Genome-Wide Identification of the Cation/Proton Antiporter (CPA) Gene Family and Functional Analysis of AtrNHX8 under Salt Stress

Amaranthus tricolor is an important vegetable, and its quality is affected by salt stress. Cation/proton antiporters (CPA) contribute to plant development and tolerance to salt stress. In this study, 35 CPA genes were identified from a genome database for A. tricolor, including 9 NHX, 5 KEA, and 21 CPA2 genes. Furthermore, in A. tricolor, the expression levels of most AtrNHX genes were higher at a low salinity level (50 or 100 mM NaCl) than in the control or 200 mM NaCl treatment. Levels of most AtrNHX genes were elevated in the stem. Moreover, AtrNHX8 was homologous to AtNHX4, which is involved in the regulation of sodium homeostasis and salt stress response. After AtrNHX8 overexpression in Arabidopsis thaliana, seed germination was better, and the flowering time was earlier than that of wild-type plants. Additionally, the overexpression of AtrNHX8 in A. thaliana improved salt tolerance. These results reveal the roles of AtrNHX genes under salt stress and provide valuable information on this gene family in amaranth.

Salt-affected cultivated lands account for a large proportion of global farmland, with more than one-third of irrigated land exhibiting some degree of salinization [8].Salt stress has become one of the most serious abiotic stresses because it can impair soil fertility and crop growth and development substantially [9], affecting crop yield and quality as well as agricultural sustainability [10,11].When plants are exposed to salt stress, they must constantly adjust their cellular ion contents to maintain K + and Na + homeostasis [12].The homeostasis of cations and pH by exchanging Na + , K + , or Li + for H + is regulated by monovalent cation-proton antiporters (CPAs) in the process of salt stress [13].CPAs can be classified into two categories: The CPA1 family (including Na + /H + NHX antiporters) and the CPA2 family (including Cation/H + (CHX) and K + efflux antiporters (KEA)).The NHX type is a particularly important CPA1 exchanger involved in Na + transport and therefore salinity tolerance [9].
Plants 2024, 13, 1701 2 of 20 The NHX gene family includes eight members (AtNHX1-8) in Arabidopsis [14].AtNHX1-4, AtNHX5-6, and AtNHX7-8 can be separated into the vacuole (Vac) subfamily, plasma membrane (PM) subfamily, and endosome nucleolus (Endo) subfamily, respectively, according to their subcellular localization [15][16][17].NHX genes may play an important role in the salt stress response and salt tolerance [9].Under salt stress, NHXs may prevent Na + in the vacuoles of most tissues from entering the cytosol [18].They regulate pH and Na + homeostasis in the cell to reduce the toxic concentration of Na + in the cytosol [19].Studies have reported the responses of various NHX genes to salt stress, such as CmoNHX1 [9] and GbNHX2 [20].BvNHX5 may interact with CBL and CIPK to enhance salt tolerance in Beta vulgaris [21].PgNHXs could play significant roles in the response to salt stress in Punica granatum [22].The LeNHX gene shows increased expression in tomato under salt stress [23].The overexpression of AtNHX1 in Arabidopsis could confer salt tolerance under NaCl stress.The overexpression of OsNHX1 in rice promotes root growth and water uptake under low salt stress [24].Upon treatment with 300 mM NaCl, the transcript levels of AtNHX4 increased initially and then decreased gradually [25].
In the study, we screened and identified members of the AtrCPA gene family from the Amaranthus tricolor genome database.AtrCPAs were comprehensively characterized.The NHX type is a particularly important CPA1 exchanger; accordingly, the expression levels of AtrNHX genes were evaluated under salt stress by qRT-PCR.Finally, the effects of the overexpression of AtrNHX8 were evaluated in Arabidopsis thaliana under salt stress.The results reveal the roles of AtrNHX genes in the response to salt stress and provide a valuable reference to further explore the functions of NHX genes in amaranth.

Identification of Cation-Proton Antiporter Gene Family Members and Analyses of Physicochemical Properties
In total, 35 CPA genes were screened from the Amaranthus genome database [26], including 9 NHXs, 5 KEAs, and 21 CPA2s.All of the genes were renamed according to the order of gene sequence IDs (Table 1).The molecular weight ranged from 25.08 kDa (AtrCPA2-7) to 135.36 kDa (AtrKEA3).The theoretical isoelectric point ranged from 4.58 (AtrCPA2-17) to 9.47 (AtrCPA2-20).All AtrNHX proteins were hydrophobic according to the grand average of hydropathy (GRAVY) value.Only AtrKEA3 and AtrKEA5 had a signal peptide.Thirty-four CPAs had typical transmembrane helix regions, other than AtrKEA2 (Supplementary Figure S1).The prediction of subcellular localization patterns showed that AtrNHX proteins may be localized in the plasma membrane or vacuole.The detailed physical and chemical properties are shown in Table 1.Thirty-five AtrCPA members were divided into three subfamilies, and most AtrCPA members in the same subfamily had similar motif compositions and domains, suggesting that these proteins have similar functions (Figure 1A-C).Motif 8 was found in all three subfamilies; its conservation suggests that it may be useful for the identification of CPAtype proteins in A. tricolor.Each subfamily had unique conserved moftis.Motifs 3,4,6,10,11,and 15 were conserved in NHX-type proteins.Motifs 8, 12, and14 were conserved in the KEA subfamily.Motifs 1, 2, 9, and 12 were conserved in CPA2-type proteins as well.The numbers and lengths of exons and introns differed among the three subfamilies of AtrCPAs (Figure 1D).The gene structures of AtrNHXs and AtrKEAs were relatively complex, while the structure was relatively simple in the AtrCPA2 subfamily.

Promoter Element Analysis
Putative promoter sequences of AtrCPAs were searched against PlantCARE to identify potential cis-acting regulatory elements, with a focus on hormone-and stress-related cis-acting elements (Figure 2).A total of 41 kinds of cis-acting elements were identified, suggesting that the expression of AtrCPAs is regulated by various mechanisms.Additionally, 162 cis-acting elements were related to hormones, including abscisic acid (ABA), auxin (IAA), gibberellin (GA), salicylic acid (SA), and methyl jasmonate (MeJA), and 501 cis-acting elements and binding sites were related to stress, including low temperature, drought, wound, and anaerobic conditions.Among these elements, the contents of antioxidant-and defense-related elements were high.

Promoter Element Analysis
Putative promoter sequences of AtrCPAs were searched against PlantCARE to identify potential cis-acting regulatory elements, with a focus on hormone-and stress-related cis-acting elements (Figure 2).A total of 41 kinds of cis-acting elements were identified, suggesting that the expression of AtrCPAs is regulated by various mechanisms.Additionally, 162 cis-acting elements were related to hormones, including abscisic acid (ABA), auxin (IAA), gibberellin (GA), salicylic acid (SA), and methyl jasmonate (MeJA), and 501 cis-acting elements and binding sites were related to stress, including low temperature, drought, wound, and anaerobic conditions.Among these elements, the contents of antioxidant-and defense-related elements were high.

Chromosomal Localization and Synteny Analysis of AtrCPAs
Based on the location data from the Amaranthus tricolor genome database, 35 AtrCPAs were mapped to 17 chromosomes, with an uneven distribution (Figure 3).Chr1, Chr 2, Chr 5, Chr 7, and Chr 11 each contained three AtrCPA members.

Chromosomal Localization and Synteny Analysis of AtrCPAs
Based on the location data from the Amaranthus tricolor genome database, 35 AtrCPAs were mapped to 17 chromosomes, with an uneven distribution (Figure 3).Chr1, Chr 2, Chr 5, Chr 7, and Chr 11 each contained three AtrCPA members.
Nine segmental duplication events involving ten AtrCPA genes were filtered out.No tandem duplication event was found in the AtrCPA genes (Figure 4A).To further study the origin and divergence of AtrCPA genes, duplication events were evaluated by comparisons between Amaranthus tricolor and Arabidopsis.A total of 12 collinear CPA gene pairs between Arabidopsis and Amaranthus tricolor were identified (Figure 4B).Only AtrNHX5, AtrNHX6, AtrNHX9, AtrKEA5, AtrCPA2-4, AtrCPA2-9, and AtrCPA2-16 had homologues in Arabidopsis, suggesting that other genes arose after the divergence of Arabidopsis.tandem duplication event was found in the AtrCPA genes (Figure 4A).To further study the origin and divergence of AtrCPA genes, duplication events were evaluated by comparisons between Amaranthus tricolor and Arabidopsis.A total of 12 collinear CPA gene pairs between Arabidopsis and Amaranthus tricolor were identified (Figure 4B).Only AtrNHX5, AtrNHX6, AtrNHX9, AtrKEA5, AtrCPA2-4, AtrCPA2-9, and AtrCPA2-16 had homologues in Arabidopsis, suggesting that other genes arose after the divergence of Arabidopsis.

Protein Interaction Analysis
To predict which proteins interact with CPAs, we used the STRING database to construct interaction networks involving Arabidopsis CPA proteins (Figure 5A-C).In the NHX subfamily, the NHX7 and NHX8 proteins showed a strong interaction with other NHX proteins in A. thaliana (Figure 5A).In the KEA subfamily (Figure 5B), the KEA2 and KEA3 proteins were predicted to interact closely with other KEA proteins in A. thaliana.In the CPA2 subfamily (Figure 5C), the CPA-2 and NHX8 proteins were predicted to interact closely with other KEA, CPA2, and NHXs in A. thaliana.Plants 2024, 13, 1701 9 of 20

Phylogenetic Analysis of NHXs
Because NHX genes may play an important role in the salt stress response and salt tolerance [9], we focused on this sub-family.NHX proteins with full-length sequences from Arabidopsis thaliana and Oryza sativa were used to construct a phylogenetic tree based on AtrNHX proteins (Figure 6).The NHX proteins were divided into three clades, clades I, II, and III, with strong support (Bootstrap = 100%).AtrNHX1 was closely related to AtNHX7 and AtNHX8 in Arabidopsis, assigned to clade I; AtrNHX2 and AtrNHX3 were closely related to AtNHX5 and AtNHX6 in Arabidopsis, assigned to clade II; and other AtrNHXs were assigned to clade III, indicating that these genes share similar functions.In particular, AtrNHX8 was closely related to AtNHX4, which is involved in the regulation of sodium homeostasis and salt stress response.

Phylogenetic Analysis of NHXs
Because NHX genes may play an important role in the salt stress response and salt tolerance [9], we focused on this sub-family.NHX proteins with full-length sequences from Arabidopsis thaliana and Oryza sativa were used to construct a phylogenetic tree based on AtrNHX proteins (Figure 6).The NHX proteins were divided into three clades, clades I, II, and III, with strong support (Bootstrap = 100%).AtrNHX1 was closely related to AtNHX7 and AtNHX8 in Arabidopsis, assigned to clade I; AtrNHX2 and AtrNHX3 were closely related to AtNHX5 and AtNHX6 in Arabidopsis, assigned to clade II; and other AtrNHXs were assigned to clade III, indicating that these genes share similar functions.In particular, AtrNHX8 was closely related to AtNHX4, which is involved in the regulation of sodium homeostasis and salt stress response.

qRT-PCR Analysis of AtrNHXs under Salt Stress
The relative expression patterns of AtrNHX genes were analyzed (Figure 7).At a low salinity level (50 mM NaCl), the expression levels of AtrNHX genes were higher than those in the control or 200 mM NaCl groups, other than AtrNHX1 and AtrNHX2, suggesting that a low salinity level is favorable for amranth growth.The expression levels of AtrNHX2, AtrNHX4, AtrNHX6, and AtrNHX8 were higher under 100 mM NaCl than in the control.

qRT-PCR Analysis of AtrNHXs under Salt Stress
The relative expression patterns of AtrNHX genes were analyzed (Figure 7).At a low salinity level (50 mM NaCl), the expression levels of AtrNHX genes were higher than those in the control or 200 mM NaCl groups, other than AtrNHX1 and AtrNHX2, suggesting that a low salinity level is favorable for amranth growth.The expression levels of AtrNHX2, AtrNHX4, AtrNHX6, and AtrNHX8 were higher under 100 mM NaCl than in the control.These results indicated that the genes play a key role in tolerance to moderate salt stress in A. tricolor.These results indicated that the genes play a key role in tolerance to moderate salt stress in A. tricolor.
Figure 7. qRT-PCR analysis of AtrNHX genes under salt stress.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

Analysis of AtrNHX Gene Expression in Different Tissues of Amaranth
A qRT-PCR analysis of NHX genes in different tissues of amaranth indicated that the relative expression levels of AtrNHX9 in leaves were higher than those in the roots.AtrNHX2, AtrNHX3, AtrNHX4, AtrNHX5, AtrNHX7, and AtrNHX9 levels in the stem were higher than those in the root.AtrNHX6 and AtrNHX8 showed high expression in the root (Figure 8).These results showed that AtrNHXs play different roles in different tissues of A. tricolor.qRT-PCR analysis of AtrNHX genes under salt stress.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

Analysis of AtrNHX Gene Expression in Different Tissues of Amaranth
A qRT-PCR analysis of NHX genes in different tissues of amaranth indicated that the relative expression levels of AtrNHX9 in leaves were higher than those in the roots.AtrNHX2, AtrNHX3, AtrNHX4, AtrNHX5, AtrNHX7, and AtrNHX9 levels in the stem were higher than those in the root.AtrNHX6 and AtrNHX8 showed high expression in the root (Figure 8).These results showed that AtrNHXs play different roles in different tissues of A. tricolor.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

Overexpression of AtrNHX8 Enhances Salt Tolerance in Arabidopsis
The seed germination rate of 35S::AtrNHX8 was significantly higher than that of the wild type (WT) under 150 mM NaCl treatment.Furthermore, seed germination in 35S::AtrNHX8 was significantly earlier compared with that of WT under NaCl treatment (Figure 9).Germination was nearly complete in 35S::AtrNHX8 and WT after 2 days and 6 days, respectively.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

Overexpression of AtrNHX8 Enhances Salt Tolerance in Arabidopsis
The seed germination rate of 35S::AtrNHX8 was significantly higher than that of the wild type (WT) under 150 mM NaCl treatment.Furthermore, seed germination in 35S::AtrNHX8 was significantly earlier compared with that of WT under NaCl treatment (Figure 9).Germination was nearly complete in 35S::AtrNHX8 and WT after 2 days and 6 days, respectively.To analyze the salt response in adult plants, 15-day-old seedlings in pots were irrigated with 150 mM NaCl solutions or water as a control.The phenotypic effects of salt treatment for 7 days are shown in Figure 10.The plant height and leaf size under salt stress in WT and 35S::AtrNHX8 lines were shorter and smaller compared with those under nonsalt stress.There were fewer leaves with salt stress than without salt (Figure 10A,B).The contents of chlorophyll a, chlorophyll b, and total chlorophyll in WT Arabidopsis thaliana under salt stress were significantly (p ≤ 0.05) lower than those without salt stress.However, the opposite results were obtained for 35S::AtrNHX8 plants.Furthermore, the chlorophyll content in 35S::AtrNHX8 plants was significantly (p ≤ 0.05) higher than that in the WT (Figure 10C).The carotenoid content in 35S::AtrNHX8 plants was significantly (p ≤ 0.05) higher than that in the WT under salt stress (Figure 10D).
AtrNHX8 gene expression levels in Arabidopsis thaliana leaves were analyzed.The expression of the AtrNHX8 gene was significantly higher in WT Arabidopsis and 35S::AtrNHX8 transformed plants under salt stress than under non-salt stress (Figure 10E).To analyze the salt response in adult plants, 15-day-old seedlings in pots were irrigated with 150 mM NaCl solutions or water as a control.The phenotypic effects of salt treatment for 7 days are shown in Figure 10.The plant height and leaf size under salt stress in WT and 35S::AtrNHX8 lines were shorter and smaller compared with those under non-salt stress.There were fewer leaves with salt stress than without salt (Figure 10A,B).The contents of chlorophyll a, chlorophyll b, and total chlorophyll in WT Arabidopsis thaliana under salt stress were significantly (p ≤ 0.05) lower than those without salt stress.However, the opposite results were obtained for 35S::AtrNHX8 plants.Furthermore, the chlorophyll content in 35S::AtrNHX8 plants was significantly (p ≤ 0.05) higher than that in the WT (Figure 10C).The carotenoid content in 35S::AtrNHX8 plants was significantly (p ≤ 0.05) higher than that in the WT under salt stress (Figure 10D).
AtrNHX8 gene expression levels in Arabidopsis thaliana leaves were analyzed.The expression of the AtrNHX8 gene was significantly higher in WT Arabidopsis and 35S::AtrNHX8 transformed plants under salt stress than under non-salt stress (Figure 10E).(E) represents the relative expression of NHX8 in Arabidopsis thaliana leaves.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

AtrNHX8 Could Promote Flowering in Arabidopsis thaliana
Under salt stress, 35S::AtrNHX8 lines at the 10-leaf period began to flower after 9 days.However, WT plants at the 21-leaf period began to flower after 18 days.Without salt stress, 35S::AtrNHX8 lines at the 10-leaf period began to flower after 9 days of treatment.WT plants at the 16-leaf period began to flower after 13 days of treatment (Figure 11).The results indicated that AtrNHX8 could promote flowering in Arabidopsis thaliana.(E) represents the relative expression of NHX8 in Arabidopsis thaliana leaves.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

AtrNHX8 Could Promote Flowering in Arabidopsis thaliana
Under salt stress, 35S::AtrNHX8 lines at the 10-leaf period began to flower after 9 days.However, WT plants at the 21-leaf period began to flower after 18 days.Without salt stress, 35S::AtrNHX8 lines at the 10-leaf period began to flower after 9 days of treatment.WT plants at the 16-leaf period began to flower after 13 days of treatment (Figure 11).The results indicated that AtrNHX8 could promote flowering in Arabidopsis thaliana.

Discussion
CPAs can be divided into two categories: The CPA1 family, which includes Na + /H + NHX antiporters, and the CPA2 family, which includes CHXs and KEAs.They play key roles in abiotic stress tolerance, especially in responses to salt stress [27].The CPA family has been characterized in several plant species, such as Arabidopsis thaliana [28], Oryza sativa [29], Brassica napus [30], and Raphanus sativus [31].In the present study, 35 CPA genes were identified in the genome of A. tricolor, including 9 NHX genes, 5 KEA genes, and 21 CPA2 genes.These CPA gene counts differed from those in other species, indicating that the CPA gene family underwent duplication events and expansions.A subcellular localization analysis of AtrCPA implied they play multiple functions in plants.
Gene functions can be predicted based on comparisons with previously characterized orthologues [32,33].Gene evolution and potential functional differences were involved in the differences in conserved motifs.Additionally, a phylogenetic analysis showed that the nine members of the NHX family in A. tricolor could be divided into three subfamilies.AtrNHX9 was paired with AtNHX3 in subclade B1.Previous studies have shown that AtNHX3 plays a role in maintaining potassium ion homeostasis during germination and seedling growth in Arabidopsis [34].AtrNHX8 was closely related to AtNHX4, which is involved in the regulation of sodium homeostasis and the salt stress response.These results verified that orthologous proteins have similar functions and that the NHX family genes are relatively conserved.
qRT-PCR analyses of AtrNHX gene family members under salt solution treatment revealed different gene expression trends.As the salt concentration increased, gene expression levels increased and then decreased, consistent with the results of previous studies [35].Upon treatment with 300 mM NaCl, the transcript levels of AtNHX4 increased and then gradually decreased; quantitative real-time PCR analyses showed similar expression patterns [25].However, the knock-out of AtNHX4 could enhance tolerance to salt stress, and Na + contents under high NaCl stress were lower than those in WT plants [25].AtrNHX2 and AtrNHX8 had the highest expression under 100 mM NaCl, and the remaining members were all expressed at 50 mM NaCl.There were differences in the responses of the AtrNHX genes under salt stress.A qRT-PCR analysis revealed that the family members were differentially expressed in various parts, with most family members expressed in the roots and stems, consistent with a previous study showing that NHX genes are highly expressed in the roots under salt stress, thereby enhancing salt tolerance [36].The results of this study provide a basis for further exploration of the NHX gene family and provide a reference for analyses of the mechanism by which NHX genes contribute to the growth of amaranth, responses to abiotic stress, and development in amaranth.
In this study, salt stress inhibited seed germination in amaranth, while 35S::AtrNHX8 Arabidopsis plants showed an increased seed germination rate, earlier germination, and improved salt tolerance.A previous study has shown that salt stress delayed seed germination and reduced the germination rate in Medicago sativa, and the germination rate and germination potential of transformed plants were significantly higher than those of WT plants under salt stress conditions [11].
Both WT Arabidopsis and 35S::AtrNHX8 transformed plants were smaller and showed a lower leaf area and leaf number under the salt stress than in the non-salt stress treatment.Furthermore, the flowering time of the transformed plants was earlier than that of WT Arabidopsis.35S::AtrNHX8-transformed plants showed higher chlorophyll contents under salt stress treatment than under non-salt stress.However, WT plants showed lower chlorophyll contents under salt stress conditions than under non-salt stress.Salt stress resulted in a decrease in the chlorophyll content in Typha domingensis Pers [37], consistent with our results.
NHX genes in the CPA (monovalent cationic reversal protein) family are widely distributed in bacteria, fungi, and higher plants and animals.The family is involved in the regulation of the cell cycle and proliferation, salt tolerance, vesicle trafficking, and growth [38].Furthermore, NHX genes regulate flower coloration [39][40][41] and flower development [42].The results of this study indicate that AtrNHX8 could promote flowering in Arabidopsis thaliana.Further studies are needed to determine the mechanism underlying the effects of AtrNHX8.
To retrieve and display the repeatedly detected association networks, protein sequences were submitted to STRING (Search Tool for Recurring Instances of Neighboring Genes) v. 11.0 (https://cn.string-db.org/).

Chromosomal Localization and Synteny Analyses
The GFF3 file that contained location data for amaranth CPA genes was downloaded from the Amaranthus tricolor Genome Database [26].The chromosomal location and results of the synteny analysis for NHX family genes were visualized using TBtools software (v.2.086).The Arabidopsis thaliana genome was downloaded from TAIR for a synteny analysis with the amaranth genome.

Sequence Alignment and Phylogenetic Analysis of NHXs
A multiple sequence alignment of the full-length amino acid sequences of NHXs from Amaranthsus tricolor, Arabidopsis thaliana, and Oryza sativa was generated using MUSCLE (Multiple Protein Sequence Alignment) within MEGA (Molecular Evolutionary Genetics Analysis) v. 11 (https://www.megasoftware.net/).Subsequently, a phylogenetic tree was constructed using the neighbor-joining method (NJ) using MEGA11 (Version 11.0.8) with 1000 bootstrap replicates, the Jones-Taylor-Thornton (JTT) model, and pairwise deletion.The NHX protein sequences in Arabidopsis thaliana and Oryza sativa were downloaded from the Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org/)and https://ricedata.cn,respectively.

Plant Material, Treatment, and qRT-PCR Analysis
Amaranth seeds of "Suxian No. 1" (supplied by the Suzhou Academy of Agricultural Sciences) were sown in culture pots in a chamber at 25 • C for 16/8 h (day/night).At the 3-leaf seedling stage, they were treated with different concentrations of NaCl (0, 50, 100, and 200 mM) in three biological replicates.The seedlings were grown in the same chamber at 25 • C and 16/8 h (D/N).The amaranth leaves were collected after 7 days of salt solution treatment.All samples were immediately frozen in liquid nitrogen and stored at −80 • C.
Total RNA was isolated from the collected samples using a kit (Yeasen, Shanghai, China) according to the manufacturer's instructions.First-strand cDNA was synthesized from 1 µg of total RNA using Recombinant M-MLV Reverse Transcriptase (TransGen Biotech, Beijing, China).Quantitative real time-PCR (qRT-PCR) was performed in optical 96-well plates using the Roche LightCycler 480 instrument (Roche, Solna, Sweden).The reactions were carried out in a 20 µL volume containing 10 µL of SYBR Premix Ex Taq, 0.8 µL of specific primers, 2 µL of diluted cDNA, and 6.4 µL of ddH 2 O.The PCR conditions were as follows: 30 s at 95 • C, 45 cycles of 10 s at 95 • C, and 20 s at 59 • C, followed by 12 s at 72 • C. AtrEF1a [46] was used as the internal reference gene.The 2 −∆∆Ct quantification method was used, and variation in expression was estimated from the three biological replicates.The primer pairs used for the qRT-PCR analysis of NHX genes are listed in Table 2.

Functional Analysis of AtrNHX8
The AtrNHX8 gene was amplified by PCR and ligated to the expression vector pCambia1301-35S-GUS.Then, the constructed recombinant vector pCambia1301-35S-AtrNHX8-GUS was transferred into Agrobacterium tumefaciens GV3101 by the freeze-thaw method [47].In addition, transgenic A. thaliana plants were obtained by the floral dip method [48].T2-generation transgenic plants were finally obtained through multi-generation selfing, GUS staining, and PCR.The primers used for vector construction and PCR detection are listed in Table 3.To better understand the mechanism by which AtrNHX8 responded to salt stress, WT and T2 generation transgenic seeds from Arabidopsis were placed in Petri dishes with 3-layer filter paper, and 15 mL of 150 mM NaCl was added to each dish.Water was used as the control.In all cases, experiments were carried out in (at least) triplicate.The germination potential and final germination rate were determined at 3 days and 7 days.
The WT and T2 generation transgenic Arabidopsis seeds were sown in culture pots.Plants at the 6-leaf stage were treated with 150 mM salt solution.After 7 days of salt treatment, the leaves of WT and transgenic Arabidopsis thaliana were collected to extract RNA for qRT-PCR, following the previously described method.The primers for the AtNHX gene used for qRT-PCR are listed in Table 3.The chlorophyll and carotenoid contents were evaluated according to Liu [2].

Figure 1 .
Figure 1.Conserved motifs and gene structure of AtrCPAs.(A) Phylogeny analysis of AtrCPAs; (B) conserved motif distribution in AtrCPA proteins; (C) conserved domains of AtrCPA proteins; (D) exon-intron structure of AtrCPAs.TBtools v. 2.086 was used to present the chart.

Figure 2 .
Figure 2. Distribution cis-acting regulatory elements in the promoter regions of AtrCPAs.TBtools v. 2.086 was used to present the chart.

Figure 2 .
Figure 2. Distribution cis-acting regulatory elements in the promoter regions of AtrCPAs.TBtools v. 2.086 was used to present the chart.

Figure 4 .
Figure 4. Syntenic analyses of CPA genes.(A) Segmental duplication events of CPA genes in Amaranthus tricolor.(B) Duplication events of NHX genes in Amaranthus tricolor and Arabidopsis.Red lines indicate CPA duplication events.Gray lines represent all synteny blocks.TBtools v. 2.086 was used to present the chart.

Figure 6 .
Figure 6.Neighbor-joining phylogenetic tree of NHX proteins from Amaranth tricolor, Arabidopsis thaliana, and Oryza sativa.NHXs were divided into three major groups.The phylogenetic tree was constructed using MEGA X.The numbers at the nodes indicate bootstrap 1000.

Figure 6 .
Figure 6.Neighbor-joining phylogenetic tree of NHX proteins from Amaranth tricolor, Arabidopsis thaliana, and Oryza sativa.NHXs were divided into three major groups.The phylogenetic tree was constructed using MEGA X.The numbers at the nodes indicate bootstrap 1000.

Figure 7 .
Figure 7. qRT-PCR analysis of AtrNHX genes under salt stress.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

Figure 8 .
Figure 8. Expression trends of AtrNHX genes in different tissues.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

Figure 8 .
Figure 8. Expression trends of AtrNHX genes in different tissues.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

Figure 9 .
Figure 9. Seed germination in Arabidopsis thaliana under salt stress.

Figure 9 .
Figure 9. Seed germination in Arabidopsis thaliana under salt stress.

Figure 10 .
Figure 10.Relative expression of GUS in Arabidopsis thaliana leaves under salt stress.(A1,A2) represent wild-type Arabidopsis before salt treatment, (A3,A4) represents wild-type Arabidopsis with 0 mM and 150 mM NaCl treatment for 7 days, respectively.(B1,B2) represent 35S::AtrNHX8 Arabidopsis before salt treatment, (B3,B4) represent 35S::AtrNHX8 Arabidopsis with 0 mM and 150 mM NaCl treatment for 7 days, respectively.(C,D) show the chlorophyll and carotenoid contents, respectively.(E)represents the relative expression of NHX8 in Arabidopsis thaliana leaves.The data are presented as mean ± standard error and were subjected to analysis of variance (ANOVA).The means were compared using the ad hoc Tukey test (p < 0.05%).Lowercase letters represent significant differences at the 0.05 level.

Table 1 .
Identification and sequence analysis of the CPA gene family in amaranth.

Table 3 .
PCR and qRT-PCR primer design for transgenic Arabidopsis thaliana.