Tamarix hispida plants were used for gene cloning, transgenic analysis and expression analysis. The seedlings of T. hispida were cultured in tissue culture bottles containing half-strength Murashige-Skoog (1/2 MS) solid medium [2% (w/v) agar] in a culture room at average temperature of 24°C with 14 h light/10 h darkness photocycle conditions. Arabidopsis thaliana (ecotype Columbia) was used for genetic transformation and functional analysis. Seven-day-old seedlings were grown in pots filled with soil and perlite in a controlled chamber (16 h light/8 h darkness photocycle; 70–75% relative humidity; 22°C).

The cDNA sequence of ThNAC13 (GenBank number: JQ974967) was identified from the transcriptomes of T. hispida (Wang et al., 2014b). Multiple sequence alignments of the deduced protein sequence of ThNAC13 together with other reported stress-responsive NACs from different plant species were deduced using ClustalW software, and the phylogenetic tree was constructed with MEGA (version 5.1) program (Tamura et al., 2011). The nuclear localization signal of ThNAC13 was predicted using the WoLFPSORT algorithm (Horton et al., 2007). The conserved motifs among the full length NAC protein sequences were predicted using MEME online program¹ (Bailey et al., 2009).

The full-length coding region of ThNAC13 (without stop codon) was cloned into the pBI121 vector containing the GFP reporter gene driven by the CaMV 35S promoter to generate the 35S:ThNAC13-GFP construct. The 35S:ThNAC13-GFP and the 35S:GFP (control) constructs were separately introduced into live onion epidermal cells using the particle bombardment method and visualized by a confocal laser scanning microscope (Wang et al., 2014a). The nuclei were stained with 4,6′-diamidino-2-phenylindole (DAPI, 10 μg.mL^-1) in phosphate-buffered saline for 3 min and analyzed.

To study the transactivation activity of ThNAC13, the full coding region and a series of truncated ThNAC13 genes were cloned into the pGBKT7 vector (Clontech, USA) to fuse with a GAL4 DNA binding domain. Fusion plasmids and the empty pGBKT7 vector (negative control) were individually transformed into the yeast strain Y2HGold and grown on SD medium lacking tryptophan (Trp; SD/-Trp) supplemented with X-α-Gal (5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside) for 2–3 days of culture at 30°C. Yeast transformation and blue/white colony assays were performed following to the manufacturer’s instructions (Clontech, USA). The transcriptional activation activities were evaluated according to the growth status of the transformants.

Two tandem copies of the NACRS with the core sequence “CGT[A/G]” and calmodulin-binding NAC (CBNAC) binding site with the core sequence “GCTT” were separately inserted into the pHIS2 vector (Clontech, USA) as the reporter constructs. The coding region of ThNAC13 gene was cloned into the pGADT7-Rec2 vector as the effector construct. The effector construct was co-transformed together with each reporter construct into the yeast strain Y187. The DNA-protein interactions were evaluated according to the growth ability of the cotransformants on SD medium lacking leucine (Leu) and tryptophan (Trp; SD/-Leu/-Trp, DDO) and SD medium without leucine (Leu), tryptophan (Trp) and histidine (His; SD/-Leu/-Trp/-His, TDO) containing 3-AT (3-amino-1,2,4-triazole).

The full coding sequence (CDS) of ThNAC13 was cloned into pROKII under the control of the CaMV 35S promoter (35S:ThNAC13) and transformed into A. thaliana according to the protocol described by Clough and Bent (1998). The positive transgenic lines were selected on kanamycin (50 mg/L) plates, and further identified by genomic DNA PCR, and the ThNAC13 expression level in leaves of each transgenic line was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The homozygous lines of T₃ generation plants were used for study.

The sense and antisense sequences of the specific coding region of ThNAC13 with 259 bp in length were cloned into the RNAi vector (pFGC5941) to generate pFGC:ThNAC13 and silence the expression of ThNAC13. Transient transformation of 6-week-old T. hispida plants was performed according to Ji et al. (2014) with some modifications. Briefly, the whole seedlings were soaked in the transformation solution [1/2 MS + 150 μM acetosyringone + 3% (w/v) sucrose + 0.01% (w/v) Tween-20, pH 5.8] with Agrobacterium tumefaciens EHA105 strain at 0.6 OD₆₀₀ and incubated with shaking at 100 r/min for 4 h at 25°C. Then, the seedlings were washed with distilled water twice and whipped with sterile facial tissue to remove excessive water. The whole seedlings were grown vertically on 1/2 MS agar medium [150 μM acetosyringone + 2.5% (w/v) sucrose, pH 5.8] in tissue culture bottles. Three types of transient transgenic T. hispida plants were generated: T. hispida overexpressing ThNAC13 (transformed with 35S:ThNAC13, OE), T. hispida RNAi-silencing ThNAC13 (transformed with pFGC:ThNAC13, IE) and plants transformed with empty pROKII vector as a control (VC). The expression of ThNAC13 in the leaves of these transformed T. hispida plants was studied using qRT-PCR.

Two representative transgenic lines overexpressing ThNAC13 and wild type (WT) Arabidopsis plants were selected for abiotic stress tolerance assays. For germination rate assays, the seeds were surface-sterilized and sowed on 1/2 MS solid medium supplemented with 100 mM NaCl, 200 mM mannitol or 2 μM ABA and incubated at 22°C. The rates of seeds were calculated to determine when the seedling opened green cotyledons. Next, 5-day-old seedlings were cultured vertically on 1/2 MS solid medium supplemented with 100 mM NaCl, 200 mM mannitol or 10 μM ABA. After 7 days of these treatments, the fresh weight and the length of primary roots of each plant were measured.

NBT and DAB in situ staining for detection of hydrogen peroxide (H₂O₂) and superoxide (O2•–) were performed. The transformed seedlings of T. hispida were exposed to 150 mM NaCl, 200 mM mannitol or 30 μM ABA for 24 h. Four-week-old soil-grown Arabidopsis plants were treated with the same concentration of NaCl, mannitol or ABA for 2 h, and approximately 10 young branches harvested from T. hispida and 10 rosette leaves harvested from Arabidopsis were incubated with nitroblue tetrazolium (NBT) or 3,3′-diaminobenzidine (DAB) solutions for staining (Zhang et al., 2011).

The transformed T. hispida plants grown on 1/2 MS solid medium supplemented with 150 mM NaCl, 200 mM mannitol or 30 μM ABA for 24 h were used to assess physiological changes. Four-week-old soil-grown Arabidopsis seedlings were treated with same concentration of NaCl, mannitol or ABA for 2 h (for H₂O₂ content measurement) and 48 h (for other physiological analyses). H₂O₂ levels were determined according to Thordal-Christensen et al. (1997). Malondialdehyde (MDA) contents, SOD and POD activities were measured according to Wang et al. (2010). Electrolyte leakage was determined following the protocol described by Ben-Amor et al. (1999). Proline contents were measured according to Bates et al. (1973). Relative chlorophyll contents were calculated using a chlorophyll analyzer (Konica Minolta, Japan). At least 20 seedlings were employed for physiological indices analysis in each sample.

Total RNA was isolated from leaves of T. hispida and Arabidopsis plants using a CTAB (hexadecyltrimethylammonium bromide) method (Chang et al., 1993). The first-strand cDNA was synthesized using the PrimeScript^TM RT reagent Kit (TaKaRa, China), and the real-time RT-PCR was performed in the Opticon 2 System (Bio-Rad, Hercules, CA, USA) following the protocol described by Wang et al. (2014b). The relative expression levels were determined using the 2^-ΔΔCT method (Livak and Schmittgen, 2001). All primer sequences used in this study are shown in Supplementary Table S1.

Each experiment was repeated independently at least three times. The error bars represent standard deviations. Statistical analyses were carried out using Excel software. The data were compared using Student’s t-test, and statistical differences were considered to be significant if P < 0.05. Asterisks indicates significant difference (^∗P < 0.05).

The full CDS of ThNAC13 is 1122 bp in length and encodes a 373 amino acid protein with a molecular weight of 40.74 kDa. Multiple sequence alignments showed that ThNAC13 contains a highly conserved DNA-binding domain at N-terminal, which is a typical NAC domain N-terminal with five subdomains (A, B, C, D, and E), and a highly variable C-terminal transcriptional regulation domain (Supplementary Figure S1). The putative nuclear localization signal of ThNAC13 was identified in the D subdomain. Phylogenetic analysis revealed that ThNAC13 shares high sequence similarities with the plant stress-responsive NAC members (Figure 1), indicating that ThNAC13 may have similar functions to these NACs. Ten conserved motifs (termed motif 1–10) were predicted based on the sequence features of NAC proteins using the MEME program, which are listed in Supplementary Table S2. Motif 1 corresponds to the A and B subdomains, and motifs 2, 3, and 4, respectively, correspond to subdomains C, D, and E.

To determine the localization of the ThNAC13 protein, the 35S:GFP (control) and 35S:ThNAC13-GFP constructs were transformed into live onion epidermal cells. The GFP fluorescence of ThNAC13-GFP was exclusively observed in the nucleus, whereas the fluorescence of GFP was distributed in the whole cells (Figure 2A). These results clearly indicate that ThNAC13 is a nuclear protein.

To determine the transcriptional activation ability of ThNAC13, the full CDS and serial deletions of the ThNAC13 CDS were cloned into the pGBKT7 vector (Figure 2B, construct 2–14). The different constructs were then transformed into the Y2HGold yeast cells and were assayed for their transcriptional activation. All transformants grew well on the SD/-Trp medium (left panel), indicating that these constructs had been successfully transformed into Y2HGold cells. On the SD/-Trp medium containing X-α-Gal (SD/-Trp/X), the cells harboring full-length CDS of ThNAC13 (construct 2, amino acid 1–373) or empty pGBKT7 (construct 1, control) were not blue (right panel), suggesting that the full CDS of ThNAC13 does not have transcriptional activation ability. However, the yeast cells containing the C-terminal putative activation domain (construct 4, amino acid 166–373) of ThNAC13 grew well and were blue, whereas the N-terminal domain (construct 3, amino acid 1–165) was not blue, suggesting that only the C-terminal has transactivation ability. With the exception of the amino acid 227–373 construct (construct 9), the other deletions of the C-terminal domain exhibited relatively weak transactivation activity. The truncated CDS with amino acids 227–373 still had transactivation activity comparable to that with the whole C-terminus, while construct 8 (amino acid 166–226) completely abolished transactivation activity. These results indicate that ThNAC13 contains the transcriptional activation domain at the region of amino acids 227–373, although the full CDS of ThNAC13 does not have transcriptional activation, which may be because the N-terminal of ThNAC13 has transcriptional repression activity.

To investigate whether ThNAC13 protein can bind to the NACRS and CBNACBS, two tandem copies of these motifs or their mutants were tested for interaction with ThNAC13 using a Y1H system. The sequences for construction of the recombinant report vectors are shown in Figure 3A, and the scheme of the reporter and effector vectors used in Y1H are shown in Figure 3B. On the SD/-Leu-/Trp medium, all transformants grew well (left panel). On the SD/-Leu-/Trp-/His medium containing 50 mM 3-AT, yeast cells carrying pGAD-ThNAC13/pHIS2-NACRS (R1–R6) grew well, while the mutants RM1 and RM2 and the negative control could not grow (Figure 3C). These results indicate that ThNAC13 can bind to both NACRS and CBNACBS but failed to bind to the mutants, which further suggest that ThNAC13 can specifically bind to the NACRS and CBNACBS motifs.

Twelve lines of T₃ homozygous ThNAC13-transformed Arabidopsis were generated by selection on medium containing kanamycin and further verified by genomic DNA PCR and qRT-PCR (Supplementary Figure S2). Two independent transgenic Arabidopsis lines (lines 7 and 12) with high transcript levels of ThNAC13 were selected for further study. We first evaluated the stress tolerance of transgenic and wild-type (WT) Arabidopsis seeds with seed germination tests. There was no difference in germination rates between WT and ThNAC13-transformed plants on 1/2 MS medium. However, the seed germination rates of ThNAC13-transformed lines were significant higher than those of WT plants under NaCl, mannitol or ABA stress (Figure 4), suggesting that overexpression of ThNAC13 improves salt and osmotic tolerances during the germination stage. Next, 5-day-old Arabidopsis seedlings were grown vertically on 1/2 MS solid medium supplemented with or without NaCl, mannitol or ABA. After growth for 7 days, the phenotype was similar between ThNAC13-transfomed and WT plants under normal conditions (Figure 5), suggesting that overexpression of ThNAC13 did not influence the growth and phenotype of transgenic plants. Under NaCl, mannitol or ABA stress conditions, the root length and fresh weight gain were significantly increased in ThNAC13-transformed plants compared with WT plants, indicating that ThNAC13 improves the tolerances to NaCl, mannitol or ABA stress.

For further investigation of the function of ThNAC13 using gain- and loss-of-function methods, T. hispida plants transiently overexpressing ThNAC13 (OE), RNAi-silenced for ThNAC13 (IE) and controls (transformed with empty pROKII, VC) were generated. The expression of ThNAC13 in these 3 transformed T. hispida plant under different stresses was studied using qRT-PCR. The transcript levels of ThNAC13 were significantly increased in OE plants and substantially decreased in IE plants compared with control (VC) plants under all the conditions, especially with salt, mannitol or ABA stress for 24 h (Supplementary Figure S3). These results indicate that the genetically transformed T. hispida plants are suitable for gain- and loss-of-function studies.

Abiotic stress can induce oxidative stress through generation of reactive oxygen species (ROS), and the accumulation of ROS damages the cell membrane by oxidizing proteins, lipids and DNA (Mittler et al., 2004). NBT and DAB staining were first used for detecting the cellular levels of O2•– and H₂O₂ in three types of transiently transformed T. hispida plants. The results showed that the transgenic Tamarix plants overexpressing ThNAC13 (OE) had the lowest O2•– and H₂O₂ accumulation, and the transgenic Tamarix plants with knockdown of ThNAC13 (IE) had the highest O2•– and H₂O₂ accumulation, although they had similar O2•– and H₂O₂ levels under normal conditions (Figures 6A,B). To confirm the results from T. hispida, we further determined the ROS accumulation in Arabidopsis plants using in situ histochemical staining with NBT and DAB. There was no obvious difference in NBT and DAB staining between ThNAC13-transformed and WT plants under normal conditions. However, compared with WT plants, the transgenic plants showed substantially reduced O2•– and H₂O₂ accumulation under salt, mannitol and ABA conditions (Figures 7A,B).

Additionally, we further measured H₂O₂ and MDA contents in transgenic T. hispida plants. There was no difference in H₂O₂ and MDA contents among OE, VC, and IE plants under normal conditions. However, under stress conditions, IE plants had the highest level, followed by IE plants, and OE had the lowest H₂O₂ levels (Figures 6C,D). Consistent with these results, transgenic Arabidopsis plants overexpressing ThNAC13 displayed low H₂O₂ and MDA contents compared with WT plants under stress conditions (Figures 7C,D). As ROS levels were decreased by expression of ThNAC13, we investigated whether SOD and POD activities were regulated by ThNAC13. Among all the studied Tamarix plants, there was no significant difference in SOD and POD activities under normal conditions. The SOD and POD activities were also markedly elevated in OE plants and reduced in IE plants compared with VC plants (Figures 6E,F). We further studied SOD and POD activities in Arabidopsis. There was no significant difference in the activities of SOD and POD between transgenic and WT plants under normal conditions. Compared with WT Arabidopsis plants, the activities of POD and SOD in ThNAC13-transfomed Arabidopsis plants were significantly increased under the different stress conditions (Figures 7E,F), which was consistent with the results obtained from T. hispida plants. Therefore, overexpression of ThNAC13 significantly activates SOD and POD activities under abiotic stress conditions.

To estimate the level of abiotic stress damage to the studied plants, several stress-related physiological changes were measured. Under normal conditions, all transgenic T. hispida plants displayed similar electrolytic leakage rates, chlorophyll and proline contents (Figures 6G–I). However, the electrolytic leakage rates in IE plants were the highest, followed by VE, and were the lowest in OE plants (Figure 6G). Meanwhile, OE plants had the highest contents of chlorophyll and proline, followed by VC plants, and IE plants had the lowest contents of chlorophyll and proline (Figures 6H,I). The electrolyte leakage rates in transgenic Arabidopsis plants overexpressing ThNAC13 were decreased compared with WT plants under salt, osmotic or ABA stress (Figure 7G). Furthermore, the contents of chlorophyll and proline in ThNAC13-transformed Arabidopsis were both significantly higher than those in WT plants (Figures 7H,I). These results are consistent with the results from T. hispida, indicating the reliability of these findings.

As the activities of POD and SOD were both markedly elevated in the Arabidopsis plants overexpressing ThNAC13, we further analyzed the transcription levels of POD and SOD genes, which were shown to have enzymatic activities in previous studies. The expression of many studied genes was highly increased in ThNAC13-transformed Arabidopsis under NaCl and mannitol treatments (Figure 8). These results suggested that ThNAC13 can induce the transcription levels of SOD and POD genes, which will enhance the activities of SOD and POD.

Most NAC proteins share a well-conserved N-terminal DNA binding domain and a diversified C-terminal domain (Puranik et al., 2012; Wang et al., 2014b). In this study, we found that the ThNAC13 protein has the conserved NAC domain at N-terminus. Multiple sequence alignments showed that ThNAC13 has high amino acid sequence identity with the reported stress-responsive NAC proteins of Arabidopsis (AtNAC019, AtNAC055, and AtNAC072) (Supplementary Figure S1), suggesting that ThNAC13 may also be a stress-responsive NAC. Previous studies have shown that NAC proteins could bind to the NACRS containg the core sequence “CGT[A/G]” (Duval et al., 2002; Tran et al., 2004; Olsen et al., 2005; Xue, 2005; Takasaki et al., 2010; Hao et al., 2011). However, Kim et al. (2007) found that the Arabidopsis transcriptional repressor CBNAC can bind to CBNACBS with a “GCTT” sequence but not the “CGT[A/G]” sequence, indicating that different NAC transcription factors may bind to different element sequences. Our study suggested that ThNAC13 can bind to the NACRS element; at the same time, it can also bind to a motif sequence containing the “GCTT,” similar to CBNAC (Figure 3C). These results suggested that ThNAC13 could bind to NACRS and CBNACBS to regulate gene expression.

Transactivation assays showed that the C-terminal of ThNAC13 has a high transactivation ability, although the full-length protein does not have this capacity (Figure 2B). However, when the N-terminal of ThNAC13 is present, the transactivation is abolished. These results indicated that the N-terminal of ThNAC13 may have a transcriptional repression domain, which leads to abolished transactivation. Previous studies also support our hypothesis. For example, the conserved hydrophobic LVFY motif was a transcription repression domain that is present in the N-terminal of GmNAC20 protein (Hao et al., 2010). We also found the LVFY motif in N-terminal of ThNAC13 (Supplementary Table S2). However, ThNAC13 could regulate the expression of POD and SOD genes (Figure 8). This is probably due to its C-terminal domain, which has high transcriptional activation activity, or the interplay among the C-terminal activation domain, the DNA-binding domain and NARD (NAC Repression Domain). Therefore, ThNAC13 may act as a transcriptional repressor or activator, depending on interactions with other transcription factors or conformational changes.

Recently, numerous reports have shown that NAC transcription factors play important roles in the response to different abiotic stresses and function as positive stress response TFs (Jeong et al., 2013; Mao et al., 2014; Fang et al., 2015). In the present study, our results showed that ThNAC13 can induce a series of physiological changes to improve tolerance to salt, osmotic and ABA stresses (Figures 4, 6, 7). These results suggested that ThNAC13 functions as a stress-responsive protein and enhances the salt and osmotic tolerance. In addition, ThNAC13 promotes tolerance to ABA, and NADPH oxidases are involved in abiotic stress tolerance and regulated by ABA, which plays an important role in ABA-mediated stomatal closure (Acharya et al., 2013). Therefore, there may be a relationship between ThNAC13 and NADPH oxidase activity, which deserves further study.

Plants are commonly exposed to various adverse environments, which result in accumulation of ROS, damage to protein synthesis and stability, cellular macromolecules and membrane lipids in plant cells and generation of oxidative stress (Wang et al., 2005). Therefore, ROS-scavenging is important for plant tolerance. Two prominent ROS species, O2•– and H₂O₂, are considered to be important signaling molecules in plant cells. In this study, biochemical staining (NBT and DAB) showed that the ROS accumulation (O2•– and H₂O₂) in transgenic Tamarix and Arabidopsis plants overexpressing ThNAC13 was lower than that in control (VC or WT) plants under abiotic stress and ABA treatment (Figures 6A,B, 7A,B). MDA, the product of lipid peroxidation caused by ROS, is also used to evaluate ROS-mediated injuries in plants (Moore and Roberts, 1998). We found that the H₂O₂ and MDA contents were both decreased by expression of ThNAC13 under abiotic stress and ABA treatment (Figures 6C,D, 7C,D). These results suggested that ThNAC13 can reduce ROS accumulation and decrease lipid peroxidation to improve tolerance to salt and osmotic stresses.

The antioxidant enzymes, SOD and POD, are known to play important roles in ROS scavenging, which influences the cellular ROS levels. We found that the activities of SOD and POD were significantly induced by ThNAC13 under different stress conditions. Furthermore, most of the studied SOD and POD genes were activated in ThNAC13 transgenic Arabidopsis plants, which may lead to increased SOD and POD activities in plants overexpressing ThNAC13 (Figures 6E,F, 7E,F, 8). These results suggest that ThNAC13 improves stress tolerance at least partly through enhancing ROS-scavenging capability, which will decrease the degree of membrane lipid peroxidation. In addition, we found that the core sequences of NACRS and CBNACBS elements were both enriched in the promoters (<1.5 kb) of the studied SODs and PODs (Supplementary Table S3), suggesting that ThNAC13 may bind to these elements to directly regulate the expression of these genes. These results suggest that ThNAC13 improves tolerance to salt and osmotic stresses by regulating the transcript levels of SODs and PODs to increase the ROS-scavenging capability.

Plant proline could protect cells by serving as an osmotic agent and also as a radical scavenger under biotic or abiotic stress. In addition, when stress is relieved, the accumulated proline could be degraded as a supply of energy for plant growth (Kavi Kishor and Sreenivasulu, 2014). In this study, proline content was positively correlated with the transcripts of ThNAC13 (Figure 6I), and Arabidopsis plants overexpressing ThNAC13 displayed increased proline contents under different stresses (Figure 7I), indicating that ThNAC13 induces the biosynthesis of proline, and the increased proline enhances both osmotic potential and ROS-scavenging capability to improve abiotic stress tolerance.