Tamarix hispida NAC Transcription Factor ThNAC4 Confers Salt and Drought Stress Tolerance to Transgenic Tamarix and Arabidopsis

Salt and drought are considered two major abiotic stresses that have a significant impact on plants. Plant NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) have been shown to play vital roles in plant development and responses to various abiotic stresses. ThNAC4, a NAC gene from Tamarix hispida involved in salt and osmotic stress tolerance, was identified and characterized in this study. According to a phylogenetic study, ThNAC4 is a member of NAC subfamily II. Subcellular localization analysis showed that ThNAC4 is located in the nucleus, and transcriptional activation experiments demonstrated that ThNAC4 is a transcriptional activator. Transgenic Arabidopsis plants overexpressing ThNAC4 exhibited improved salt and osmotic tolerance, as demonstrated by improved physiological traits. ThNAC4-overexpressing and ThNAC4-silenced T. hispida plants were generated using the transient transformation method and selected for gain- and loss-of-function analysis. The results showed that overexpression of ThNAC4 in transgenic Tamarix and Arabidopsis plants increased the activities of antioxidant enzymes (SOD, POD, and GST) and osmoprotectant (proline and trehalose) contents under stress conditions. These findings suggest that ThNAC4 plays an important physiological role in plant abiotic stress tolerance by increasing ROS scavenging ability and improving osmotic potential.


Introduction
Plants grow in dynamic environments in which factors such as drought, salt, extreme temperature, and biotic stresses frequently constrain growth and development. Among these stresses, salt and drought are two major stresses that have a significant impact on plants. To combat and survive these stresses, plants have developed several adaptive mechanisms involving signal transduction and gene expression regulation.
Transcription factors (TFs) are DNA-binding proteins that regulate the expression of downstream genes as trans-acting elements via specific binding to the cis-acting elements present in the promoter region of the target genes [1]. Many transcription factors, such as AP2/ERF, bZIP, MYB, WRKY, and NAC, have been found to play critical roles in the plant response to various abiotic stimuli [2][3][4][5][6]. Among them, NAC (NAM, ATAF1/2, and CUC2) TFs are one of the largest plant-specific TF families that play important roles in the regulation of plant abiotic stress responses. For instance, in Arabidopsis, ANAC019, ANAC055, and ANAC072 are induced by drought, salinity, and abscisic acid (ABA), and overexpression of these NAC genes confers enhanced drought tolerance in transgenic Arabidopsis [7,8]. OsNAC5, OsNAC6, and OsNAC10, three ABA-dependent NAC TFs

Cloning and Bioinformatics Analysis of ThNAC4 from Tamarix hispida
The open reading frame (ORF) of ThNAC4 is 1158 bp in length and is predicted to encode a protein of 387 amino acid residues with a molecular weight of 42.65 kDa. Multiple sequence alignment analysis revealed that ThNAC4 and NAC proteins from other plant species shared a highly conserved binding domain at the N-terminus, which is the basic characteristic of the NAC family ( Figure S1). A phylogenetic tree was constructed to study the evolutionary relationships between ThNAC4 and 94 other NACs from Arabidopsis. This analysis showed that ThNAC4 is closely related to two Arabidopsis NAC proteins, ANAC080/ATNAC4 (AT5G07680) and ANAC100/ATNAC5 (AT5G61430), from NAC subfamily II (Figure 1), suggesting that ThNAC4 should also belong to NAC subfamily II.

ThNAC4 Is Localized in the Nucleus and Exhibits Transactivation Activity
The N-terminus of NAC family proteins has a highly conserved domain that might serve as a nuclear localization signal (NLS). Numerous NACs are located in the nucleus of cells [22,23]. To determine whether ThNAC4 is located in the nucleus, we transformed ThNAC4-GFP fusion proteins into onion epidermal cells by the Agrobacterium-mediated transformation method, and 35S-GFP functioned as a control. As shown in Figure 2A, free GFP was distributed uniformly in the cytoplasm, whereas the ThNAC4-GFP fusion proteins were detected in the nuclei. This finding indicates that ThNAC4 is a nuclear protein and supports the idea that ThNAC4 functions as a transcription factor.
Transcriptional activation ability was investigated according to Hu et al. [24]. The entire CDS or serial deletions of the ThNAC4 CDS were combined with pGBKT7 plasmids and transformed into Y2HGold cells to detect their transactivation activity ( Figure 2B). All transformants could grow normally on the SD/-Trp plates, showing that they had been transformed into Y2HGold cells. The yeast cells containing the full-length CDS of ThNAC4 grew well and appeared blue on the SD/-Trp/-His/X-a-Gal plates, suggesting that ThNAC4 functions as a transcriptional activator. Furthermore, we noticed that the yeast cells transformed with the predicted C-terminal activation domain (amino acids 182-386) of ThNAC4 thrived and became blue, whereas cells expressing the N-terminal domain (amino acids 1-193) of ThNAC4 were not blue, showing that the C-terminal domain of ThNAC4 has transactivation activity. In addition, analysis of the truncated CDSs of ThNAC4 suggested that ThNAC4 contains the transcriptional activation domain at the region of amino acids 175-291. The N-terminus of NAC family proteins has a highly conserved domain that might 95 serve as a nuclear localization signal (NLS). Numerous NACs are located in the nucleus 96 of cells [22,23]. To determine whether ThNAC4 is located in the nucleus, we transformed 97 ThNAC4-GFP fusion proteins into onion epidermal cells by the Agrobacterium-mediated 98 transformation method, and 35S-GFP functioned as a control. As shown in Figure 2A, free 99 GFP was distributed uniformly in the cytoplasm, whereas the ThNAC4-GFP fusion pro-100 teins were detected in the nuclei. This finding indicates that ThNAC4 is a nuclear protein 101 and supports the idea that ThNAC4 functions as a transcription factor. 102 103 Figure 1. Phylogenetic analysis of ThNAC4 and NAC proteins from Arabidopsis. The phylogenetic relationship of ThNAC4 and NAC proteins from Arabidopsis. ThNAC4 and the Arabidopsis NACs were aligned; the unrooted NJ tree was constructed using MEGA 7.

Constitutive Expression of ThNAC4 Enhances Salt and Osmotic Resistance in Transgenic Arabidopsis
To reveal the molecular function of ThNAC4 in vivo under salt and osmotic stress, we obtained seven independent T3 homozygous Arabidopsis lines overexpressing ThNAC4. Three independent transgenic Arabidopsis lines (OE2, OE8, and OE9) with high transcript levels of ThNAC4 were selected for further experiments ( Figure S2). Under normal growth conditions, the seed germination, growth phenotype, fresh weight, and root length were not different between transgenic Arabidopsis and wild-type (WT) plants ( Figure 3). However, after treatment with NaCl (150 mmol/L) and mannitol (200 mmol/L), the three tested transgenic lines were greener and exhibited less wilting than WT plants, and they displayed significantly improved seed germination rates, root length, and fresh weight ( Figure 3B-F). In addition, the chlorophyll contents were similar among the three transgenic lines and WT plants under normal conditions. However, under salt and osmotic stress conditions, all transgenic lines had significantly higher chlorophyll levels than WT plants ( Figure 3G). Moreover, the water loss assay indicated that the water loss rates of transgenic Arabidopsis were lower than those of WT plants ( Figure 3H). These results together indicated that the constitutive expression of ThNAC4 conferred enhanced tolerance to NaCl and mannitol treatment in transgenic plants. Transcriptional activation ability was investigated according to Hu et al. [24]. The 110 entire CDS or serial deletions of the ThNAC4 CDS were combined with pGBKT7 plasmids 111 and transformed into Y2HGold cells to detect their transactivation activity ( Figure 2B). All 112 transformants could grow normally on the SD/-Trp plates, showing that they had been 113 transformed into Y2HGold cells. The yeast cells containing the full-length CDS of ThNAC4 114 grew well and appeared blue on the SD/-Trp/-His/X-a-Gal plates, suggesting that 115 ThNAC4 functions as a transcriptional activator. Furthermore, we noticed that the yeast 116 cells transformed with the predicted C-terminal activation domain (amino acids 182-386) 117 of ThNAC4 thrived and became blue, whereas cells expressing the N-terminal domain 118 (amino acids 1-193) of ThNAC4 were not blue, showing that the C-terminal domain of 119 ThNAC4 has transactivation activity. In addition, analysis of the truncated CDSs of 120 ThNAC4 suggested that ThNAC4 contains the transcriptional activation domain at the 121 region of amino acids 175-291. Abiotic stresses can lead to oxidative damage due to the increased production of reactive oxygen species (ROS). Therefore, plants need to activate antioxidative systems to cope with oxidative damage. To determine the accumulation of two main ROS species, superoxide anion (O 2 − ) and hydrogen peroxide (H 2 O 2 ), in transgenic and WT Arabidopsis plants under osmotic and salt stresses, nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining were performed. There were no significant differences in O 2 − and H 2 O 2 levels between transgenic lines and WT plants under normal growth conditions. However, both the O 2 − and H 2 O 2 accumulation levels were lower in transgenic lines than in WT plants under both osmotic and salt stress conditions ( Figure 4A,B). We determined the level of cell death in leaves under salt and osmotic stress conditions using Evans blue staining. The results showed that the level of cell membrane damage was lower in the transgenic lines than in the WT plants under both salt and osmotic stress conditions ( Figure 4C). The malondialdehyde (MDA) content was analyzed to investigate membrane lipid peroxidation levels. Likewise, the transgenic Arabidopsis accumulated less MDA than WT under salt and osmotic stresses ( Figure 4D). In addition, the activities of the two main antioxidant enzymes, superoxide dismutase (SOD) and peroxidase (POD), induced by NaCl and mannitol treatments were analyzed in transgenic and WT plants. The activities of SOD and POD were significantly higher in transgenic lines than in WT plants under both salt and osmotic stresses ( Figure 5E,F). Glutathione S-transferases (GSTs) are involved in catalyzing detoxification processes in plant development and plant stress responses, including GSH-dependent POD reactions that scavenge toxic organic hydroperoxides [25]. In this study, we also measured GST activity in transgenic and WT Arabidopsis plants. GST activity in all transgenic plants was higher than that in WT plants under salt and osmotic stress conditions ( Figure 4G), indicating that overexpression of ThNAC4 enhanced GST activity in transgenic Arabidopsis. These results indicated that overexpression of ThNAC4 could improve ROS scavenging enzyme activity to lower ROS accumulation and limit cell damage in transgenic Arabidopsis under salt and osmotic stress conditions. Three-week-old seedlings in soil were treated with 150 mM NaCl or 200 mM mannitol for one week. Photographs were taken, and the chlorophyll content and water loss were measured. The error bars represent the standard deviations of the mean measurements, which were calculated from three independent experiments. * indicates a significant difference (p < 0.05) between transgenic lines and WT plants.
We next studied whether ThNAC4 is involved in osmotic protectant and compatible solute accumulation under salt and osmotic stress conditions. The proline and trehalose contents were measured in comparison to the WT and transgenic Arabidopsis. The results showed that there was no distinct difference in proline and trehalose contents between transgenic and WT Arabidopsis plants under normal growth conditions. However, under conditions of salt and osmotic stresses, the contents of these osmolytes were significantly higher in transgenic Arabidopsis plants than in WT plants ( Figure 4H,I), suggesting that ThNAC4 plays a role in the regulation of osmotic protectant and compatible solute biosynthesis. from three independent experiments. * indicates a significant difference (p < 0.05) between transgenic lines and WT plants. 189 We next studied whether ThNAC4 is involved in osmotic protectant and compatible 190 solute accumulation under salt and osmotic stress conditions. The proline and trehalose 191 contents were measured in comparison to the WT and transgenic Arabidopsis. The results 192 showed that there was no distinct difference in proline and trehalose contents between 193 transgenic and WT Arabidopsis plants under normal growth conditions. However, under 194 conditions of salt and osmotic stresses, the contents of these osmolytes were significantly 195 higher in transgenic Arabidopsis plants than in WT plants ( Figure 4H, I), suggesting that 196 ThNAC4 plays a role in the regulation of osmotic protectant and compatible solute bio-197 Additionally, we studied the genes involved in SOD, POD, and trehalose biosynthesis in transgenic and WT Arabidopsis plants with NaCl and mannitol treatments using the qRT-PCR method. The results showed that the expression of many studied genes was highly increased in ThNAC4-transformed Arabidopsis under NaCl and mannitol treatments ( Figure 5). These results indicate that ThNAC4 can increase the transcription of the SOD, POD, TPS, and TPP genes, which may contribute to increased SOD and POD activities and trehalose content under salt and drought stress.
Plants 2021, 10, x FOR PEER REVIEW 9 of 18 highly increased in ThNAC4-transformed Arabidopsis under NaCl and mannitol treatments ( Figure 5). These results indicate that ThNAC4 can increase the transcription of the SOD, POD, TPS, and TPP genes, which may contribute to increased SOD and POD activities and trehalose content under salt and drought stress.

Transient Overexpression or Knockdown of ThNAC4 in T. hispida Plants
To further study the function of ThNAC4 using gain-and loss-of-function methods, we generated ThNAC4-overexpressing (OE), RNAi-silenced ThNAC4 (IE), and control plants (transformed with empty pROKII, VC) and examined the expression of ThNAC4 using the qRT-PCR method. The expression of ThNAC4 was significantly increased in the OE plants but was significantly decreased in the IE plants compared to those in the control (VC) plants ( Figure S3). These results indicated that ThNAC4 had been successfully overexpressed or knocked down and was suitable for further study.

ThNAC4 Affects ROS Scavenging Capability
ROS accumulation is a substantial indicator of the degree of stress tolerance. Therefore, NBT and DAB staining were used to determine two major ROS species, superoxide anion (O 2 -) and hydrogen peroxide (H 2 O 2 ), in these transgenic T. hispida plants. Under salt and osmotic stress conditions, compared with the control plants, OE T. hispida plants exhibited reduced NBT and DAB staining, while IE T. hispida plants displayed significantly increased NBT and DAB staining ( Figure 6A). Moreover, the MDA contents were measured in transgenic T. hispida plants. Under normal growth conditions, OE T. hispida plants had lower MDA levels than control and IE T. hispida plants. When exposed to salt or osmotic stress, IE T. hispida plants had significantly higher MDA levels, whereas OE plants had significantly lower MDA levels than control plants ( Figure 6B). In addition, OE T. hispida plants also displayed higher chlorophyll contents than control and IE plants ( Figure 6C).
We further studied the activities of three primary ROS scavenging enzymes, SOD, POD, and GST, in the three types of transiently transformed T. hispida plants. Under salt and osmotic stress conditions, the OE plants had significantly higher SOD, POD, and GST activities than the control and IE plants ( Figure 6D,E). These results indicate that ThNAC4 expression enhances ROS scavenging capability by increasing the activities of antioxidant enzymes.

Analysis of Proline and Trehalose Contents
The proline and trehalose contents were measured in all transgenic T. hispida plants to study whether ThNAC4 is involved in the biosynthesis of these osmolytes in salt or osmotic stress environments. As shown in Figure 6G,H, there was no significant difference in proline and trehalose contents between the three types of transgenic T. hispida seedlings under normal conditions. However, under salt and osmotic stress conditions, the OE T. hispida plants had significantly higher proline and trehalose levels, followed by the IE and control plants. These results are consistent with the results from transgenic Arabidopsis, indicating that overexpression of ThNAC4 increases osmolyte contents under salt and osmotic stresses.

258
Salt and drought are two major stresses that have a significant impact on plants. 259 When exposed to drought stress, plants display a reduced photosynthetic rate and lower 260 chlorophyll content. Furthermore, long-term drought stress also leads to oxidative dam-261 age because of increased ROS accumulation, which disrupts cellular physiological home-262 ostasis [26]. The first stage of salinity stress restricts plant growth in the form of osmotic 263 stress, which is then followed by ion toxicity, which causes various physiological changes 264 in plant cells [27]. 265 Plants have developed various defense mechanisms to combat the negative impacts 266 of abiotic stress, and transcriptional control of gene expression plays an important role in 267

Discussion
Salt and drought are two major stresses that have a significant impact on plants. When exposed to drought stress, plants display a reduced photosynthetic rate and lower chlorophyll content. Furthermore, long-term drought stress also leads to oxidative damage because of increased ROS accumulation, which disrupts cellular physiological homeostasis [26]. The first stage of salinity stress restricts plant growth in the form of osmotic stress, which is then followed by ion toxicity, which causes various physiological changes in plant cells [27].
Plants have developed various defense mechanisms to combat the negative impacts of abiotic stress, and transcriptional control of gene expression plays an important role in this process. Plant-specific NAC TFs are one of the key regulators in plant responses to various abiotic stresses, and their function has been studied in a variety of plant species [28]. However, few studies have investigated the abiotic tolerance function of NAC TFs in halophytic woody plants. Therefore, identifying and characterizing novel stress-responsive NAC TFs from halophytes such as T. hispida can provide more insight into this special group of transcription factors.
Previously, a total of 21 full-length NAC genes were identified in T. hispida. qRT-PCR analysis revealed that these ThNAC genes are all expressed in the roots, stems, and leaves of T. hispida in response to salinity, drought, heavy metal, or ABA stimuli, suggesting that they play important roles in the abiotic stress response and are involved in ABAdependent stress-signaling pathways [21]. Therefore, we selected ThNAC4 for further research. Multiple sequence alignment analysis indicated that ThNAC4 contains a classic NAC domain that is the basic characteristic of the NAC family. Based on phylogenetic tree analysis, ThNAC4 falls into NAC subfamily II ( Figure 1) and is most similar to ATNAC4 (AT5G07680) gene in Arabidopsis, which responds to biotic and abiotic stresses [29,30]. According to previous studies, ThNAC4 was included in NAC subfamily I in T. hispida, which contains ThNAC13 and ThNAC7. Moreover, these NAC genes were found to enhance tolerance to salt and osmotic stress by improving the osmotic potential and increasing ROS scavenging [31,32]. Therefore, we decided to further study the role of ThNAC4 in salt and osmotic stresses.
Transcriptional activation assays were performed to investigate whether ThNAC4 activated transcription and to identify the activation domain. The results suggested that ThNAC4 is a transcriptional activator and that the transcriptional activation domain is located in a region containing amino acids 175-291 of ThNAC4 (Figure 2).
Previous research has shown that NAC TFs play important roles in the response to both salinity and drought and identified increased expression of genes associated with tolerance to these stresses [28,32,33]. Therefore, three independent ThNAC4 transgenic Arabidopsis lines (OE2, OE5, and OE9) and three types of transiently transformed T. hispida (OE, IE, and VC) were used to analyze the molecular function of ThNAC4 under salt and osmotic stresses. Growth phenotype, seed germination, root length, fresh weight, and chlorophyll content are important markers for evaluating stress tolerance in plants. Our results showed that overexpression of ThNAC4 significantly improved these physiological parameters in both transgenic Arabidopsis and T. hispida plants (Figures 3 and 6), suggesting that ThNAC4 plays a positive role in plant tolerance to salt and osmotic stress.
Reactive oxygen species (ROS) are involved in the oxidation of requisite biomolecules and act as a signal under abiotic stress conditions [34]. However, plants accumulate excessive ROS when exposed to an adverse environment, which causes imbalances in homeostasis at the cellular level and, eventually, cell death. Therefore, antioxidant defense systems play an important role in plant abiotic stress resistance [35]. According to a previous study, NAC TFs generally share a positive relationship with the activation of plant antioxidant systems [14,17]. We found that overexpression of ThNAC4 could reduce excess ROS accumulation under salt and osmotic stress conditions (Figures 4 and 6). Moreover, the transcription levels of SOD and POD genes were induced by ThNAC4 in transgenic Arabidopsis, which corresponded with the activities of SOD and POD, respectively. Plant GSTs are involved in protecting plants against different abiotic stress conditions [25]. A previous study overexpressing SINAC2 in Arabidopsis showed enhanced salt and drought tolerance with alterations in glutathione metabolism [36]. In this study, overexpression of ThNAC4 in Arabidopsis and T. hispida increased the content of GST under both salt and osmotic stress conditions, suggesting that it may also be involved in the detoxification of excess ROS in plants. These results suggested that ThNAC4 increases stress tolerance by at least partially reducing ROS accumulation and membrane lipid peroxidation by enhancing the activities of antioxidant enzymes and ROS-scavenging capability.
The resilience of woody halophytes mainly relies on osmoregulation. The production and dislocation of osmolytes as part of the osmoregulatory process is critical for moderate stress tolerance [37]. Under stress conditions, many plants accumulate proline as a nontoxic and protective osmolyte [38]. It contributes to the stabilization of subcellular structures, buffering cellular redox potential, maintaining photosynthetic activity, scavenging free radicals, modulating cellular functions, and even triggering gene expression [39]. Trehalose is a typical organic osmolyte that is effectively involved in plant abiotic stress tolerance. It is a natural nonreducing sugar that plays a key role as a carbon source, an osmoprotectant, and a stabilizing molecule in plants [40]. Trehalose is synthesized in plants in a twostep process. Trehalose phosphate synthase (TPS) catalyzes the formation of T6P from UDP-glucose and glucose-6-phosphate. T6P is dephosphorylated by trehalose phosphate phosphatase (TPP) to generate trehalose. In Arabidopsis thaliana, there are 11 genes encoding TPSs (AtTPS1-11) and 10 genes encoding TPPs (AtTPPA-J) [41]. Our results showed that overexpression of ThNAC4 significantly increased the proline and trehalose contents in transgenic Arabidopsis plants under salt and osmotic stress conditions (Figure 4). At the same time, transcripts of ThNAC4 were positively correlated with the proline and trehalose contents ( Figure 6). In addition, we further investigated the expression levels of TPS and TPP genes related to trehalose biosynthesis in Arabidopsis. ThNAC4 overexpression in Arabidopsis plants increased the transcription levels of many studied genes under NaCl and mannitol treatments, which may play important roles in the biosynthesis of trehalose when the plant is exposed to salt and osmotic stresses (Figures 4 and 5). These findings suggested that ThNAC4 may activate proline and trehalose biosynthesis to increase the contents of these osmolytes, thereby enhancing plant abiotic stress tolerance by increasing osmotic potential and ROS-scavenging capability.

Plant Materials and Growth Conditions
Tamarix hispida seeds were cultured in tissue culture bottles with half-strength Murashige-Skoog (1/2 MS) solid medium [2% (w/v)] in a greenhouse at 24 • C, 70-75% relative humidity, and a 14 h light/10 h dark photoperiod. Arabidopsis thaliana Columbia (Col-0) seeds were germinated on 1/2 MS solid medium plates. After stratification in the dark at 4 • C for two days, the plates were placed in a greenhouse for germination. Oneweek-old seedlings were transferred from the plates to pots containing a 4:1:1 mixture of soil/vermiculite/perlite and grown in the greenhouse at 22 • C, 70-75% relative humidity, and a 16 h light/8 h dark photoperiod.

Cloning and Sequence Analysis of ThNAC4
The cDNA sequence of ThNAC4 (GenBank number: JQ974958) was identified from the transcriptomes of T. hispida [21]. CLUSTALX1.81 was used to perform multiple sequence alignments of ThNAC4 and 12 NAC proteins from different species. The phylogenetic tree of ThNAC4 and 94 NAC proteins from Arabidopsis was generated with MEGA7 software.

Subcellular Localization Analysis of the ThNAC4 Protein
The full-length coding region of ThNAC4 without the termination codon was fused with the N-terminus of green fluorescent protein (GFP) in the pBI121 vector driven by the CaMV 35S promoter to generate the 35S-ThNAC4-GFP construct. The GFP gene driven by a 35S promoter (35S-GFP) was used as a control. The two kinds of plasmids were transformed into onion epidermal cells by the Agrobacterium-mediated transformation method [42]. The onion epidermal cells were treated on MS medium plates in the dark at 25 • C for 48 h and visualized using confocal laser scanning microscopy (LSM800, Zeiss, Jena, Germany).

Transactivation Activity Analysis in Yeast
Transactivation activity analysis was performed according to Hu et al. [24]. To investigate the transactivation activity of ThNAC4, the entire coding region and a series of truncated ThNAC4 genes were cloned into the pGBKT7 vector (Clontech, Palo Alto, CA, USA) and fused with a GAL4 DNA-binding domain. The yeast strain Y2H (Clontech, Palo Alto, CA, USA) was transformed with eight constructs and the pGBKT7 vector (negative control). The transformed strains were confirmed by sequencing and streaked on SD/-Trp/-His/X-α-gal plates. The trans-activation activities were evaluated based on their growth status. The plates were incubated for 3-5 days at 30 • C. Three independent experiments were performed.

Plasmid Construction and Plant Transformation
To obtain ThNAC4-overexpressing plants, the open reading frame (ORF) of ThNAC4 was inserted into pROKII and stably transformed into Arabidopsis thaliana by using the floral dip method [43]. The highly efficient transient transformation of T. hispida plants was performed according to Ji et al. [44]. We constructed three types of transgenic T. hispida

Stress Tolerance Assays
Three T3 generation lines (Line OE2, OE8, and OE9) of ThNAC4-transformed Arabidopsis plants were employed in the salt and drought stress tolerance assays. For the germination rate assay, the seeds of wild-type (WT) and transgenic Arabidopsis plants were surface sterilized and sown on 1/2 MS medium (as a control) and 1/2 MS medium containing 150 mM NaCl or 200 mM mannitol. The plates were chilled at 4 • C in the dark for 2 days for stratification and were transferred to the greenhouse for 7 days. The germination rates of the transgenic and WT lines were calculated. For the stress tolerance experiment, seeds were sown on 1/2 MS medium for 7 days for germination and then transferred into 1/2 MS medium or 1/2 MS medium supplied with 150 mM NaCl or 200 mM mannitol. After seven days, the root length and fresh weight were measured, and photographs of the seedlings were taken. Arabidopsis seeds were planted in the soil after five days of culture in 1/2 MS plates. Three-week-old seedlings were watered with 150 mM NaCl or 200 mM mannitol for one week, and the chlorophyll contents and water loss of the plants were determined. The chlorophyll contents of detached leaves were measured as described by Lichtenthaler [45]. Three independent experiments were performed.

Physiological Analysis of the Stress Response
For histochemical staining analysis, the transformed T. hispida seedlings were treated with 150 mM NaCl or 200 mM mannitol for 24 h. Four-week-old Arabidopsis plants grown in soil were watered with 150 mM NaCl or 200 mM mannitol solution for 2 h. The leaves of T. hispida or Arabidopsis plants were stained with nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) using the methods of Zhang et al. [46] and Fryer et al. [47], respectively. Evans blue staining for examination of cell death was performed as described by Kumar et al. [48]. For physiological assays, T. hispida seedlings were cultured for 48 h in 1/2 MS or 1/2 MS with 150 mM NaCl or 200 mM mannitol, and Arabidopsis grown in the soil for four weeks was watered with 150 mM NaCl or 200 mM mannitol solution for 48 h. The malondialdehyde (MDA) content was determined using an MDA content measuring kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The superoxide dismutase (SOD), peroxidase (POD), and glutathione S-transferase (GST) activities were determined using SOD, POD, and GST enzyme activity measuring kits, respectively (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Proline was measured using a proline content measuring kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Trehalose content was measured using a trehalose content measuring kit (Suzhou Keming Bioengineering Institute, Suzhou, China). Three independent experiments were performed.

Real-Time Quantitative RT-PCR Assay
T. hispida total RNA was isolated using a CTAB (hexadecyltrimethylammonium bromide) method with slight adjustments described by Chang et al. [49] Arabidopsis total RNA was isolated using the Plant Total RNA Extraction Kit. The Primescript RT Reagent Kit was used to reverse-transcribe total RNA (1 µg) into cDNA using oligo (dT) primers, which were then diluted to 100 µL as a PCR template. The MJ Research OptionTM2 instrument (Bio-Rad, Hercules, CA, USA) was used for real-time PCR. To normalize the number of templates used in PCR for T. hispida analysis, α-tubulin (GenBank number: FJ618518), β-tubulin (GenBank number: FJ618519), and β-actin (GenBank number: FJ618517) were used as the internal references. Tub2 (GenBank number: AT5G62690) and Act7 (GenBank number: AT5G09810) were used as the internal references for Arabidopsis analysis. RT-PCR was performed using GoTaq qPCR Master Mix and the Step One Real-Time PCR System (TransGen Biotech, Beijing, China). The reaction system contained 2 µL of cDNA template, 0.5 µM of each primer, and 10 µL of SYBR Green Real-time PCR Master Mix in a 20 µL volume. The amplification was conducted at the following temperatures: 94 • C for 30 s, 45 cycles of 94 • C for 12 s, 58 • C for 30 s, 72 • C for 40 s, and 80 • C for 1 s for plate reading. Three biological replicates were conducted, and the expression levels were calculated using the 2 −∆∆CT method [50]. Three independent experiments were performed. Supplementary Table S1-S8 lists all the primer sequences used in this study.

Statistical Analysis
Data analyses were conducted using SPSS statistical software (Version 19). Significance was calculated using Student's t test, with a threshold of p < 0.05. * represents p < 0.05 in the figures. Figures were created using GraphPad Prism 5.

Conclusions
NAC TFs play an important role in plant responses to abiotic stress. In this study, a stress-responsive NAC gene from T. hispida, ThNAC4, was cloned and functionally characterized. According to our results, ThNAC4 functions as a transcription factor that positively regulates salt and osmotic tolerance by reducing ROS accumulation and membrane damage by increasing antioxidant enzyme activities, ROS-scavenging ability, and osmolyte accumulation. Although this study contributed to fundamental understanding of the role of ThNAC4 in the T. hispida response to salt and drought stresses, further research is still required to fully understand the detailed molecular mechanism of ThNAC4 in the complex antioxidative system and metabolic pathways under abiotic stress.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/plants11192647/s1, Figure S1: Multiple sequence alignment analysis of NAC proteins from 12 other species. Figure S2: Quantitative RT-PCR analysis of ThNAC4 expression in the WT and 9 homozygous overexpression lines (Line1-9) of ThNAC4-transformed Arabidopsis. Figure S3: Expression of ThNAC4 in the different kinds of transgenic T. hispida plants. Table S1: Primers used for cloning of the ThNAC4 gene. Table S2: Primers used in constructing recombinant plasmid pROKII-ThNAC4. Table S3: Primers used in constructing recombinant plasmid pFGC5941-ThNAC4. Table S4: Primers used in constructing recombinant plasmid pBI121-ThNAC4-GFP. Table S5: Gene-specific primers used in real-time PCR. Table S6: Primer sequences of SOD, POD and Trehalose synthase genes used in real-time PCR. Table S7: Primer sequences used in construction pGBKT7-ThNAC4 vector. Table S8: Primer sequences used in construction effector of pGADT7-rec2-ThNAC4.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.