A Transcription Factor SlNAC10 Gene of Suaeda liaotungensis Regulates Proline Synthesis and Enhances Salt and Drought Tolerance

The NAC (NAM, ATAF1/2, and CUC2) transcription factors are one of the largest families of transcription factors in plants and play an important role in plant development and the response to adversity. In this study, we cloned a new NAC gene, SlNAC10, from the halophyte Suaeda liaotungensis K. The gene has a total length of 1584 bp including a complete ORF of 1107 bp that encodes 369 amino acids. The SlNAC10-GFP fusion protein is located in the nucleus and SlNAC10 has a transcription activation structural domain at the C-terminus. We studied the expression characteristics of SlNAC10 and found that it was highest in the leaves of S. liaotungensis and induced by drought, salt, cold, and abscisic acid (ABA). To analyze the function of SlNAC10 in plants, we obtained SlNAC10 transgenic Arabidopsis. The growth characteristics and physiological indicators of transgenic Arabidopsis were measured under salt and drought stress. The transgenic Arabidopsis showed obvious advantages in the root length and survival rate; chlorophyll fluorescence levels; and the antioxidant enzyme superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and the proline content was higher than that of the wild-type (WT) Arabidopsis, whereas the relative electrolyte leakage and malondialdehyde (MDA) content were lower than those of the wild-type Arabidopsis. We explored the regulatory role of SlNAC10 on proline synthesis-related enzyme genes and found that SlNAC10 binds to the AtP5CS1, AtP5CS2, and AtP5CR promoters and regulates their downstream gene transcription. To sum up, SlNAC10 as a transcription factor improves salt and drought tolerance in plants possibly by regulating proline synthesis.


Introduction
In recent years, with increasingly serious environmental pollution, plants have been subjected to more and more abiotic stresses, which have seriously affected their growth and development. Upon sensing a stress signal, plants increase their resistance to the stressful environment. Transcription factors (TFs) play a key role in the conversion of stress signal perception into stress-responsive gene expression during signal transduction processes [1]. TFs make up approximately 7% of the coding part of the plant transcriptome. A typical TF is comprised of four parts: a conserved DNA-binding part, a variable transcriptional regulatory part, an oligomeric part, and a nuclear localization signal (NLS) for protein entry into the nucleus [2]. In plants, TFs usually belong to large gene families such as the NAC (no apical meristem (NAM), Arabidopsis transcriptional activator factor (ATAF), cup-shaped cotyledon (CUC)) superfamily [3]. Plant NAC proteins contain a highly conserved DNAbinding (NAC) domain at the N-terminal and a variable transcriptional regulatory domain at the C-terminal [2]. The N terminus consists of nearly 160 amino acid residues divided into five major conserved sub-regions named A to E [4]. revealed that the transgenic plants had a significant survival advantage under salt and drought stress compared to wild-type plants. In addition, proline content was increased in SlNAC10 transgenic Arabidopsis under unfavorable conditions so we investigated the role of SlNAC10 in the regulation of genes related to proline synthesis and found that SlNAC10 could bind to the promoters of the AtP5CS1, AtP5CS2, and AtP5CR genes and regulate the transcription of their downstream genes. The above findings suggest that the SlNAC10 transcription factor enhanced plant stress tolerance by regulating proline synthesis, which helps us to further study the NAC transcription factor and its stress tolerance mechanism in salt-tolerant plants of S. liaotungensis.

SlNAC10 Encodes a Protein Containing the NAC Domain
Based on the EST sequence of SlNAC10 in the transcriptome database, the cDNA sequence of SlNAC10 (GenBank NO. KJ933532.1) was obtained by PCR amplification and 3 and 5 RACE. SlNAC10 has a total length of 1584 bp, including a complete ORF of 1107 bp that encodes 369 amino acids, with ATG as the start codon and TAG as the stop codon. Conserved domains program analysis showed that SlNAC10 had a conserved NAM domain containing the typical five subdomains of A, B, C, D, and E ( Figure 1A). The SlNAC10 sequence was analyzed using the Blastn program on the NCBI (http://www.ncbi.nlm.nih. gov, accessed on 21 May 2022) website, and a phylogenetic tree was constructed using MEGA 7.0. SlNAC10 had a high similarity to AtNAC19, AtNAM, and AtATAF, which suggested that SlNAC10 belonged to the NAM subfamily ( Figure 1B).

SlNAC10 Is a Stress-Responsive Gene
The expression levels of SlNAC10 in several organs of S. liaotungensis, as well as the response to various abiotic stressors and ABA stimulation, were determined by qRT-PCR. We found that the SlNAC10 gene was expressed in different organs, with the highest expression in leaves ( Figure 2A). Under drought stress, SlNAC10 gene expression was highest at 12 h ( Figure 2B); under salt stress, expression was significantly higher at 0.5 h and 24 h ( Figure 2C); under low-temperature stress at 4 • C, expression was highest at 3 h and higher at 24 h ( Figure 2D); and under ABA treatment, expression was significantly higher at 6 h than other periods ( Figure 2E). In response to drought, high salt, low-temperature stress, and ABA treatment, SlNAC10 was recognized as a stress-responsive gene.

SlNAC10 Is Located in the Nucleus and Has Transcriptional Activation Activity
The pEGAD-SlNAC10-GFP and pEGAD-GFP plasmids were injected into onion epidermal cells for transient expression. The SlNAC10-GFP protein was found only in the nucleus under laser confocal microscopy with 488 nm excitation light, whereas the GFP protein produced by the control pEGAD vector was found in the nucleus and cytoplasm of the cells (Figure 3). The results indicated that the SlNAC10 protein was localized in the nucleus.       The full-length coding sequence, N-terminal domain, and C-terminal domain of SlNAC10 were inserted into the pGBKT7 vector ( Figure 4A) and then the pGBKT7-SlNAC10-F (full length), pGBKT7-SlNAC10-N (N-terminal), pGBKT7-SlNAC10-C (C-terminal), and pGBKT7 empty vectors were transformed into yeast cells AH109. Each yeast cell grew normally on an SD/-Trp medium. Only pGBKT7-SlNAC10-F and pGBKT7-SlNAC10-C were able to grow normally on the SD/-Trp-His-Ade medium. β-galactosidase activity displayed a 6 of 18 blue color for pGBKT7-SlNAC10-F and pGBKT7-SlNAC10-C ( Figure 4B). The above results indicate that SlNAC10 has a transcriptional activation role and the transcriptional activation domain is at the C-terminus.
The full-length coding sequence, N-terminal domain, and C-terminal domain of SlNAC10 were inserted into the pGBKT7 vector ( Figure 4A) and then the pGBKT7-SlNAC10-F (full length), pGBKT7-SlNAC10-N (N-terminal), pGBKT7-SlNAC10-C (C-terminal), and pGBKT7 empty vectors were transformed into yeast cells AH109. Each yeast cell grew normally on an SD/-Trp medium. Only pGBKT7-SlNAC10-F and pGBKT7-SlNAC10-C were able to grow normally on the SD/-Trp-His-Ade medium. β-galactosidase activity displayed a blue color for pGBKT7-SlNAC10-F and pGBKT7-SlNAC10-C ( Figure  4B). The above results indicate that SlNAC10 has a transcriptional activation role and the transcriptional activation domain is at the C-terminus.
Root length and survival rates are often used as indicators of plant response to abiotic stresses. The determination of the root length of Arabidopsis seedlings grown on different MS media showed that the primary root lengths of all Arabidopsis seedlings were essentially the same when cultured in normal MS; in MS + 150 mM NaCl and MS + 200 mM Dmannitol, the primary root lengths of both the L1 and L2 lines were longer than those of the VT1, VT2, and WT lines ( Figure 5A,B). The survival rate of Arabidopsis thaliana under different treatments showed that the L1 and L2 lines survived significantly better than the VT1, VT2, and WT lines under salt stress treatment. The leaves of the VT1, VT2, and WT lines showed a loss of green color at 21 d of treatment and the VT1, VT2, and WT lines showed extensive mortality at 28 d of treatment with a survival rate of only about 30% ( Figure 5C,E). Under drought treatment, the leaves of Arabidopsis thinned and slowly deepened in color as the stress time increased and then began to shrivel and even die. The results of the survival rate of the lines after 3 d of rehydration showed that the L1 and L2 lines had a survival rate of over 50%, whereas only about 30% of the seedlings of the VT1, VT2, and WT lines continued to grow ( Figure 5D,F).
Root length and survival rates are often used as indicators of plant response to abiotic stresses. The determination of the root length of Arabidopsis seedlings grown on different MS media showed that the primary root lengths of all Arabidopsis seedlings were essentially the same when cultured in normal MS; in MS + 150 mM NaCl and MS + 200 mM Dmannitol, the primary root lengths of both the L1 and L2 lines were longer than those of the VT1, VT2, and WT lines ( Figure 5A,B). The survival rate of Arabidopsis thaliana under different treatments showed that the L1 and L2 lines survived significantly better than the VT1, VT2, and WT lines under salt stress treatment. The leaves of the VT1, VT2, and WT lines showed a loss of green color at 21 d of treatment and the VT1, VT2, and WT lines showed extensive mortality at 28 d of treatment with a survival rate of only about 30% ( Figure 5C,E). Under drought treatment, the leaves of Arabidopsis thinned and slowly deepened in color as the stress time increased and then began to shrivel and even die. The results of the survival rate of the lines after 3 d of rehydration showed that the L1 and L2 lines had a survival rate of over 50%, whereas only about 30% of the seedlings of the VT1, VT2, and WT lines continued to grow ( Figure 5D,F).  crease in the Fv/Fm values, whereas the WT, VT1, and VT2 lines did not recover chlorophyll fluorescence and the Fv/Fm values were almost all zero, indicating that Arabidopsis had died ( Figure 6B,D). Plants suffer varying degrees of damage to their membrane systems in adverse environments. Relative conductivity correlates with the amount of electrolyte leakage, allowing an indirect measure of plasma membrane permeability, and the amount of MDA in plants can reveal the extent of membrane lipid peroxidation and thus indirectly detect plant stress tolerance. The relative conductivity and MDA content of all lines of Arabidopsis were low in normal culture; when subjected to salt and drought stress, the relative conductivity and MDA content of all lines increased to varying degrees and were lower in the L1 and L2 lines than in the WT, VT1 and VT2 lines ( Figure 7A,B). These results indicated that the SlNAC10 transgenic Arabidopsis membrane system was less damaged under stress treatment.
Plants produce large amounts of reactive oxygen species under adverse conditions which can be scavenged by antioxidant enzymes. Based on an analysis of antioxidant enzyme activities, it was found that SOD, POD, and CAT activities were low in Arabidopsis of all plants grown in normal culture. When salt and drought stresses were encountered SOD, POD, and CAT activities increased to different degrees in all Arabidopsis lines. Additionally, SOD, POD, and CAT activities were significantly higher in the L1 and L2 lines than in the WT, VT1, and VT2 lines ( Figure 7C-E). Plants suffer varying degrees of damage to their membrane systems in adverse environments. Relative conductivity correlates with the amount of electrolyte leakage, allowing an indirect measure of plasma membrane permeability, and the amount of MDA in plants can reveal the extent of membrane lipid peroxidation and thus indirectly detect plant stress tolerance. The relative conductivity and MDA content of all lines of Arabidopsis were low in normal culture; when subjected to salt and drought stress, the relative conductivity and MDA content of all lines increased to varying degrees and were lower in the L1 and L2 lines than in the WT, VT1 and VT2 lines ( Figure 7A,B). These results indicated that the SlNAC10 transgenic Arabidopsis membrane system was less damaged under stress treatment.
Plants produce large amounts of reactive oxygen species under adverse conditions, which can be scavenged by antioxidant enzymes. Based on an analysis of antioxidant enzyme activities, it was found that SOD, POD, and CAT activities were low in Arabidopsis of all plants grown in normal culture. When salt and drought stresses were encountered, SOD, POD, and CAT activities increased to different degrees in all Arabidopsis lines. Additionally, SOD, POD, and CAT activities were significantly higher in the L1 and L2 lines than in the WT, VT1, and VT2 lines ( Figure 7C-E).
Proline is one of the most important osmoregulatory substances in plants. In our study, there was no statistically significant difference between the lines in the proline levels in Arabidopsis plants grown in normal culture. However, the proline content in all Arabidopsis lines increased to different degrees after salt and drought stress treatments, with the L1 and L2 lines having significantly higher proline content compared to the WT, VT1, and VT2 lines ( Figure 7F), meaning that SlNAC10 was able to improve the tolerance of Arabidopsis to adversity by accumulating proline. It is worth noting that proline accumulation is not the only mechanism that enhances plant stress resistance; there may be other mechanisms that act synergistically with proline and this will be the direction of our future research. Proline is one of the most important osmoregulatory substances in plants. In our study, there was no statistically significant difference between the lines in the proline levels in Arabidopsis plants grown in normal culture. However, the proline content in all Arabidopsis lines increased to different degrees after salt and drought stress treatments, with the L1 and L2 lines having significantly higher proline content compared to the WT, VT1, and VT2 lines ( Figure 7F), meaning that SlNAC10 was able to improve the tolerance of Arabidopsis to adversity by accumulating proline. It is worth noting that proline accumulation is not the only mechanism that enhances plant stress resistance; there may be other mechanisms that act synergistically with proline and this will be the direction of our future research.

SlNAC10 Upregulates the Expression of Proline Synthesis-Related Enzyme Genes
As a result of our study, we found that proline content was increased in SlNAC10 transgenic Arabidopsis thaliana under adverse conditions and the promoters of the proline

SlNAC10 Upregulates the Expression of Proline Synthesis-Related Enzyme Genes
As a result of our study, we found that proline content was increased in SlNAC10 transgenic Arabidopsis thaliana under adverse conditions and the promoters of the proline synthesis-related enzyme genes P5CS1, P5CS2, and P5CR all contained NAC binding sites (CGTG/A). Using qRT-PCR, we examined the expression of the AtP5CS1, AtP5CS2, and AtP5CR genes in the wild-type (WT) and transgenic plants (L1). The expression of the AtP5CS1, AtP5CS2, and AtP5CR genes remained essentially the same in the WT and L1 Arabidopsis plants under unstressed treatments, whereas the expression of each gene was higher in the L1 lines than in the WT lines under salt and drought stress, with the highest expression of AtP5CR (Figure 8). These results indicate that the expression of the AtP5CS1, AtP5CS2, and AtP5CR genes is induced by salt and drought in SlNAC10 transgenic Arabidopsis. expression of AtP5CR (Figure 8). These results indicate that the expression of the AtP5CS1, AtP5CS2, and AtP5CR genes is induced by salt and drought in SlNAC10 transgenic Arabidopsis. This was followed by a rapid examination of protein-DNA interactions using a yeast single-hybrid assay. Yeast growth can be inhibited by Aureobasidin A (AbA) and the pAbAi vector contains the AUR1-C and URA3 reporter genes. If SlNAC10 can bind to the bait element inserted at the pAbAi multiple cloning site, the AUR1-C reporter gene can be activated to allow the yeast to grow on an AbA-containing medium. In order to prevent the leakage of other yeast and reporter genes from affecting the results of the experiment, the AbA concentration that inhibited the growth of the bait yeast was screened and the final AbA concentration of 400 ng/mL was determined as the background expression level for the bait yeast. The constructed pGADT7-SlNAC10 recombinant vector and pGADT7 were then transferred into each bait yeast receptor cell and coated on different AbA concentrations of the medium. The results showed that the pGADT7-SlNAC10 vector could grow on the SD/-Leu medium at AbA concentrations of 0ng/mL and 400ng/mL, whereas the pGADT7 empty bait yeast strain failed to grow on the SD/-Leu medium at AbA concentrations of 400 ng/mL, demonstrating that SlNAC10 was able to bind to the AtP5CS1, AtP5CS2, and AtP5CR promoters (Figure 9).

Discussion
NAC transcription factors are one of the largest families of transcription factors in plants and play an important role in the transcription regulation of plant adaptation to environmental stresses. In our previous study, we found more than 20 NAC genes (SlNACs) in S. liaotungensis, of which SlNAC1, SlNAC2, SlNAC7, and SlNAC8 have all been shown to play important roles in the adaptation to abiotic stresses [33][34][35][36]. In this study, This was followed by a rapid examination of protein-DNA interactions using a yeast single-hybrid assay. Yeast growth can be inhibited by Aureobasidin A (AbA) and the pAbAi vector contains the AUR1-C and URA3 reporter genes. If SlNAC10 can bind to the bait element inserted at the pAbAi multiple cloning site, the AUR1-C reporter gene can be activated to allow the yeast to grow on an AbA-containing medium. In order to prevent the leakage of other yeast and reporter genes from affecting the results of the experiment, the AbA concentration that inhibited the growth of the bait yeast was screened and the final AbA concentration of 400 ng/mL was determined as the background expression level for the bait yeast. The constructed pGADT7-SlNAC10 recombinant vector and pGADT7 were then transferred into each bait yeast receptor cell and coated on different AbA concentrations of the medium. The results showed that the pGADT7-SlNAC10 vector could grow on the SD/-Leu medium at AbA concentrations of 0ng/mL and 400ng/mL, whereas the pGADT7 empty bait yeast strain failed to grow on the SD/-Leu medium at AbA concentrations of 400 ng/mL, demonstrating that SlNAC10 was able to bind to the AtP5CS1, AtP5CS2, and AtP5CR promoters (Figure 9). expression of AtP5CR (Figure 8). These results indicate that the expression of the AtP5CS1, AtP5CS2, and AtP5CR genes is induced by salt and drought in SlNAC10 transgenic Arabidopsis. This was followed by a rapid examination of protein-DNA interactions using a yeast single-hybrid assay. Yeast growth can be inhibited by Aureobasidin A (AbA) and the pAbAi vector contains the AUR1-C and URA3 reporter genes. If SlNAC10 can bind to the bait element inserted at the pAbAi multiple cloning site, the AUR1-C reporter gene can be activated to allow the yeast to grow on an AbA-containing medium. In order to prevent the leakage of other yeast and reporter genes from affecting the results of the experiment, the AbA concentration that inhibited the growth of the bait yeast was screened and the final AbA concentration of 400 ng/mL was determined as the background expression level for the bait yeast. The constructed pGADT7-SlNAC10 recombinant vector and pGADT7 were then transferred into each bait yeast receptor cell and coated on different AbA concentrations of the medium. The results showed that the pGADT7-SlNAC10 vector could grow on the SD/-Leu medium at AbA concentrations of 0ng/mL and 400ng/mL, whereas the pGADT7 empty bait yeast strain failed to grow on the SD/-Leu medium at AbA concentrations of 400 ng/mL, demonstrating that SlNAC10 was able to bind to the AtP5CS1, AtP5CS2, and AtP5CR promoters (Figure 9).

Discussion
NAC transcription factors are one of the largest families of transcription factors in plants and play an important role in the transcription regulation of plant adaptation to environmental stresses. In our previous study, we found more than 20 NAC genes (SlNACs) in S. liaotungensis, of which SlNAC1, SlNAC2, SlNAC7, and SlNAC8 have all been shown to play important roles in the adaptation to abiotic stresses [33][34][35][36]. In this study,

Discussion
NAC transcription factors are one of the largest families of transcription factors in plants and play an important role in the transcription regulation of plant adaptation to environmental stresses. In our previous study, we found more than 20 NAC genes (SlNACs) in S. liaotungensis, of which SlNAC1, SlNAC2, SlNAC7, and SlNAC8 have all been shown to play important roles in the adaptation to abiotic stresses [33][34][35][36]. In this study, we cloned a new NAC transcription factor, SlNAC10, from S. liaotungensis and demonstrated that this gene improved tolerance to salt and drought stress in transgenic Arabidopsis by directly regulating proline synthesis-related enzyme gene expression and proline synthesis.
The full length of the SlNAC10 cDNA was obtained from the EST sequence in the transcriptome database. The multiple sequence alignment showed that the N terminus of the gene had high similarity to the N terminus of other NACs and had a conserved NAC structural domain, which was identified as a NAC transcription factor. By constructing a phylogenetic tree, SlNAC10 was shown to be highly similar to AtNAC19, AtNAM, and AtATAF (Figure 1), with AtNAM having a role in stress resistance [37], and it is hypothesized that SlNAC10 may also have a role in the response to stress resistance.
Typical transcription factors have a nuclear localization signal. For example, the MaNAC1-MaNAC5 proteins in bananas were distributed in the nucleus and MaNAC6 was distributed throughout the entire cell [38]. The ChNAC1 transcription factor of the Chinese dwarf cherry (Cerasus humilis) was localized to the cell nucleus [39]. The subcellular localization analysis in this study showed that SlNAC10-GFP was distributed in the nucleus (Figure 3). Because transcription factors often have transcription activation functional regions that regulate downstream genes at the transcription level, many studies have examined the transcription activation activity of the NAC transcription factors, for example, the Brassica napus protein BnNAC2 has transcription activation activity but BnNAC5 has no transcription activation activity [40]. A novel Miscanthus NAC gene MlNAC12 has a transactivation activity in the C-terminus [41]. Our study of the transactivation activity of SlNAC10 revealed that the C-terminus of SlNAC10 has transcriptional activation activity, demonstrating that SlNAC10 is a transcription factor and plays a role in the transcription of genes (Figure 4).
An increasing number of studies have reported the involvement of NAC transcription factors in plant responses to abiotic stresses, including salt, drought, and cold. For example, GmNAC06 transcript levels were the highest in soybean cotyledons and GmNAC06 expression was significantly increased in leaves and roots after induction by salt, ABA, cold, and PEG [42]. The expression level of TaNAC29 was higher in wheat leaves and the transcription of TaNAC29 increased after salt, PEG6000, H 2 O 2, and ABA treatments [43]. In this study, SlNAC10 was induced by various abiotic stress treatments and the expression was significantly different in the roots, stems, and leaves of S. liaotungensis (Figure 2). In addition, we focused on the role of SlNAC10 in response to salt and drought stresses. Longer primary root lengths and higher survival rates in SlNAC10 transgenic Arabidopsis under salt and drought conditions were observed, presumably because SlNAC10 enhanced the ability of transgenic Arabidopsis to withstand stress ( Figure 5). This is similar to the way that OsNAC6-overexpressing rice lines show higher drought tolerance and OsNAC6 improves adaptation to adversity by increasing the number of roots and expanding the root diameter [44]. RhNAC31 overexpression is associated with increased cold tolerance in Arabidopsis, conferring a higher survival rate [45]. We subsequently found that the leaves of SlNAC10 transgenic Arabidopsis thaliana under salt and drought stress treatments retained more green color to maintain photosynthetic activity, resulting in a higher survival rate for transgenic Arabidopsis, whereas non-transgenic Arabidopsis wilted, yellowed, and died. The Fv/Fm values of Arabidopsis were measured under stress treatment and it was found that the transgenic Arabidopsis maintained higher photosynthetic activity under stress conditions, indicating that the SlNAC10 gene contributes to plant stress tolerance ( Figure 6). The strength of photosynthesis directly affects the growth and development of Arabidopsis plants. The magnitude of the photosynthetic capacity is limited by its own photosynthetic system, for example, the transgenic lines of banana cultivar Rasthali overexpressing MusaNAC042 can maintain higher chlorophyll levels and better Fv/Fm values in stressful environments [46]. The Fv/Fm images and chlorophyll content revealed that the WT plants were significantly damaged compared with the CmNAC1-EE plants under salt stress [47].
In an adverse environment, the relative conductivity and MDA content can reflect the integrity of the plasma membrane. The antioxidant enzyme system is the mainstay of enzymatic defense in an adverse environment and is responsible for the removal of reactive oxygen species from the body. In addition, stress treatments can cause an increase in osmoregulatory substances and proline is one of the most important osmoregulatory substances in the plant. Studies have shown that transgenic Arabidopsis with high expression of TtNAC2A has greater water retention, weaker oxidative stress, lower electrolyte leakage, and higher proline content compared to wild-type plants [48]. Chrysanthemum (Dendranthema morifolium) DgNAC1 overexpressing showed lower electrical conductivity under salt stress, whereas the transgenic chrysanthemum showed reduced accumulation of MDA and reactive oxygen species; increased SOD, CAT, and POD activities; and increased proline content, enhancing the resistance of chrysanthemum to salt stress [49]. The LpNAC13 overexpression plants had increased antioxidant enzyme activities and proline content, and decreased MDA content under salt conditions [50]. In our study, salt and drought stresses in all lines of Arabidopsis caused an increase in MDA content and relative electrolyte leakage in the plants but to a lesser extent in the transgenic Arabidopsis, indicating that the membranes of the transgenic Arabidopsis with the SlNAC10 gene were less damaged and more resistant to the adverse environment. Furthermore, the transgenic Arabidopsis had higher antioxidant enzyme activity and proline content under stress conditions, which fully coordinated the enzymatic defense system in the plant and regulated the content of osmoregulatory substances, and could better adapt to the adverse environment, which was also a reflection of the enhanced stress resistance of the transgenic Arabidopsis with the SlNAC10 gene ( Figure 7).
Plants accumulate and produce large amounts of proline under adverse environmental conditions and the accumulation of proline reflects the plant's resistance to unfavorable conditions. Other studies have found that under salt stress, proline content was increased in ScPIP2-1-overexpressing Arabidopsis and the AtP5CS1 and AtP5CS2 genes were highly expressed, demonstrating the enhanced salt tolerance of transgenic Arabidopsis [51]. Overexpression of the cotton GhMYB4 gene leads to proline accumulation through the upregulation of AtP5CS and AtP5CR expression, ultimately leading to enhanced salt tolerance and drought resistance in transgenic Arabidopsis [52]. The yeast single-hybrid assay can measure the interaction between the DNA and plant transcription factors. For example, the ability of CmCyC2c to bind to the ClCyC2f promoter was demonstrated by a yeast single hybridization assay, providing clues that CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in C. morifolium [53]. Yeast single hybridization also confirmed the binding of CsATAF1 to the CsABI5, CsCu-ZSOD, and CsDREB2C promoters, providing evidence that CsATAF1 may be a transcriptional activator regulating CsCu-ZnSOD, CsABI5, and CsDREB2C [54]. In this study, we found that SlNAC10 could enhance the resistance to abiotic stresses of transgenic Arabidopsis by up-regulating the expression of proline synthesis-related enzyme genes ( Figure 8). In addition, SlNAC10 was suggested to bind to the promoters of the AtP5CS1, AtP5CS2, and AtP5CR genes and regulate the transcription of their downstream genes by a yeast single hybridization assay (Figure 9).
In summary, our study shows that SlNAC10 can improve the tolerance of transgenic Arabidopsis to salt and drought stresses. Therefore, we believe that SlNAC10 has a potential utility as a natural novel resource for the cultivation of new crop varieties in saline or arid regions.

Plant Material and Treatment
Suaeda liaotungensis leaves and seeds were collected in Yingchengzi, Dalian, Liaoning, China. The leaves were frozen in liquid nitrogen and stored in a refrigerator at −80 • C for the cloning of the SlNAC10 gene. Seeds were stored in a refrigerator at 4 • C and seedlings were raised in a greenhouse (25 • C, 16 h of light/8 h of dark, 50-65% humidity) for the study of the SlNAC10 expression profile.
Arabidopsis thaliana (Columbia ecotype) was used for the gene transformation and phenotypic analysis. Arabidopsis seeds were grown on MS solid medium for 10 days at 22 • C with constant light, and seedlings were transplanted in a 1:1 soil mixture of humus and vermiculite and cultured in a greenhouse (23 • C, 16 h of light/8 h of dark).

Cloning and Sequence Analysis of the SlNAC10 Gene
RNAiso Plus (Takara, Beijing, China) was used to extract RNA from S. liaotungensis leaves, and total RNA was used as a template for reverse transcription into cDNA using PrimeScript ® RT Master Mix Perfect Real Time (Takara, Beijing, China). To obtain a partial fragment of SlNAC10, primers (Table S1, EST-F and EST-R) were designed based on the EST sequence of SlNAC10. The primers (Table S1, 3 -Outer, 3 -Inner and 5 -Outer, 5 -Inner) were then used to perform 3 , 5 RACE to obtain the cDNA sequence of SlNAC10. Finally, the full length of SlNAC10 was obtained by amplification using primers (Table S1, full length-F and full length-R) and was cloned into a pEASY-T1 vector (TransGen Biotech, Beijing, China).
We performed a BLAST search of the NCBI database (http://www.ncbi.nlm.nih.gov) to identify the sequences homologous to SlNAC10 and performed multiple sequence comparisons using DNAMAN software (version 6.0.40, LynnonBiosoft, CA, USA). Using MEGA software (version 7.0, Mega Limited, Auckland, New Zealand), a phylogenetic tree was then constructed from the NACs of other plants, which were found to have high similarity to SlNAC10. DNAMAN was used for the multiple sequence alignment and the NJ method in the MEGA 7.0 software was used to construct the evolutionary trees.

Growth Assay of SlNAC10 Transgenic Arabidopsis under Salt and Drought Stress
The coding region of SlNAC10 was cloned into a pBI121 vector. The pBI121-SlNAC10 and pBI121 were transformed into Agrobacterium tumefaciens GV3101 by the freeze-thaw method and then into Arabidopsis thaliana (Columbia ecotype) by the floral dipping method, respectively. Homozygous transgenic Arabidopsis lines were obtained by screening on an MS medium containing 100 mg/L kanamycin and determined by PCR and semi-quantitative RT-PCR. Relative gene expression was calculated using the 2 −∆∆CT method [55].
Subsequently, the measurements of the primary root length and survival ratio of Arabidopsis thaliana under salt stress and drought stress were carried out using T3 homozygous transgenic Arabidopsis and wild-type (WT) plants. Seeds of the above Arabidopsis lines were sown in MS solid medium with 0, 150 mM NaCl, and 200 mM D-mannitol. After 7 d, plants of equal growth were transferred to square Petri dishes containing MS + 150 mM NaCl and MS + 200 mM D-mannitol medium and cultured vertically for 8 d. The main root lengths were measured. Arabidopsis thaliana cultured normally for one week with almost the same growth were treated with stress (salt stress was applied by pouring 1 L of 300 mM NaCl solution every 7 d for 21 d and after 21 d of drought stress, the plants were re-watered for 3 d). The number of survivors of each plant was then counted and the survival rate was calculated.

Determination of Physiological Indicators of SlNAC10 Transgenic Arabidopsis Thaliana
One-week-old wild-type and transgenic Arabidopsis seedlings were selected and treated with salt stress (1 L of 300 mM NaCl solution every 7 d for 21 d) and drought stress (no watering for 21 d and re-watering for 3 d). The Fv/Fm and photochemical parameters were measured every 7 d starting from before the stress treatment using a chlorophyll fluorescence imager (IMAGING-PAM, Walz GmbH, Bad Waldsee, Germany) to take photographs.
The above well-grown Arabidopsis lines were subjected to 15 d of salt stress (1 L of 300 mM NaCl solution every 7 d) and drought stress (no watering). The relative conductivity in leaves was determined using a conductivity meter (DDS-11A, Shanghai Pengshun Technology Co., Ltd., Shanghai, China) [56]. In addition, the MDA content and the CAT, SOD, and POD activities, as well as the proline content, of the leaves were also measured [26].

Expression of P5CS1, P5CS2, and P5CR in Arabidopsis thaliana Overexpressing the SlNAC10 Gene
When the Arabidopsis seedlings of the WT and trans-SlNAC10 type had grown 10 leaves, they were transferred, respectively, to a one-half MS liquid medium, one-half MS liquid medium containing 200 mM NaCl, and one-half MS liquid medium containing 10% PEG for 6 h. The true leaves of the seedlings from different plants were then cut and RNA was extracted using RNAiso Plus (TAKARA, Beijing, China). TransScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix were used for reverse transcription. Primers (Table S1, AtP5CS1-qF, AtP5CS1-qR, AtP5CS2-qF, AtP5CS2-qR, AtP5CR-qF, AtP5CR-qR) for qRT-PCR were designed based on the sequences of AtP5CS1 (GenBank: AT2G39800), AtP5CS2 (GenBank: AT3G55610), and AtP5CR (GenBank: AT5G14800). Atactin2 (GenBank: NM_112764) was used as the internal reference gene and primers (Table S1, AtActin-qF and AtActin-qR) were designed based on its sequence. Relative gene expression was calculated using the 2 −∆∆CT method.

Analysis of the Interaction between SlNAC10 and the P5CS1, P5CS2, and P5CR Promoters
The core regions of the AtP5CS1, AtP5CS2, and AtP5CR promoters were predicted using BDGP: Neural Network Promoter Prediction online software, and then the promoter core regions were amplified using primers (Table S1, AtP5CS1-F, AtP5CS1-R, AtP5CS2-F, AtP5CS2-R, AtP5CR-F, AtP5CR-R). Yeast bait vectors pAbAi-P5CS1, pAbAi-P5CS2, and pAbAi-P5CR were constructed and linearized, transformed into Y1H Gold receptor cells using the Quick Easy Yeast Transformation Mix kit, and then assayed for background expression levels of the bait yeast reporter genes. Relative gene expression was calculated using the 2 −∆∆CT method [55].
The pGADT7-SlNAC10 vector was constructed by amplifying the SlNAC10 gene using primers (Table S1, SlNAC10-F and SlNAC10-R) and cloning it into the pGADT7 vector. The bait yeast strain was prepared as the receptor cells, and the pGADT7-SlNAC10 plasmid was transformed into the receptor cells of the bait yeast strain and coated in an SD/-Leu medium with an AbA concentration of 0 ng/mL and AbA concentration of 400 ng/mL, which was incubated at 30 • C for 3 to 5 days at a constant temperature with pGADT7 as the control, to verify whether the transcription factor SlNAC10 interacts with the promoters AtP5CS1, AtP5CS2, and AtP5CR.

Statistical Analysis
Statistical analysis was performed by ANOVA using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) and p < 0.05 was considered significant. Each experiment was performed in triplicate (transcriptional analysis n = 3; primary root length and survival analysis n = 60; physiological analysis n = 10).

Conclusions
In this study, a new NAC transcription factor gene, SlNAC10, was cloned and identified as a transcription activator from the halophyte S. liaotungensis. Through a comprehensive analysis of SlNAC10 in S. liaotungensis and transgenic Arabidopsis thaliana at the molecular, morphological, and physiological biochemistry levels, SlNAC10 was confirmed to participate in abiotic stress responses and enhance salt and drought tolerance in plants.
Proline is an important osmotic regulator in plants and can improve plant resistance under stressful conditions. Our study revealed that the proline content in SlNAC10 transgenic Arabidopsis was increased and the expression of proline synthesis-related enzyme genes (AtP5CS1, AtP5CS2, and AtP5CR) were upregulated under salt and drought stress. Moreover, SlNAC10 can bind to the AtP5CS1, AtP5CS2, and AtP5CR promoters and regulate the transcription of their downstream genes. Our results demonstrate that SlNAC10 directly regulates the expression of proline synthesis-related enzyme genes and increases the proline synthesis, thus enhancing the salt and drought tolerance in plants. Therefore, we believe that SlNAC10 can be studied in more depth to produce new varieties of stress-resistant crops ( Figure 10). Funding: This research was funded by grants from the National Natural Science Foundation of Figure 10. Technology roadmap of this study.