amino acid sequence of protein was compared by DNAMAN software, and the phylogenetic tree was constructed by MEGA7 to observe the relationship between PutNAR2.1 and NAR2.1 of other species.

Real-time quantitative PCR analysis for PutNAR2.1 expression in P. tenuiflora

The expression pattern of PutNAR2.1 under salt stress in P. tenuiflora was examined. Seeds were sown onto half-strength MS medium. The seedlings of one-month age were subjected to various stress treatments (300 mM NaCl, 100 mM Na₂CO₃ or 150 mM NaHCO₃) for 0, 6, 12, 24 or 36 h. The PutNAR2.1 expression in P. tenuiflora seedlings under different stress treatments was detected by RT-qPCR analysis, total RNA was isolated from P. tenuiflora and cDNA was synthesized. Subsequently RT-qPCR analyses were carried out by SYBR green (Takara, Tokyo, Japan) and IQ5 real-time PCR equipment (Bio-Rad, Hercules, CA, USA) with the steps: 95℃ for 30 s, 30 cycles of 95℃ for 5 s, and 55℃ for 30 s. The next steps were added to melt-curve analysis: 95℃ for 15 s, followed by continuously increased from 60 to 95℃. The PutAct2 gene expression was used as control. The forward primer sequence was ActinF: 5′-GGTAACATTGTGCTCAGTGGTGG-3′ and reverse primer sequence was ActinR: 5′-AACGACCTTAATCTTCATGCTGC -3′). PutNAR2.1 RT-qPCR primers were designed by Quant Prime Tool.

To examine the expression pattern of the PutNAR2.1 in P. tenuiflora among the different organs, total RNA was extracted from roots, shoots, leaves, flowers and seeds respectively and cDNA was synthesized. Subsequent RT-qPCR procedure was same as above.

Construction of expression vectors and transformation

The open reading frame (ORF) of the PutNAR2.1 gene was amplified from pMD18T-PutNAR2.1 plasmid DNA with BamH I forward primer 5'-GGATCCATGGCTCGGCAAGGAA-3' (restriction site underlined for all restriction enzymes) and XhoI reverse primer 5-' CTCGAGTTAGTTGTTCTTCTTGCG-3'. The PCR amplified fragment with BamH I and XhoI was recovered and then ligated to the corresponding restriction enzyme site of the pYES2, pGEX-6p-3 and pBI121 vector, respectively. It was finally verified by double enzyme digestion and binary expression vector generating the plasmid pYES2-PutNAR2.1, pGEX-PutNAR2.1 and pBI121-PutNAR2.1. The pGEX-PutNAR2.1 and pGEX-6p-3 plasmid transformed E. Coli BL21 cells were used for the expression of PutNAR2.1 fusion protein in E. coli BL21, and plasmid pGEX-6p-3 was used as the control.

The plasmid DNAs of pYES2-PutNAR2.1 and pYES2 were transformed into the yeast strain INVSC1 (Saccharomyces cerevisiae) using the electric impulse method. The plasmid DNAs of pBI121-PutNAR2.1 was transformed into the Agrobacterium tumefaciens strain EHA105 (Takara, Tokyo, Japan) by electro-transformation, and the A. thaliana (ecotype Columbia) was infected with floral dip method (Clough and Bent 1998).

Salt stress-resistance experiments

Control strain pGEX-6p-3 vector and the transformant strain expressing PutNAR2.1 were grown in Luria-Bertani (LB) liquid medium at 37℃ until the absorbance at 600 nm was 0.5 (OD₆₀₀=0.5). Expression of the PutNAR2.1 protein was induced by 1 mM IPTG for 1 h, and separately added with 0.8 M NaCl, 0.1 M Na₂CO₃ or 0.2 M NaHCO₃. The concentrations were chosen based on the information available from a large number of previous experiments. All the strains grow well in the selected concentration). The cultures were grown with rotary shaking (160 rpm) at 37℃ for 1, 2, 3, 4 and 5 h. The growth rate of strains was monitored by absorbance change at 600 nm using Spectrometer. Data are preliminary for three replicate experiments.

The pYES2-PutNAR2.1 transgenic yeast and pYES2 were cultivated in Yeast extract peptone dextrose medium (YPD) overnight at 30℃. When the bacterial culture concentration reached OD₆₀₀ = 0.6, culture solutions with serial dilutions (10, 10^-1, 10^-2, 10^-3 and 10^-4) were dripped onto YPD agar plates with no treatment (CK), 0.6 M NaCl, 0.8 M NaCl, 10 mM Na₂CO₃, 20 mM Na₂CO₃, 30 mM Na₂CO₃, 20 mM NaHCO₃, 40 mM NaHCO₃ or 60 mM NaHCO₃, respectively.

To observed the effects of seeds germination of T3 generation homozygous transgenic lines #1, #3 and #5 A. thaliana and wild-type (Fig. 7), disinfected seeds were directly placed on half-strength MS agar medium supplemented with nothing else (CK), 100 mM NaCl, 125 mM NaCl, 150 mM NaCl, 3 mM Na₂CO₃, 5 mM Na₂CO₃, 7 mM Na₂CO₃, 3 mM NaHCO₃, 5 mM NaHCO₃ or 7 mM NaHCO₃. The experiment was performed in triplicate independently and photographed after 14 days.

Two-week-old seedlings with similar size were arranged onto half-strength MS medium supplemented with nothing and numerous stress (125 mM NaCl, 150 mM NaCl, 175 mM NaCl, 3 mM Na₂CO₃, 5 mM Na₂CO₃, 7 mM Na₂CO₃, 3 mM NaHCO₃, 5 mM NaHCO₃ or 7 mM NaHCO₃,). Each stress treatment (included control) had three replications mentioned above. The Petri plates were vertically positioned in order to visualize the root growth. Plants were photographed after stress treatment for 7 days.

Statistical analysis

All treatments were performed in triplicates and data were treated for analysis of variance using SPSS for Windows version 11.5.

Results

Bioinformatics analysis of PutNAR2.1

The ORF of PutNAR2.1 was obtained from the cDNA in the P. tenuiflora. The full-length sequence of PutNAR2.1 was 597 bp and encoded 199 amino acids; it contained the conserved domains of the NAR2.1 gene family (Fig. 1). The alignment of PutNAR2.1 amino acid sequence illustrated that it had the highest similarity (88.94%) with TaNAR2.1 protein from T. aestivum (Fig. 2). Phylogenetic tree analysis was used to compare PutNAR2.1 protein with others known homologous NAR2.1 protein from a variety of plants, which revealed that PutNAR2.1 was most closely related to TaNAR2.1 from Triticum aestivum (Fig. 3).

RT-qPCR analysis for PutNAR2.1 expression in P. tenuiflora

The expression of PutNAR2.1 under different salt stresses was analyzed by RT-qPCR. The results verified that the expression of PutNAR2.1 was increased gradually and reached its the highest at 24 h under 300 mM NaCl, 100 mM Na₂CO₃ or 150 mM NaHCO₃, nearly 2.3 times higher than untreated (0 h) under 300 mM NaCl in (Fig. 4A), almost 2.7 times higher than untreated (0 h) under 100 mM Na₂CO₃ in (Fig. 4B), over 3.1 times higher than untreated (0 h) under 150 mM NaHCO₃ in (Fig. 4C). PutNAR2.1 had the highest expression in roots, followed by leaves and seeds, but was quite less in shoots and flowers (Fig. 4D).

PutNAR2.1 expression in E. coli cells under salt stress

To investigate the salt response of PutNAR2.1 in E. coli, the E. coli growth with only pGEX vector and transformants expressing pGEX-PutNAR2.1 were compared. PutNAR2.1 expressing strain and the pGEX vector were inoculated into LB liquid medium, adding different salt stresses respectively when both cell density was measured as OD₆₀₀=0.5.

Fig. 1: Analyzing the conservative domain of PutNAR2.1 in the NCBI database

Fig. 3: Phylogenetic tree of 10 selected plant NAR2.1 protein. The MEGA7 program was used for the construction of phylogenetic trees. Bar represents 0.2 amino acid substitutions per site

Fig. 4: Real-time Quantitative PCR analysis for PutNAR2.1 expression in P. tenuiflora. (A) PutNAR2.1 expression at different times under 300 mM NaCl. (B) PutNAR2.1 expression at different times under 100 mM Na₂CO₃. (C) PutNAR2.1 expression at different times under 150 mM NaHCO₃. (D) PutNAR2.1 expression in different organs of P. tenuiflora

Fig. 2: Alignment of PutNAR2.1 deduced amino acid sequence with other plant species NAR2.1 protein. The amino acid sequence of this transcript had the similarity with that of the TaNAR2.1 protein (GenBank: AAV35210.1, 88.94%) from Triticum aestivum, BdNAR2.1 protein (XP_003575282.1, 84.08%) from Brachypodium distachyon, AtaNAR2.2 protein (XP_020163322.1, 85.43%) from Aegilops tauschii subsp. Tauschii, HvNAR2.1 protein (AAP31850.1, 86.43%) from Hordeum vulgare subsp. Vulgare, DoNAR2.1 protein (OEL38054.1, 71.51%) from Dichanthelium oligosanthes, PmNAR2.1 (GenBank: RLM78325.1, 70.39%) protein from Panicum miliaceum, SiNAR2.1 protein (XP_004952978.1, 65.84%) from Setaria italica, OsNAR2.1 protein (XP_015623791.1, 65.12%) from Oryza sativa Japonica Group, ZmNAR2.1 protein (GenBank: AAY40796.1, 67.05%) from Zea mays, SbNAR2.1 protein (XP_002454118.1 , 68.00%)

Fig. 5: The bacterial concentration of pGEX and pGEX-PutNAR2.1 at different times in OD 600 nm under salt stresses. CK: no salt treatment

Fig. 6: Growth of PutNAR2.1 transgenic yeast cells under salt stress. Ten-fold dilutions of yeast cells containing pYES2 (upper line) and pYES2-PutNAR2.1 vector (lower line) were spotted onto solid YPG media supplemented with the indicated stresses. No treatment is a control (CK)

Sorghum bicolor

When both of the stains inoculated in LB liquid medium (CK), the OD₆₀₀ values of the control strain and the transgenic strain after culture for 1 h were 0.68 and 0.67, respectively, both of them had the maximum OD₆₀₀ of 2.0 after 5 h of incubation (Fig. 5A). Under 0.8 M NaCl treatment, the OD₆₀₀ values of the control strain was decreased to 0.57. However, the transgenic strain did not decrease after 1 h incubation, while the OD₆₀₀ values of the control strain and the transgenic strain after 5 h of culture were noted as 1.38 and 1.62, respectively (Fig. 5B). Under the treatment of 0.1 M Na₂CO₃, the OD₆₀₀ values of the control strain and the transgenic strain were 0.29 and 0.48 after 1 h, the OD₆₀₀ values were 0.69 and 1.45 after 5 h, respectively (Fig. 5C). Under 0.2 M NaHCO₃ treatment, control and transgenic strain were cultured, with OD₆₀₀ values of 0.41 and 0.65 after 1 h, these values were 0.98 and 1.67 after 5 h, respectively (Fig. 5D).

PutNAR2.1 transgenic yeast response to salinity

Salt stress types induced expression of PutNAR2.1 in transgenic yeast was investigated. The growth of PutNAR2.1 transgenic yeast cell and pYES2 was compared at five serial dilutions for different salt treatments (corresponding to five columns in each panel in Fig. 6). The control was no treatment (CK). The growth of both pYES2 (upper line) and pYES2-PutNAR2.1 vector (lower line) transgenic yeasts showed no significant difference. However, the growth of yeasts had the most drastic change when salt treatment was applied. The transgenic yeasts grew significantly better than control in 0.8 M NaCl, 20 mM Na₂CO₃ and 40 mM NaHCO₃ treatments. When the concentration was increased to 30 mM Na₂CO₃ or 60 mM NaHCO₃ the transgenic yeasts grew as before, but non-transgenic yeasts could hardly grow.

Identification of PutNAR2.1 transgenic A. thaliana plant

Fig. 7: Identification of transgenic A. thaliana lines by contrasting the expression of PutNAR2.1 in the wild-type and transgenic lines using RT-qPCR. WT: wild-type. #1–#7: PutNAR2.1 transgenic A. thaliana lines

Fig. 8: Seed germination in A. thaliana wild-type, PutNAR2.1 transgenic plants under different stresses. Seed germination on medium supplemented with 0 mM (CK), 100 mM NaCl, 125 mM NaCl, 150 mM NaCl, 3 mM NaHCO₃, 5 mM NaHCO₃ and 7 mM NaHCO₃, 3 mM Na₂CO₃, 5 mM Na₂CO₃ and 7 mM Na₂CO₃.WT: Wild-type A. thaliana. #1, #3 and #5: PutNAR2.1 transgenic lines

The expression of PutNAR2.1 transgenic A. thaliana plant was identified via RT-qPCR analysis (Fig. 7). The expression level of PutNAR2.1 in seven randomly selected T3 transgenic A. thaliana was higher than its wild-type counterparts. PutNAR2.1 expression in the transgenic lines #1- #7 had 31, 29, 42, 25, 30, 12 and 13 times higher than that in wild-type plants, respectively. Among transgenic lines, #1, #3 and #5 indicated higher expression level of PutNAR2.1, and were selected for further research.

The T3 PutNAR2.1 transgenic A. thaliana which exhibited higher levels of PutNAR2.1 (#1, #3, #5) and wild-type seeds were placed on half-strength MS supplemented with no stress (CK) and salt stresses (Fig. 8). Seeds of transgenic and wild-type plants exhibited no difference when they were germinated on half-strength MS directly (CK). With 100 mM NaCl, 3 mM NaHCO₃ or 5 mM NaHCO₃ treatment, seeds of transgenic plants had bigger leaves than the wild-type, but germination of both was similar. Transgenic plant seeds were germinated 1–2 days earlier than wild-type on the medium containing 125 mM NaCl, 3 mM Na₂CO₃ 5 mM Na₂CO₃ or 7 mM NaHCO₃, and the transgenic A. thaliana growth was obviously better. The germination of the wild-type plants was suppressed significantly under 150 mM NaCl and 7 mM Na₂CO₃; a few seeds did not germinate and the leaves of wild-type seedlings were severely curled with light color. However, all the seeds of the transgenic A. thaliana lines germinated and remained green. These results demonstrated that transgenic lines had significantly higher salt tolerance compared to wild-type plants.

Fig. 9: Seedlings growth between A. thaliana wild-type and PutNAR2.1 transgenic plants under different stresses. Seedlings growth on half-strength MS medium supplemented with 0 mM (CK), 125 mM NaCl, 150 mM NaCl, 175 mM NaCl, 3 mM NaHCO₃, 5 mM NaHCO₃ and 7 mM NaHCO₃, 3 mM Na₂CO₃, 5 mM Na₂CO₃ and 7 mM Na₂CO₃. WT: Wild-type A. thaliana. #1, #3 and #5: PutNAR2.1 transgenic lines

Furthermore, PutNAR2.1 transgenic line and wild-type grown on half-strength MS medium with no treatment (CK) or salt stresses for 2 weeks were tested at the seedling stage (Fig. 9). Under normal growth condition, the PutNAR2.1 transgenic lines and wild-type seedlings showed no significant morphological or developmental abnormalities. Under increased NaCl treatment, in the wide-type plants, the leaf margins turned brown and the color became darker. When seedlings were grown on half-strength MS medium containing Na₂CO₃ stress, the cotyledons of wild-type seedlings were smaller compared with PutNAR2.1 transgenic lines and most wild-type leaves turned white. Data further showed that PutNAR2.1 was induced by salt stress and exhibited a positive response to salt stress. Therefore, the PutNAR2.1 gene is involved in the response to salt stress, expression of PutNAR2.1 gene can increase plant tolerance to salt stress.

Discussion

Reportedly P. tenuiflora is one of the few plants that can survive on saline-alkali land (Zhang et al. 2013). The gene related to salt and alkali stress was cloned from the P. tenuiflora and the study of its gene function is helpful to explore the molecular mechanism of salt and alkali resistance of the species (Ye et al. 2019).

The NAR2 protein is mostly studied as nitrogen transport (Yan et al. 2011; Chen et al. 2019). In recent years, NAR2.1 gene has been found to be related to stress tolerance and NAR2.1 was described as novel potato drought-responsive genes (Pieczynski et al. 2018). NaCl salinity reduces the absorption of nitrogen by roots (Rubinigg et al. 2003; Yousif et al. 2010). PutNAR2.1 transcript level was upregulated significantly in transcriptome analyses of P. tenuiflora treated with water extracts from the saline-alkali soils. In this study, the possibility of PutNAR2.1 participating in regulation of the response to salt stress was examined by using PutNAR2.1 E. coli transformants, PutNAR2.1 transgenic yeasts and PutNAR2.1 transgenic A. thaliana lines.

The PutNAR2.1 has been cloned from P. tenuiflora. The amino acid sequence of PutNAR2.1 had the similarity with other plant species. Drought stress can induce OsNAR2.1 high expression (Chen et al. 2019). In this research, the expression of PutNAR2.1 started to increase gradually at 6 h and reached the highest value at 24 h in P. tenuiflora under 300 mM NaCl, 100 mM Na₂CO₃ or 150 mM NaHCO₃ stress, indicated its gradual induction by salt stress. NAR2 was mainly expressed in root (Orsel et al. 2002; Lupini et al. 2016; Luo et al. 2018), while the highest expression of the PutNAR2.1 was also found in roots of P. tenuiflora, under salt stresses. Thus PutNAR2.1 may play a defense role when roots are exposed to salt stress.

It takes a long time to identify the related functions after the gene is transferred into the plant. The effect of salt stress on the growth of control strain and PutNAR2.1 expressing strain was examined in LB medium. The OD₆₀₀ value of the PutNAR2.1 expressing strain after 5 h culture was higher than that of the control strain. It is indicated that PutNAR2.1 can protect E. coli to against the salt stress from the environment. However, the prokaryotes may not have the function of protein completely consistent with that in eukaryotes. The growth of control yeasts was inhibited by NaCl, Na₂CO₃ or NaHCO₃ stress, while PutNAR2.1 transgenic yeast grew well. These results revealed that the function of PutNAR2.1 in eukaryotes was similar to that in the prokaryotes.

Chen et al. (2019) reported that OsNAR2.1 overexpressing plant line increased the grain yield by about 26.6% compared to wild-type in limited irrigation conditions. To observe PutNAR2.1 response to salt stress, comparison was made for seeds germination and seedlings growth between transgenic lines and wild-type counterparts. The germination and seedlings growth of wild-type were quite more reduced than that of transgenic lines under salt stress. PutNAR2.1 transgenic line showed better growth compared with wild-type under stress of various salts at different plant development stages. The data proved that expression of PutNAR2.1 helped the plants to resist the salt stress better.

Nitrate uptake was closely related to plant growth and development, and the ability of nitrate uptake by roots was decreased under salt stress. PutNAR2.1 is the key gene for nitrate uptake and transport in P. tenuiflora. Under salt stress, P. tenuiflora can regulate the expression of NAR to improve the ability of nitrate uptake and utilization, thus enhanced the resistance to salt stress.

Conclusion

Tolerance of E. coli, yeast and plant to salt stresses could be greatly enhanced with the high expression of PutNAR2.1. Based on the previous research progress of NAR2.1 gene and the results of this experiment, it is conjectured that PutNAR2.1 may be used as an auxiliary protein to participate in the high-affinity nitrogen absorption system of plants under different salinity treatment. Consequently, PutNAR2.1 overexpression can help plants to resist abiotic stress in the environment, but determination of specific working mechanism needs further studies.

Acknowledgments

This work was supported by the Heilongjiang Province Nature Science Foundation (LH2019C011), Key Laboratory Open Fund of Saline-alkali Vegetation Ecology Restoration (SAVER1701) and the National Natural Science Foundation of China (No. 31070616, No. 31500317).

Author Contributions

JSM planned the experiments and contributed reagents/materials/analysis tools; ZGQ, CSY, XY and HH performed the experiments; ZGQ statistically analyzed the data and made illustrations; JSM and ZGQ wrote the manuscript.

References

Al-Maskri A, L Al-Kharusi, H Al-Miqbali (2010). Effects of salinity stress on growth of lettuce (Lactuca sativa) under closed-recycle nutrient film technique. Intl J Agric Biol 12:377‒380

Chen J, T Qi, Z Hu, X Fan, L Zhu, MF Iqbal, X Yin, G Xu, X Fan (2019). OsNAR2.1 positively regulates drought tolerance and grain yield under drought stress conditions in rice. Front Plant Sci 10; Article 197

Clough SJ, AF Bent (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735‒743

Rubinigg M, F Posthumus, M Ferschke, JTM Elzenga, I Stulen (2003). Effects of NaCl salinity on ¹⁵N-nitrate fluxes and specific root length in the halophyte Plantago maritima. Plant Soil 250:201‒213

Kotur Z, AD Glass (2015). A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high-affinity nitrate influx in Arabidopsis thaliana. Plant Cell Environ 38:1490‒1502

Laugier E, E Bouguyon, A Mauries, P Tillard, A Gojon, L Lejay (2012). Regulation of high-affinity nitrate uptake in roots of Arabidopsis depends predominantly on posttranscriptional control of the NRT2.1/NAR2.1 transport system. Plant Physiol 158:1067‒1068

Li W, Y Wang, M Okamoto, NM Crawford, MY Siddiqi, AD Glass (2007). Dissection of the AtNRT2.1: AtNRT2.2 inducible high-affinity nitrate transporter gene cluster. Plant Physiol 143:425‒433

Luo B, J Chen, L Zhu, S Liu, B Li, H Lu, G Ye, G Xu, X Fan (2018). Overexpression of a high-affinity nitrate transporter OsNRT2.1 increases yield and manganese accumulation in rice under alternating wet and dry condition. Front Plant Sci 9; Article 1192

Lupini A, F Mercati, F Araniti, AJ Miller, F Sunseri, MR Abenavoli (2016). Nar2.1/nrt2.1 functional interaction with NO₃^- and H⁺ fluxes in high-affinity nitrate transport in maize root regions. Plant Physiol Biochnol 102:107‒114

Munns R, M Tester (2008). Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651‒681

Okamoto M, A Kumar, W Li, Y Wang, MY Siddiqi, NM Crawford, AD Glass (2006). High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1. Plant Physiol 140:1036‒1046

Orsel M, A Krapp, F Daniel-Vedele (2002). Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression. Plant Physiol 129:886‒896

Orsel M, F Chopin, Q Leleu, SJ Smith, A Krapp, F Daniel-Vedele, AJ Miller (2006). Characterization of a two-component high affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction. Plant Physiol 142:1304‒1317

Pieczynski M, A Wyrzykowska, K Milanowska, D Boguszewska-Mankowska, B Zagdanska, W Karlowski, A Jarmolowski, Z Szweykowska-Kulinska (2018). Genomewide identification of genes involved in the potato response to drought indicates functional evolutionary conservation with Arabidopsis plants. Plant Biotechnol J 16:603‒614Quesada A, A Galvan, E Fernandez (1994). Identification of nitrate transporter genes in Chlamydomonas reinhardtii. Plant J 5:407‒419

Tong Y, JJ Zhou, Z Li, AJ Miller (2005). A two‐component high‐affinity nitrate uptake system in barley. Plant J 41:442‒450

Wei XX (2016). The Identification on Drought-Resistance of 15 Puccinellia germplasm materials in seedling stage. Pratacult Anim Husban 5:18‒24

Yan M, X Fan, H Feng, AJ Miller, Q Sheng, G Xu (2011). Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3 nitrate transporters to provide uptake over high and low concentration ranges. Plant Cell Environ 34:1360‒1372

Yang Y, Y Guo (2018). Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytol 217:523‒539

Ye X, H Wang, X Cao, X Jin, F Cui, Y Bu, H Liu, W Wu, T Takano, S Liu (2019). Transcriptome profiling of Puccinellia tenuiflora during seed germination under a long-term saline-alkali stress. BMC Genomics 20; Article 589

Yousif BS, NT Nguyen, Y Fukuda, H Hakata, Y Okamoto, Y Masaoka, H Saneoka (2010). Effect of salinity on growth, mineral composition, photosynthesis and water relations of two vegetable crops; New Zealand spinach (Tetragonia tetragonioides) and water spinach (Ipomoea aquatica). Intl J Agric Biol 12:211‒216

Yu J, S Chen, Q Zhao, T Wang, C Yang, C Diaz, G Sun, S Dai (2011). Physiological and proteomic analysis of salinity tolerance in Puccinellia tenuiflora. J Proteome Res 10:3852‒3870

Zhang X, L Wei, Z Wang, T Wang (2013). Physiological and molecular features of Puccinellia tenuiflora tolerating salt and alkaline-salt stress. J Integr Plant Biol 55:262‒276

Zhou JJ, E Fernandez, A Galvan, AJ Miller (2000). A high affinity nitrate transport system from Chlamydomonas requires two gene products. FEBS Lett 466: 225‒227