Effects of Arbuscular Mycorrhizal Fungus on Sodium and Chloride Ion Channels of Casuarina glauca under Salt Stress

Casuarina glauca is an important coastal protection forest species, which is exposed to high salt stress all year round. Arbuscular mycorrhizal fungi (AMF) can promote the growth and salt tolerance of C. glauca under salt stress. However, the effects of AMF on the distribution of Na+ and Cl− and the expression of related genes in C. glauca under salt stress need to be further explored. This study explored the effects of Rhizophagus irregularis on plant biomass, the distribution of Na+ and Cl−, and the expression of related genes in C. glauca under NaCl stress through pot simulation experiments. The results revealed that the mechanisms of Na+ and Cl− transport of C. glauca under NaCl stress were different. C. glauca took a salt accumulation approach to Na+, transferring Na+ from roots to shoots. Salt accumulation of Na+ promoted by AMF was associated with CgNHX7. The transport mechanism of C. glauca to Cl− might involve salt exclusion rather than salt accumulation, and Cl− was no longer transferred to shoots in large quantities but started to accumulate in roots. However, AMF alleviated Na+ and Cl− stress by similar mechanisms. AMF could promote salt dilution of C. glauca by increasing biomass and the content of K+, compartmentalizing Na+ and Cl− in vacuoles. These processes were associated with the expression of CgNHX1, CgNHX2-1, CgCLCD, CgCLCF, and CgCLCG. Our study will provide a theoretical basis for the application of AMF to improve salt tolerance in plants.


Introduction
Salt stress is one of the most severe abiotic stresses that the world is facing [1]. Approximately 800 million hectares of land and approximately 20% of irrigated land in the world are under some degree of salt stress [2,3]. The ionic toxicity of salt stress can induce osmotic stress in plants, leading to imbalanced nutrition and even plant death [4]. NaCl stress is one of the most extensive and damaging salt stresses [5,6]. However, halophytes can grow under high salt stress [7,8]. There are three methods related to their salt tolerance under salt stress. Salt exclusion is common in pseudohalophytes. Their roots rarely absorb salt or mainly accumulate the absorbed salt in the roots, so the ion concentration in the leaves is significantly lower than that in the roots within a certain concentration range [9,10]. Salt excretion is common in recretohalophytes. They have salt glands, which can actively secrete the absorbed salt to the surface of the stems and leaves for the rain to wash away [11]. Salt dilution is common in euhalophytes. They reduce salt concentration by rapidly growing cells to absorb water, increasing osmotic adjustment substances, and compartmentalizing salt in vacuoles [12,13]. It would be interesting to explore the mechanism of salt tolerance 1.
To explore the influence of mycorrhization on the growth and distribution of Na + and Cl −.

2.
To explore the influence of mycorrhization on the gene expression of the CgNHX and CgCLC families. 3.
To explore whether these genes are related to Na + and Cl − distribution. Figure S1 shows the colonization of arbuscule, hypha, vesicle, and spore in mycorrhizal C. glauca under NaCl stress. With a change in NaCl concentration from 0 to 600 mM, arbuscular colonization decreased from 39% to 14%, vesicle and spore colonization increased from 34% to 57%, and hyphal colonization exhibited no significant change.

Effect of Rhizophagus irregularis on Mycorrhizal Colonization and Plant Biomass under NaCl Stress
Under NaCl stress, the biomass of C. glauca differed between plants with R. irregularis inoculation and those without. Figure S2 shows that NaCl stress inhibited the growth of C. glauca and R. irregularis inoculation alleviated this inhibition ( Figure S2a). NaCl stress reduced the fresh weight, ground diameter, and height of C. glauca ( Figure S2b-d).
The inoculation with R. irregularis could promote the growth of C. glauca regardless of the presence of NaCl stress, specifically by increasing the fresh weight, plant height, and ground diameter of C. glauca ( Figure S2b-d). These results show that R. irregularis inoculation can promote the growth and salt tolerance of C. glauca under NaCl stress.

Effect of R. irregularis on Na + , K + , and Cl − Status under NaCl Stress
The effects of R. irregularis inoculation on the Na + and Cl − contents under NaCl stress were consistent, and both were increased. Therefore, the contents in the soil were reduced (Figure 1a-d). The Na + and Cl − concentrations of C. glauca were increased by NaCl stress. Instead, under NaCl stress, inoculation with R. irregularis decreased the Na + and Cl − concentrations of shoots and roots (Figure 1e,f). The K + content of C. glauca was reduced by NaCl stress, but inoculation with R. irregularis increased the K + content of shoots and roots under NaCl stress (Figure 1g). The Na + /K + of C. glauca was increased by NaCl stress. Nevertheless, under NaCl stress, R. irregularis inoculation decreased the Na + /K + of shoots and roots by 8% and 24%, respectively (Figure 1h).  Figure S1 shows the colonization of arbuscule, hypha, vesicle, and spore in mycorrhizal C. glauca under NaCl stress. With a change in NaCl concentration from 0 to 600 mM, arbuscular colonization decreased from 39% to 14%, vesicle and spore colonization increased from 34% to 57%, and hyphal colonization exhibited no significant change.

Effect of Rhizophagus irregularis on Mycorrhizal Colonization and Plant Biomass under NaCl Stress
Under NaCl stress, the biomass of C. glauca differed between plants with R. irregularis inoculation and those without. Figure S2 shows that NaCl stress inhibited the growth of C. glauca and R. irregularis inoculation alleviated this inhibition ( Figure S2a). NaCl stress reduced the fresh weight, ground diameter, and height of C. glauca ( Figure S2b-d). The inoculation with R. irregularis could promote the growth of C. glauca regardless of the presence of NaCl stress, specifically by increasing the fresh weight, plant height, and ground diameter of C. glauca ( Figure S2b-d). These results show that R. irregularis inoculation can promote the growth and salt tolerance of C. glauca under NaCl stress.

Effect of R. irregularis on Na + , K + , and Cl − Status under NaCl Stress
The effects of R. irregularis inoculation on the Na + and Cl − contents under NaCl stress were consistent, and both were increased. Therefore, the contents in the soil were reduced (Figure 1a-d). The Na + and Cl − concentrations of C. glauca were increased by NaCl stress. Instead, under NaCl stress, inoculation with R. irregularis decreased the Na + and Cl − concentrations of shoots and roots (Figure 1e,f). The K + content of C. glauca was reduced by NaCl stress, but inoculation with R. irregularis increased the K + content of shoots and roots under NaCl stress (Figure 1g). The Na + /K + of C. glauca was increased by NaCl stress. Nevertheless, under NaCl stress, R. irregularis inoculation decreased the Na + /K + of shoots and roots by 8% and 24%, respectively ( Figure 1h). irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. The data are the means ± standard errors (n = 3). Different capital letters indicate significant differences between means at the p < 0.05 level by Tukey's test. Data of roots from Wang et al. [31] in the same project. The content of K + in C. glauca. (h) The Na + /K + of C. glauca. NM, nonmycorrhizal; AM, inoculated with R. irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. The data are the means ± standard errors (n = 3). Different capital letters indicate significant differences between means at the p < 0.05 level by Tukey's test. Data of roots from Wang et al. [31] in the same project. The effects of NaCl stress on the TF of Na + and Cl − were different, increasing the TF of Na + but decreasing that of Cl − . The effects of R. irregularis inoculation on the Na + and Cl − TF under NaCl stress were consistent, and both were increased ( Figure 2). The effects of NaCl stress on the TF of Na + and Cl − were different, increasing the TF of Na + but decreasing that of Cl − . The effects of R. irregularis inoculation on the Na + and Cl − TF under NaCl stress were consistent, and both were increased ( Figure 2). irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. The data are the means ± standard errors (n = 3). Different capital letters indicate significant differences between means at the p < 0.05 level by Tukey's test.

Effect of R. irregularis on Gs and Tr under NaCl Stress
To explore whether the increase of TF of Na + and Cl − caused by R. irregularis inoculation is related to stomatal conductance (Gs) and transpiration rate (Tr), we investigated the effect of R. irregularis on Gs and Tr under NaCl Stress. The results showed that NaCl stress decreased Gs and Tr. Under NaCl stress, compared with no inoculation, inoculation with R. irregularis increased Gs and Tr by 31% and 26%, respectively. (Figure 3). The Tr in leaves of C. glauca in different treatments. NM, nonmycorrhizal; AM, inoculated with R. irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. The data are the means ± standard errors (n = 3). * indicates significant differences between means at the p < 0.05 level by one-way ANOVA, and ** indicates significant differences between means at the p < 0.01 level by one-way ANOVA.

Effect of R. irregularis on Expression of CgNHXs and CgCLCs under NaCl Stress
To explore whether the effect of R. irregularis inoculation on Na + , K + , and Cl − status is related to Na + and Cl − transport-related genes, we determined the expression of CgNHXs and CgCLCs.
A total of five full-length CgNHX genes and five full-length CgCLC genes were obtained by cloning. These full-length translated protein sequences were compared with NHX and CLC proteins of known species such as Arabidopsis thaliana and Zea mays, and a phylogenetic tree was constructed to initially determine their potential functions. As shown in Figure 4a, the five NHX proteins were divided into three branches. Among them, CgNHX1, CgNHX2-1, and CgNHX2-2 were classified into the same branch with a closer affinity to AtNHX1 and AtNHX2. CgNHX6 was classified into the second branch with closer affinity to AtNHX6. CgNHX7 was classified into the third branch with a closer The TF of Cl − in C. glauca. NM, nonmycorrhizal; AM, inoculated with R. irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. The data are the means ± standard errors (n = 3). Different capital letters indicate significant differences between means at the p < 0.05 level by Tukey's test.

Effect of R. irregularis on Gs and Tr under NaCl Stress
To explore whether the increase of TF of Na + and Cl − caused by R. irregularis inoculation is related to stomatal conductance (Gs) and transpiration rate (Tr), we investigated the effect of R. irregularis on Gs and Tr under NaCl Stress. The results showed that NaCl stress decreased Gs and Tr. Under NaCl stress, compared with no inoculation, inoculation with R. irregularis increased Gs and Tr by 31% and 26%, respectively. (Figure 3). The effects of NaCl stress on the TF of Na + and Cl − were different, increasing the TF of Na + but decreasing that of Cl − . The effects of R. irregularis inoculation on the Na + and Cl − TF under NaCl stress were consistent, and both were increased ( Figure 2). irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. The data are the means ± standard errors (n = 3). Different capital letters indicate significant differences between means at the p < 0.05 level by Tukey's test.

Effect of R. irregularis on Gs and Tr under NaCl Stress
To explore whether the increase of TF of Na + and Cl − caused by R. irregularis inoculation is related to stomatal conductance (Gs) and transpiration rate (Tr), we investigated the effect of R. irregularis on Gs and Tr under NaCl Stress. The results showed that NaCl stress decreased Gs and Tr. Under NaCl stress, compared with no inoculation, inoculation with R. irregularis increased Gs and Tr by 31% and 26%, respectively. (Figure 3). The Tr in leaves of C. glauca in different treatments. NM, nonmycorrhizal; AM, inoculated with R. irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. The data are the means ± standard errors (n = 3). * indicates significant differences between means at the p < 0.05 level by one-way ANOVA, and ** indicates significant differences between means at the p < 0.01 level by one-way ANOVA.

Effect of R. irregularis on Expression of CgNHXs and CgCLCs under NaCl Stress
To explore whether the effect of R. irregularis inoculation on Na + , K + , and Cl − status is related to Na + and Cl − transport-related genes, we determined the expression of CgNHXs and CgCLCs.
A total of five full-length CgNHX genes and five full-length CgCLC genes were obtained by cloning. These full-length translated protein sequences were compared with NHX and CLC proteins of known species such as Arabidopsis thaliana and Zea mays, and a phylogenetic tree was constructed to initially determine their potential functions. As shown in Figure 4a, the five NHX proteins were divided into three branches. Among them, CgNHX1, CgNHX2-1, and CgNHX2-2 were classified into the same branch with a closer affinity to AtNHX1 and AtNHX2. CgNHX6 was classified into the second branch with closer affinity to AtNHX6. CgNHX7 was classified into the third branch with a closer The Tr in leaves of C. glauca in different treatments. NM, nonmycorrhizal; AM, inoculated with R. irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. The data are the means ± standard errors (n = 3). * indicates significant differences between means at the p < 0.05 level by one-way ANOVA, and ** indicates significant differences between means at the p < 0.01 level by one-way ANOVA.

Effect of R. irregularis on Expression of CgNHXs and CgCLCs under NaCl Stress
To explore whether the effect of R. irregularis inoculation on Na + , K + , and Cl − status is related to Na + and Cl − transport-related genes, we determined the expression of CgNHXs and CgCLCs.
A total of five full-length CgNHX genes and five full-length CgCLC genes were obtained by cloning. These full-length translated protein sequences were compared with NHX and CLC proteins of known species such as Arabidopsis thaliana and Zea mays, and a phylogenetic tree was constructed to initially determine their potential functions. As shown in Figure 4a, the five NHX proteins were divided into three branches. Among them, CgNHX1, CgNHX2-1, and CgNHX2-2 were classified into the same branch with a closer affinity to AtNHX1 and AtNHX2. CgNHX6 was classified into the second branch with closer affinity to AtNHX6. CgNHX7 was classified into the third branch with a closer affinity to AtNHX7. As shown in Figure 4b, the five CLC proteins were divided into four branches. Among them, CgCLCB was divided into the first branch, which is more closely related to AtCLCB. CgCLCC and CgCLCG were divided into the second branch, which is more closely related to AtCLCC and AtCLCG. CgCLCD was divided into the third branch, which is more closely related to AtCLCD. CgCLCF was divided into the fourth branch, which is more closely related to AtCLCF. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 14 affinity to AtNHX7. As shown in Figure 4b, the five CLC proteins were divided into four branches. Among them, CgCLCB was divided into the first branch, which is more closely related to AtCLCB. CgCLCC and CgCLCG were divided into the second branch, which is more closely related to AtCLCC and AtCLCG. CgCLCD was divided into the third branch, which is more closely related to AtCLCD. CgCLCF was divided into the fourth branch, which is more closely related to AtCLCF.   The patterns of CgNHX expression differed between shoots and roots. In shoots, the expression of CgNHX1 and CgNHX2-1 was upregulated by R. irregularis inoculation. The expression of CgNHX2-2 and CgNHX6 was upregulated by NaCl stress but downregulated by R. irregularis inoculation under NaCl stress. In addition, the expression of CgNHX7 was downregulated by NaCl stress. In roots, the expression of CgNHX1, CgNHX2-1, and CgNHX2-2 was upregulated by R. irregularis inoculation under NaCl stress. The expression of CgNHX6 was upregulated by NaCl stress. Under NaCl stress, the expression of CgNHX7 was upregulated by R. irregularis inoculation ( Figure 5).  The patterns of CgNHX expression differed between shoots and roots. In shoot expression of CgNHX1 and CgNHX2-1 was upregulated by R. irregularis inoculation expression of CgNHX2-2 and CgNHX6 was upregulated by NaCl stress but down lated by R. irregularis inoculation under NaCl stress. In addition, the expressio CgNHX7 was downregulated by NaCl stress. In roots, the expression of CgN CgNHX2-1, and CgNHX2-2 was upregulated by R. irregularis inoculation under stress. The expression of CgNHX6 was upregulated by NaCl stress. Under NaCl stres expression of CgNHX7 was upregulated by R. irregularis inoculation ( Figure 5).
The patterns of CgCLC expression differed between shoots and roots. In shoot expression of CgCLCB was downregulated by NaCl stress. The expression of CgCLCC upregulated by NaCl stress but downregulated by R. irregularis inoculation under stress. In addition, the expression of CgCLCD, CgCLCF, and CgCLCG was upregulat R. irregularis inoculation under NaCl stress. In roots, the expression of CgCLCB CgCLCD was downregulated by NaCl stress, whereas the expression of CgCLCC, CgC and CgCLCG was upregulated by NaCl stress. R. irregularis inoculation downregu the expression of CgCLCC but upregulated CgCLCF and CgCLCG under NaCl stress ure 5). Figure 5. Effects of NaCl stress and R. irregularis inoculation on the expression of CgNHX CgCLCs in shoots and roots. NM, nonmycorrhizal; AM, inoculated with R. irregularis; 0, no stress; 600, 600 mM NaCl stress. Different capital letters indicate significant differences be means at the p < 0.05 level by Tukey's test.

Correlation Analysis of Gene Expression and Physiological Indicators of C. glauca unde NaCl Stress
To explore the correlation between the effect of R. irregularis inoculation on ph logical indicators and the Na + , Cl − transport-related genes under NaCl stress, we correlation analysis between them under salt stress.
Opposite to CgNHX6, the correlation analysis results of the expression of CgNH shoots and roots and CgNHX2-2 in roots were all positively correlated with mycorr colonization, biomass, and the content of Na + , K + , except that the expression of CgN Figure 5. Effects of NaCl stress and R. irregularis inoculation on the expression of CgNHXs and CgCLCs in shoots and roots. NM, nonmycorrhizal; AM, inoculated with R. irregularis; 0, no NaCl stress; 600, 600 mM NaCl stress. Different capital letters indicate significant differences between means at the p < 0.05 level by Tukey's test.
The patterns of CgCLC expression differed between shoots and roots. In shoots, the expression of CgCLCB was downregulated by NaCl stress. The expression of CgCLCC was upregulated by NaCl stress but downregulated by R. irregularis inoculation under NaCl stress. In addition, the expression of CgCLCD, CgCLCF, and CgCLCG was upregulated by R. irregularis inoculation under NaCl stress. In roots, the expression of CgCLCB and CgCLCD was downregulated by NaCl stress, whereas the expression of CgCLCC, CgCLCF, and CgCLCG was upregulated by NaCl stress. R. irregularis inoculation downregulated the expression of CgCLCC but upregulated CgCLCF and CgCLCG under NaCl stress ( Figure 5).

Correlation Analysis of Gene Expression and Physiological Indicators of C. glauca under NaCl Stress
To explore the correlation between the effect of R. irregularis inoculation on physiological indicators and the Na + , Cl − transport-related genes under NaCl stress, we did a correlation analysis between them under salt stress.
Opposite to CgNHX6, the correlation analysis results of the expression of CgNHX1 in shoots and roots and CgNHX2-2 in roots were all positively correlated with mycorrhizal colonization, biomass, and the content of Na + , K + , except that the expression of CgNHX1 in shoots was negatively correlated with the concentration of Na + and that in roots was negatively correlated with Na + /K + . The expression of CgNHX2-1 in shoots was positively correlated with mycorrhizal colonization and the content of K + , and that in roots was only positively correlated with mycorrhizal colonization. The expression of CgNHX7 in roots was positively correlated with mycorrhizal colonization and biomass but was negatively correlated with the concentration of Na + , the content of Na + in soil, and Na + /K + (Figure 6).
in shoots was negatively correlated with the concentration of Na + and that in roots was negatively correlated with Na + /K + . The expression of CgNHX2-1 in shoots was positively correlated with mycorrhizal colonization and the content of K + , and that in roots was only positively correlated with mycorrhizal colonization. The expression of CgNHX7 in roots was positively correlated with mycorrhizal colonization and biomass but was negatively correlated with the concentration of Na + , the content of Na + in soil, and Na + /K + (Figure 6).
Correlation analysis showed that the expression of CgCLCB in shoots was positively correlated with the concentration of Cl − and negatively correlated with the TF of Cl − . However, the expression of CgCLCC in shoots was positively correlated with the concentration of Cl − and negatively correlated with other indicators, which was the opposite of the expression of CgCLCD in shoots. The expression of CgCLCC in roots was negatively correlated with mycorrhizal colonization, fresh weight, and Cl − content. Both the expression of CgCLCF in shoots and roots were positively correlated with fresh weight and the content of Cl − . In addition, the expression of CgCLCF in roots was also positively correlated with mycorrhizal colonization. The correlation analysis results of CgCLCF in roots were consistent with those of CgCLCG in roots ( Figure 6).

Effects of R. irregularis Inoculation and NaCl Stress on Colonization and Biomass
Salt stress inhibited plant growth and AMF growth in the same way [48,49]. Soil salinity had a negative correlation with the intensity of mycorrhizal colonization on the one hand, and a positive correlation with spore density on the other hand. We believed that Correlation analysis showed that the expression of CgCLCB in shoots was positively correlated with the concentration of Cl − and negatively correlated with the TF of Cl − . However, the expression of CgCLCC in shoots was positively correlated with the concentration of Cl − and negatively correlated with other indicators, which was the opposite of the expression of CgCLCD in shoots. The expression of CgCLCC in roots was negatively correlated with mycorrhizal colonization, fresh weight, and Cl − content. Both the expression of CgCLCF in shoots and roots were positively correlated with fresh weight and the content of Cl − . In addition, the expression of CgCLCF in roots was also positively correlated with mycorrhizal colonization. The correlation analysis results of CgCLCF in roots were consistent with those of CgCLCG in roots ( Figure 6).

Effects of R. irregularis Inoculation and NaCl Stress on Colonization and Biomass
Salt stress inhibited plant growth and AMF growth in the same way [48,49]. Soil salinity had a negative correlation with the intensity of mycorrhizal colonization on the one hand, and a positive correlation with spore density on the other hand. We believed that these results reflect the assistance of R. irregularis to plants resisting NaCl stress [22]. Sporulation was considered as a resistance behavior exhibited by mycorrhization against high salt concentrations [21].
The most significant and common effect of salt on plants is growth inhibition, since plants need to absorb water and ions to maintain osmotic pressure, synthesize osmotic adjustment substances, and compartmentalize harmful ions to survive. These processes require energy, resulting in less energy required for growth and lower biomass [50,51]. AMF could increase energy by enhancing the ability of plants to absorb water, synthesize osmotic adjustment substances, and compartmentalize harmful ions, thereby promoting plant growth [52,53]. Additionally, we regarded the significant increase of C. glauca biomass caused by R. irregularis inoculation as an important indicator of the ability of plants to tolerate salinity stress [53,54].

Effects of R. irregularis Inoculation and NaCl Stress on Na + , K + , and Cl − Uptake
Many studies have focused only on the tolerance mechanism of Na + under salt stress, and have assumed that Na + can represent NaCl. However, our results showed that the change trends of Na + and Cl − TF were different under NaCl stress, reflecting the different salt tolerance mechanisms of the two. For Na + , the Na + TF increased under NaCl stress, which meant that the tolerance mechanism of Na + in C. glauca might be salt accumulation, and hence a large amount of Na + accumulated in the shoots [55,56]. The shoots were then protected by salt dilution, increasing tissue water content, or compartmentalizing Na + to the vacuole [13,57]. However, for Cl − , the TF of Cl − was significantly decreased under NaCl stress. At the same time, the Cl − content in shoots was still greater than in roots. This meant that under the stress of 600 mM NaCl, the tolerance mechanism of C. glauca to Cl − might involve salt exclusion rather than salt accumulation [58]. Perhaps Cl − is more toxic to shoots than Na + for C. glauca, or the concentration of Cl − in shoots reaches the tolerance threshold, but Na + concentration does not [59,60].
Although the mechanisms of C. glauca tolerance to Na + and Cl − were different, the mitigation mechanisms of R. irregularis inoculation to their stress were similar. The upward transport of Na + and Cl − improved by AMF was caused by transpiration flow in plants under salt stress [61]. The transpiration flow was caused by increases in Gs and Tr, which were attributed to R. irregularis inoculation. AMF could promote salt dilution of halophytes by increasing plant biomass and the content of osmotic adjustment substances, compartmentalizing Na + and Cl − [62,63]. Due to the increase in biomass caused by R. irregularis inoculation, mycorrhizal C. glauca could simultaneously maintain high contents and low concentrations of Na + and Cl − . This is a common feature of AMF-associated plants under salt stress [26,64]. Compared with nonmycorrhizal C. glauca, mycorrhizal C. glauca absorbed larger amounts of Na + and Cl − from the soil but maintained lower concentrations of Na + and Cl − to protect themselves from excessive damage. Meanwhile, mycorrhizal plants had lower Na + /K + to regulate the dynamic equilibrium of ions and improve salt tolerance [65,66]. Therefore, even if increased Gs and Tr promotes the upward transport of Na + and Cl − , the mycorrhizal C. glauca does not suffer severe ion damage.

Effects of R. irregularis Inoculation and NaCl Stress on Gene Expression
Roots are the first plant part to be stressed during salt stress. The AM symbiosis increased the saline stress tolerance of Robinia pseudoacacia through the upregulation of SOS1/NHX7 in roots, which enhanced the exclusion of Na + from root cells [67]. This indicated that R. irregularis inoculation promoted Na + efflux from roots and prevented Na + poisoning. Meanwhile, R. irregularis inoculation decreased the Na + content in the soil and increased TF of Na + , implying that possibly that most of the Na + efflux in roots upon induction of CgNHX7 by R. irregularis inoculation was transferred to the shoots rather than the soil. Meanwhile, under NaCl stress, the expression of CgNHX7 decreased in shoots but increased in roots. This is consistent with our previous notion (see above) that R. irregularis inoculation might promote the salt accumulation of Na + in shoots. We mentioned earlier that AMF could promote salt dilution of halophytes by increasing plant biomass and the content of osmotic adjustment substances, compartmentalizing Na + .
So, are these processes co-regulated with the expression of CgNHXs?, since vacuolar NHXs, NHX1 and NHX2 could promote the transport of Na + to vacuoles [53]. Hence, the upregulation of CgNHX1 and CgNHX2-1 caused by R. irregularis inoculation might have alleviated the inhibitory effects of Na + by transporting it to vacuoles. Furthermore, similar to our correlation analysis results, NHX1 and NHX2 had the same functions in growthpromoting and the stabilization of K + in vacuoles [35,68]. Therefore, the upregulation of CgNHX1 and CgNHX2-1 by R. irregularis inoculation might not only increase the biomass of mycorrhizal C. glauca to reduce Na + toxicity by bio-dilution, but also stabilize the cellular environment by increasing the K + content in shoots and roots.
Which CgCLCs can mycorrhizal C. glauca use for salt dilution? The expression of Cg-CLCC in C. glauca under NaCl stress was higher than in those without NaCl stress treatment, and plants with high Cl − toxicity protect themselves by upregulating the expression of CLCC to transport Cl − into vacuoles [69]. Another CLC gene, AtCLCG, is involved in salt tolerance by altering Cl − homeostasis in mesophyll cells and Cl − compartmentalization into the vacuole. This gene is co-expressed with AtCLCC in guard cells during stomatal regulation [47]. Although R. irregularis inoculation downregulated CgCLCC under salt stress, it upregulated CgCLCG, which might promote the transfer of Cl − into mesophyll cell vacuoles, thereby improving tolerance of mycorrhizal C. glauca against NaCl stress, and promoting its growth. In addition, CLCD, an anion transporter involved in compartments and trafficking, might be related to growth and Cl − transfer into the trans-Golgi network [33]. The function of another CLC gene, CgCLCF, might be similar to that of CgCLCD, which was correlated with Cl − transport and growth. The increase in biomass and Cl − compartmentalization upon R. irregularis inoculation might have been related to CgCLCD and CgCLCF induction.

Experimental Design and Materials
This study was a two-factor experiment: NaCl stress (0 mM NaCl and 600 mM NaCl) and AMF inoculation (no inoculation with AMF and inoculation with Rhizophagus irregularis). There were four treatments, and three biological replicates for each treatment. Each replicate contained 12 pots, for a total of 144 pots.
C. glauca seeds were sterilized and germinated into seedlings, and the best-growing plant was selected from the seedlings to prepare a batch of tissue culture seedlings, which were used as experimental materials [31]. R. irregularis was multiplied by Lycopersicon esculentum Mill and then triturated, and 40 g of inoculum was added to each pot for R. irregularis inoculation treatment, and 40 g of sterilized inoculum was added to each pot for no AMF treatment. Seedlings were grown in greenhouse at temperatures ranging from 25-35 • C and relative humidity ranging from 50-70%.

Plant Harvest
The stomatal conductance (Gs) and transpiration rate (Tr) were measured with a Li-6400 portable open flow gas-exchange system (Li-Cor Inc., Lincoln, NE, USA) between 8:30 to 11:30 a.m. The method and parameter settings were those of Sheng et al. [70].
After the shoots were weighed for total fresh weight, they were divided into three parts. The first part was used to calculate the water content, the second was used for elemental concentration determination, and the third was used to extract RNA. After weighing the total fresh weight of roots, the grouping and uses were the same as those of shoots, except that a part was separated to measure AMF colonization.

Mycorrhizal Colonization
Refer to Phillips and Hayman [71] and Giovannetti and Mosse [72] for the determination method of AMF colonization.

Plant Biomass and Water Content
The total fresh weight of shoots and roots was weighed at harvest time. The plant height was measured with a tape measure, and the ground diameter was measured with a digital caliper (Hengliang, Shanghai, China). The part of the sample used to measure the water content was weighed fresh and then dried at 65 • C after fixation at 105 • C to measure the dry weight. The water content was calculated using the dry and fresh weights. Total dry weight was calculated using water content and total fresh weight.

Na + , K + , and Cl − Status
After being ground, the part of the sample used to measure the Na + and K + concentration was extracted with HNO 3 and a microwave digestion instrument (Milestone ETHOS, Milan, Italy). The elements were measured by atomic absorption spectrophotometer (PerkinElmer, Waltham, MA, USA). The measurement method of Cl − concentration was that of Matsushita and Matoh [73] and Zhan et al. [74]. The contents of the elements were calculated by the concentration and total dry weight. The extraction method of elements in the soil was that of Bao [75]. The method for measuring elements in soil was the same as above. The transfer factor (TF) of Na + and Cl − was obtained by dividing the shoot content by the root content. . Reverse transcription of RNA was done using the TIANScript RT Kit (TIANGEN Bio, Beijing, China) to obtain cDNA. The sequences of CgNHXs and CgCLCs were obtained from the assembly of the C. glauca transcriptome sequenced by Illumina HiSeq technology (PRJNA690646). The full-length gene was cloned using the SMARTer ® RACE 5 /3 Kit (Takara Bio, Beijing, China). The primers used for qRT-PCR were designed with the NCBI Primer-BLAST tool (Table S1). qRT-PCR was conducted using a CF96X real-time PCR system (Bio-Rad, Hercules, CA, USA). The specific reaction system and steps were consistent with previous research [31]. There were three biological replicates for each treatment, and two technical replicates for each biological replicate. The relative quantity of transcripts was determined using the 2 −∆∆C T method [76].

Conclusions
Under NaCl stress, the tolerance mechanisms of C. glauca to Na + and Cl − were different. For Na + , salt accumulation was adopted, and Na + absorbed from the soil was transferred from roots to shoots. This process was promoted by R. irregularis inoculation and was related to Gs, Tr, and CgNHX7 expression. The Cl − tolerance mechanism involved salt exclusion, and Cl − absorbed from soil was no longer transferred to shoots in large quantities but started accumulating in roots. However, the mechanism of R. irregularis inoculation alleviating Na + and Cl − stress was similar, promoting growth, increasing osmotic adjustment substances, and ionic compartmentalization. Therefore, mycorrhizal C. glauca could not only absorb large amounts of Na + and Cl − from the soil to remediate it but also mitigate Na + and Cl − stress in plants. The process of R. irregularis inoculation alleviating Na + and Cl − stress was correlated with the expression of CgNHX1, CgNHX2-1, and CgCLCD, CgCLCF, CgCLCG. Future studies may focus on the specific effects of R. irregularis inoculation on these genes and the detailed Na + and Cl − transfer pathways in C. glauca under NaCl exposure.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author.