The Poplar (Populus trichocarpa) Dehydrin Gene PtrDHN-3 Enhances Tolerance to Salt Stress in Arabidopsis

Dehydrin (DHN), a member of the late embryogenesis abundant protein (LEA) family, was recently found to play a role in physiological responses to salt and drought stress. In this study, we identified and cloned the PtrDHN-3 gene from Populus trichocarpa. The PtrDHN-3 protein encoded 226 amino acids, having a molecular weight of 25.78 KDa and an isoelectric point of 5.18. It was identified as a SKn-type DHN and was clustered with other resistance-related DHN proteins. Real-time fluorescent quantitative PCR showed that transcription levels of PtrDHN-3 were induced by mannitol stress, and more significantly by salt stress. Meanwhile, in a yeast transgenic assay, salt tolerance increased in the PtrDHN-3 transgenic yeast, while the germination rate, fresh weight and chlorophyll content increased in PtrDHN-3-overexpressing transgenic Arabidopsis plants (OE) under salt stress. Significant increases in expression levels of six antioxidant enzymes genes, and SOD and POD enzyme activity was also observed in the OE lines, resulting in a decrease in O2- and H2O2 accumulation. The proline content also increased significantly compared with the wild-type, along with expression of proline synthesis-related genes P5CS1 and P5CS2. These findings suggest that PtrDHN-3 plays an important role in salt resistance in plants.


Introduction
Extremely harsh environmental conditions, such as high and low temperatures, salinealkali conditions and drought, have a severe effect on plant growth and development, and are the main causes of plant mortality worldwide [1]. To survive adversity, plants have therefore evolved a series of mechanisms aimed at resisting stress. For example, salinity, high and low temperatures, drought, and other abiotic stresses cause water stress within the plant, in turn inducing a series of physiological reactions, such as increases in the contents of proline, soluble protein, carbohydrates, and organic acids, and a loss of lipid membranes [2]. These reactions are also thought to alter or induce the production of additional proteins, and all of these metabolic changes are regulated by gene expression [3,4]. Identification of the genes related to abiotic stress is therefore an important focus of research into the molecular mechanisms of plant stress resistance.
Dehydrins (DHNs) are members of the late embryogenesis abundant protein (LEA) family [5] which accumulate in the late stages of embryogenesis when water content in seeds declines, or in response to various stressors. DHN is clustered into the group II of highly hydrophilic proteins and has been discovered in a large number of plant species [6,7]. DHN usually contains a highly conserved N-terminal Y segment [T/V]D[E/Q]YGNP), a C-terminal K segment (EKKGIMDKIKEKLPG), and a central S segment (serine-track) [8,9]. Studies indicate that the K segments are the functional core parts of DHNs that mediate cellular stress tolerance or maintain enzyme activity [10]. S segments are generally thought to affect the localization or function of DHNs by phosphorylation [11], while the Y-segment is composed of aromatic conserved tyrosine residues the function of which remains unclear [12,13]. According to the number and position of these conserved domains, they can

Characterization of Cis Elements in the PtrDHN-3 Gene Promoter
The 1500-bp upstream sequence of the initiator codon ATG in the Ptr was selected for analysis of cis-acting elements in the gene promoter. Predic showed a number of cis-acting elements related to stress responses (

Characterization of Cis Elements in the PtrDHN-3 Gene Promoter
The 1500-bp upstream sequence of the initiator codon ATG in the PtrDHN-3 gene was selected for analysis of cis-acting elements in the gene promoter. Prediction analysis showed a number of cis-acting elements related to stress responses (Figure 2), such as an MYB binding site (MBS), ABA response element (ABRE), MYC recognition site, DRE recognition site, and general transcription factor binding sites such as TATA box.

Analysis of PtrDHN-3 Expression under Abiotic Stress
To determine the response of PtrDHN-3 to abiotic stress, qRT-PCR analysis was used to obtain expression profiles under respective treatment with 100 mM NaCl and 200 mM mannitol. Under mannitol stress ( Figure 3A), the transcripts of PtrDHN-3 were first induced in the roots at 6 h followed by the stems and leaves at 12 h, with a peaking at 12 h in all tissues. Peak transcription levels in the leaves were 14 times greater than those of the control. These results indicate that PtrDHN-3 plays a role in the response to mannitolinduced drought stress. Meanwhile, treatment with NaCl caused a rapid increase in transcription levels of PtrDHN-3 in the roots, stems and leaves, with a notable increase in the leaves. Levels were highest at 24 h, at 41 times that of the control ( Figure 3B). Since the

Analysis of PtrDHN-3 Expression under Abiotic Stress
To determine the response of PtrDHN-3 to abiotic stress, qRT-PCR analysis was used to obtain expression profiles under respective treatment with 100 mM NaCl and 200 mM mannitol. Under mannitol stress ( Figure 3A), the transcripts of PtrDHN-3 were first induced in the roots at 6 h followed by the stems and leaves at 12 h, with a peaking at 12 h in all tissues. Peak transcription levels in the leaves were 14 times greater than those of the control. These results indicate that PtrDHN-3 plays a role in the response to mannitolinduced drought stress. Meanwhile, treatment with NaCl caused a rapid increase in transcription levels of PtrDHN-3 in the roots, stems and leaves, with a notable increase in the leaves. Levels were highest at 24 h, at 41 times that of the control ( Figure 3B). Since the

PtrDHN-3 Improves Salt Tolerance in Yeast
To verify the potential function of PtrDHN-3 in salt stress, pYES2-PtrDHN-3 recombinant plasmids were transferred into the INVSC1 yeast strain. An empty pYES2 vector was used as a control. Under control conditions, there were no obvious differences in growth between the control and PtrDHN-3 transgenic cells. Meanwhile, under NaCl stress, growth of the PtrDHN-3 transgenic yeast was greater than that of the control at each dilution (10, 100, and 1000). Notably, the survival rate of transgenic yeast cells diluted 100-fold was much greater than that of control ( Figure 4).

Overexpression of PtrDHN-3 Increases Salt Tolerance in Arabidopsis
To confirm the role of PtrDHN-3 in salt tolerance, PtrDHN-3-overexpressing Arabidopsis plants (OE) were constructed. Agrobacterium-mediated transformation of PtrDHN-3 in ten Arabidopsis lines was confirmed by PCR ( Figure S1). Two T3 homozygous lines (OE-8 and OE-10) were then randomly selected for analysis of salt tolerance. When grown on 1/2 MS medium, no obvious differences in germination rates were observed between the transgenic and WT lines. However, on 1/2 MS medium containing 100 mM NaCl, germination rates were about three times greater in the transgenic lines than the WT ( Figure  5A,B). Fresh weights were also significantly greater in the OE lines compared to the WT

PtrDHN-3 Improves Salt Tolerance in Yeast
To verify the potential function of PtrDHN-3 in salt stress, pYES2-PtrDHN-3 recombinant plasmids were transferred into the INVSC1 yeast strain. An empty pYES2 vector was used as a control. Under control conditions, there were no obvious differences in growth between the control and PtrDHN-3 transgenic cells. Meanwhile, under NaCl stress, growth of the PtrDHN-3 transgenic yeast was greater than that of the control at each dilution (10, 100, and 1000). Notably, the survival rate of transgenic yeast cells diluted 100-fold was much greater than that of control ( Figure 4). response to salt stress was greater than that to drought, subsequent analyses focused on the function of PtrDHN-3 in salt stress.

PtrDHN-3 Improves Salt Tolerance in Yeast
To verify the potential function of PtrDHN-3 in salt stress, pYES2-PtrDHN-3 recombinant plasmids were transferred into the INVSC1 yeast strain. An empty pYES2 vector was used as a control. Under control conditions, there were no obvious differences in growth between the control and PtrDHN-3 transgenic cells. Meanwhile, under NaCl stress, growth of the PtrDHN-3 transgenic yeast was greater than that of the control at each dilution (10, 100, and 1000). Notably, the survival rate of transgenic yeast cells diluted 100-fold was much greater than that of control ( Figure 4).

Overexpression of PtrDHN-3 Increases Salt Tolerance in Arabidopsis
To confirm the role of PtrDHN-3 in salt tolerance, PtrDHN-3-overexpressing Arabidopsis plants (OE) were constructed. Agrobacterium-mediated transformation of PtrDHN-3 in ten Arabidopsis lines was confirmed by PCR ( Figure S1). Two T3 homozygous lines (OE-8 and OE-10) were then randomly selected for analysis of salt tolerance. When grown on 1/2 MS medium, no obvious differences in germination rates were observed between the transgenic and WT lines. However, on 1/2 MS medium containing 100 mM NaCl, germination rates were about three times greater in the transgenic lines than the WT ( Figure  5A,B). Fresh weights were also significantly greater in the OE lines compared to the WT

Overexpression of PtrDHN-3 Increases Salt Tolerance in Arabidopsis
To confirm the role of PtrDHN-3 in salt tolerance, PtrDHN-3-overexpressing Arabidopsis plants (OE) were constructed. Agrobacterium-mediated transformation of PtrDHN-3 in ten Arabidopsis lines was confirmed by PCR ( Figure S1). Two T3 homozygous lines (OE-8 and OE-10) were then randomly selected for analysis of salt tolerance. When grown on 1/2 MS medium, no obvious differences in germination rates were observed between the transgenic and WT lines. However, on 1/2 MS medium containing 100 mM NaCl, germination rates were about three times greater in the transgenic lines than the WT ( Figure 5A,B). Fresh weights were also significantly greater in the OE lines compared to the WT under salt stress ( Figure 5C). Under control conditions, no significant differences in growth phenotypes were observed between the transgenic and WT plants; however, under salt stress, growth was better in the transgenic compared to the WT plants, and rosette leaves were healthier looking ( Figure 5D). Chlorophyll contents decreased under salt stress in both the OE and WT plants; however, the decrease was greater in the WT.
Plants 2022, 11, x FOR PEER REVIEW under salt stress ( Figure 5C). Under control conditions, no significant differe growth phenotypes were observed between the transgenic and WT plants; howev der salt stress, growth was better in the transgenic compared to the WT plants, and leaves were healthier looking ( Figure 5D). Chlorophyll contents decreased und stress in both the OE and WT plants; however, the decrease was greater in the WT and salt str mM NaCl) conditions. (B) Seed germination rates, and analyses of (C) fresh weights, (D) phenotypes, and (E) chlorophyll contents. WT and OE Plants were grown on 1/2 MS mediu trol) or 1/2 MS medium plus 100 mM NaCl for two weeks for analysis of seed germination ra analysis of fresh weights, WT and OE plants germinated on 1/2 MS medium plates for fi were transferred to 1/2 MS medium plates with or without 100 mM NaCl. Measurements w tained after continuous cultivation for seven days. For observations of growth phenotypes, old T3 transgenic and WT Arabidopsis seedlings grown in pots were treated with 100 m solution for 10 days then their phenotypes were photographed. The chlorophyll content o was measured. Asterisks indicate a significant difference compared with the WT plants at p

PtrDHN-3 Scavenges ROS
Under normal growth conditions, Diaminobenzene (DAB) and Nitrotetra Blue chloride (NBT) staining revealed similar accumulation of H2O2 and O2 ·-betw transgenic and WT Arabidopsis plants ( Figure 6A). However, under salt stress cond accumulation of H2O2 and O2 -decreased in the transgenic compared with WT pla pecially at 12 h, and blue and brown staining were detected in the leaves.
Analysis of antioxidant enzymes activity showed that SOD and POD activitie significantly higher in the transgenic compared to WT plants under salt stress 6B,C). Meanwhile, qRT-PCR analysis of SOD and POD gene expression revealed nificant differences between the OE and WT plants under non-stress conditions   Supplementary Table S1. Three independent biological repeats were performed per treatment, and each experiment consisted of at least 9 transformed seedlings. * Indicates a significant difference according to Student's t test at p < 0.05 compared with the WT plants.

PtrDHN-3 Improves Biosynthesis of Proline under Salt Stress
Studies have shown that proline content can be used to measure salt tolerance in plants [28]. To determine whether PtrDHN-3 induces proline biosynthesis, proline contents were measured in the transgenic and WT Arabidopsis. Under non-stress conditions, proline contents were similar between the transgenic and WT plants; however, under salt stress, contents increased significantly in the transgenic plants ( Figure 7A). Expression of proline biosynthesis-related genes P5CS1 and P5CS2 has also been studied in relation to salt stress [29]. In this study, no obvious differences were observed between the transgenic and WT lines under normal conditions; however, under salt stress, transcript levels of both genes increased in the transgenic plants ( Figure 7B).  Supplementary Table S1. Three independent biological repeats were performed per treatment, and each experiment consisted of at least 9 transformed seedlings. * Indicates a significant difference according to Student's t test at p < 0.05 compared with the WT plants.
Analysis of antioxidant enzymes activity showed that SOD and POD activities were significantly higher in the transgenic compared to WT plants under salt stress ( Figure 6B,C). Meanwhile, qRT-PCR analysis of SOD and POD gene expression revealed no significant differences between the OE and WT plants under non-stress conditions ( Figure 6D), but a significant increase under salt stress in the transgenic plants ( Figure 6E).

PtrDHN-3 Improves Biosynthesis of Proline under Salt Stress
Studies have shown that proline content can be used to measure salt tolerance in plants [28]. To determine whether PtrDHN-3 induces proline biosynthesis, proline contents were measured in the transgenic and WT Arabidopsis. Under non-stress conditions, proline contents were similar between the transgenic and WT plants; however, under salt stress, contents increased significantly in the transgenic plants ( Figure 7A). Expression of proline biosynthesis-related genes P5CS1 and P5CS2 has also been studied in relation to salt stress [29]. In this study, no obvious differences were observed between the transgenic and

Discussion
Considerable research has been carried out into the role of DHN genes in cold stress resistance [20]. Meanwhile, in more recent years, DHN has also been found to play a role in drought resistance and salt tolerance [23,25]. However, few studies have examined the role of this gene in trees. A previous study in poplar (Populus trichocarp) revealed that PtrDHN genes are related to abiotic stress responses [30]. Therefore, in this study, the function of PtrDHN3 in response to stress was investigated further.
Phylogenetic tree analysis showed that PtrDHN-3 is clustered with a number of stress-resistant genes including IpDHN, OesDHN, StDHN and CcDHN ( Figure 1A), the functions of which in relation to abiotic stress have been previously identified [31][32][33]. Meanwhile, multiple sequence alignment revealed that PtrDHN-3 is a SKn-type DHN protein ( Figure 1B). The functions of different types of DHN proteins differ, together with their responses to stress. For example, studies have shown that SKn-type DHNs are a type of acid DHN that are induced by multiple stresses [15][16][17]. In this study, analysis of the promoter region of the PtrDHN-3 gene revealed a variety of elements related to stress, such as an MYB binding site (MBS), ABA response element (ABRE), MYC recognition site and DRE recognition site ( Figure 2). These sequence characteristics therefore suggest that PtrDHN-3 is involved in abiotic stress responses in poplar.
In general, genes related to abiotic stress resistance are induced under stress conditions. In order to verify whether this is the case with PtrDHN-3, responses to salt and drought stress were examined. Transcript levels of PtrDHN-3 were induced under both mannitol and NaCl treatment; however, the response was greater under salt stress, especially in the leaves (Figure 3), suggesting an important role in salt stress tolerance. This was subsequently confirmed via histochemical staining, and analyses of SOD activity, POD activity, and chlorophyll and proline contents.
Yeast is an important tool for screening genes involved in abiotic stress resistance. For example, transference of ThvhAC1 gene from Tamarix hispida into yeast cells improved tolerance and survival of transgenic yeast under abiotic stress [34], while overexpression of TdSHN1 (Durum wheat gene) improved tolerance to Cd, Cu, and Zn stress in transgenic yeast [35]. In this study, PtrDHN-3 improved tolerance to salt stress in the transformed yeast cells (Figure 4), although there was no obvious difference under mannitol-induced drought stress (data not shown).
To verify the function of PtrDHN-3 in salt tolerance, PtrDHN-3 overexpression was induced in Arabidopsis. Analysis showed that under NaCl stress conditions, the transgenic Arabidopsis plants had a greater germination rate, fresh weight and chlorophyll content than the WT. Various germination tests and phenotype analyses can be used to show

Discussion
Considerable research has been carried out into the role of DHN genes in cold stress resistance [20]. Meanwhile, in more recent years, DHN has also been found to play a role in drought resistance and salt tolerance [23,25]. However, few studies have examined the role of this gene in trees. A previous study in poplar (Populus trichocarp) revealed that PtrDHN genes are related to abiotic stress responses [30]. Therefore, in this study, the function of PtrDHN3 in response to stress was investigated further.
Phylogenetic tree analysis showed that PtrDHN-3 is clustered with a number of stress-resistant genes including IpDHN, OesDHN, StDHN and CcDHN ( Figure 1A), the functions of which in relation to abiotic stress have been previously identified [31][32][33]. Meanwhile, multiple sequence alignment revealed that PtrDHN-3 is a SKn-type DHN protein ( Figure 1B). The functions of different types of DHN proteins differ, together with their responses to stress. For example, studies have shown that SKn-type DHNs are a type of acid DHN that are induced by multiple stresses [15][16][17]. In this study, analysis of the promoter region of the PtrDHN-3 gene revealed a variety of elements related to stress, such as an MYB binding site (MBS), ABA response element (ABRE), MYC recognition site and DRE recognition site ( Figure 2). These sequence characteristics therefore suggest that PtrDHN-3 is involved in abiotic stress responses in poplar.
In general, genes related to abiotic stress resistance are induced under stress conditions. In order to verify whether this is the case with PtrDHN-3, responses to salt and drought stress were examined. Transcript levels of PtrDHN-3 were induced under both mannitol and NaCl treatment; however, the response was greater under salt stress, especially in the leaves (Figure 3), suggesting an important role in salt stress tolerance. This was subsequently confirmed via histochemical staining, and analyses of SOD activity, POD activity, and chlorophyll and proline contents.
Yeast is an important tool for screening genes involved in abiotic stress resistance. For example, transference of ThvhAC1 gene from Tamarix hispida into yeast cells improved tolerance and survival of transgenic yeast under abiotic stress [34], while overexpression of TdSHN1 (Durum wheat gene) improved tolerance to Cd, Cu, and Zn stress in transgenic yeast [35]. In this study, PtrDHN-3 improved tolerance to salt stress in the transformed yeast cells (Figure 4), although there was no obvious difference under mannitol-induced drought stress (data not shown).
To verify the function of PtrDHN-3 in salt tolerance, PtrDHN-3 overexpression was induced in Arabidopsis. Analysis showed that under NaCl stress conditions, the transgenic Arabidopsis plants had a greater germination rate, fresh weight and chlorophyll content than the WT. Various germination tests and phenotype analyses can be used to show the role of resistance genes in salt tolerance [36]. The present results suggest that PtrDHN-3 functions in salt tolerance by improving germination and reducing tissue dehydration and photosynthetic damage under stress conditions ( Figure 5).
When subjected to abiotic stress, plants produce a large amount of ROS, thereby inducing oxidative harm. Wheat DHN was found to improve salt tolerance in transgenic Arabidopsis thaliana by eliminating ROS [37], while cellular accumulation of IpDHN was found to improve salt tolerance in transgenic Arabidopsis, possibly by activating the oxygen-scavenging system [27]. DAB and NBT staining are commonly used to determine the contents of H 2 O 2 and O 2 - [38], and in this study, were used to identify the function of PtrDHN3 in ROS scavenging ( Figure 6A). The results showed that overexpression of PtrDHN3 reduced the accumulation of both H 2 O 2 and O 2 under salt stress conditions, suggesting that PtrDHN3 improves salt tolerance by reducing ROS accumulation. Plants possess an antioxidant enzyme system that protects cells from oxidative damage by scavenging ROS. SOD and POD play an important role in ROS detoxification and are therefore crucial factors in abiotic stress resistance. In this study, a significant increase in SOD and POD activity were observed under salt stress in the PtrDHN-3 transgenic plants ( Figure 6B,C), thereby functioning to reduce ROS accumulation. Expression levels of SOD and POD genes were also examined, revealing a significant increase in the transgenic component compared to WT plants under salt stress ( Figure 6E). Consistent with this, previous knock-outs of CaDHN4 and CaDHN5 inhibited expression of manganese superoxide dismutase (MnSOD) and peroxidase (POD) genes, causing an increase in severe wilting and ROS accumulation in the leaves of Arabidopsis [39,40]. Taken together, these results suggest that PtrDHN-3 enhances salt tolerance in the transgenic plants by inducing the expression of SOD and POD, improving the activities of SOD and POD, and thereby reducing ROS accumulation.
Plants often accumulate proline under abiotic stress conditions, and proline is therefore thought to play an important role in plant cell adaptation to osmotic stress [41]. We therefore determined the expression of two important proline synthesis-related genes, P5CS1 and P5CS2, as well as the content of proline in transgenic Arabidopsis (Figure 7). Under salt stress conditions, PtrDHN-3 induced an increase in the expression of P5CS1 and P5CS2, and improved proline biosynthesis in the transgenic compared to WT plants. These findings suggest that overexpression of PtrDHN-3 improves tolerance to salt stress by inducing the synthesis of proline. Consistent with this, the role of DHN in regulating proline synthesis to confer salt tolerance has also been shown in Australian wild rice [42].

Populus Trichocarpa Cultivation and Stress Treatment
Four-week-old tissue culture seedlings of P. trichocarpa were transplanted into pots and cultivated in a greenhouse for two months under 14 h light/10 h dark, 70-75% relative humidity, and an indoor temperature of 25 • C. Two-month-old seedlings were then treated with 100 mM NaCl or 200 mM mannitol for 6, 12, 36, 24, 48, or 72 h. Well-watered seedlings were used as a control. Root, stem, and leaf samples from at least three seedlings per treatment were then harvested and pooled for real-time quantitative RT-PCR (qRT-PCR) analysis.

RNA Isolation and qRT-PCR
PtrDHN-3 RNA was isolated using pBIOZOL Plant Total RNA extraction reagent (Bioflux, Beijing, China). cDNA was then synthesized from the total RNA using a Reverse Transcription Kit (Takara Japan, Osaka, Japan).
Takara quantitative PCR enzyme (Takara Japan) was used for the qRT-PCR reactions, all of which were performed in triplicate as follows: 94 • C for 30 s followed by 45 cycles of 94 • C for 12 s, 58 • C for 30 s and 72 • C for 45 s. Detection was carried out using the Roche LightCycler 480 II (Jena q Tower 3G, Jena, Germany), and relative abundance was determined based on the 2 −∆∆CT method [45]. All experiments were conducted with three biological replicates.

Salt Stress Tolerance Assays in Yeast
Analysis of salt tolerance in yeast was carried out using a yeast expression vector. PCR primers were designed based on the CDS sequence of the PtrDHN-3 gene as follows: forward primer 5'-ACTCACTATAGGGAATATTA ATGGCTGAGGAAAACAAGAGC-3' and reverse primer 5'-TAATTACATGATGCGGCCCTCTACTGGGAAGCACTCTCCT-3'. The PtrDHN-3 gene was transferred into the yeast expression vector pYES2 (Carlsbad, CA, USA) using the Infusion enzyme (Zhongmei Taihe, c5891-50, Taihe, China). The constructed expression vector pYES2-PtrDHN-3 was then transformed into INVSC1 yeast cells, with an empty pYES2 plasmid used as a control. The transformed cells were cultivated on an SC-U medium containing 2% glucose in an incubator at 30 • C for three days. Positive clones were then identified by PCR detection. Single-clone cells containing the recombinant DNA (pYES2-PtrDHN-3) and control vector cells were then incubated in an SC-U medium with shaking until an OD of 0.6 was reached. Yeast cells were then diluted 10-, 100-and 1000-fold and spotted on an SC-U medium. For the salt tolerance assay, yeast cells were re-suspended in 3 M NaCl for 24 h then spread on an an SC-U medium and inverted in an incubator at 30 • C for three days. Images were then obtained (Canon EOS 7D MARKII, Japan). As a control, the cultured yeast cells were spotted on an SC-U medium without NaCl treatment.

Salt Stress Tolerance Assays in Arabidopsis
For analysis of salt stress tolerance, seeds of two transgenic Arabidopsis lines (OE-8, OE-10) and wild-type (WT) plants were sown on a 1/2 MS solid medium containing 100 mM NaCl. The germination rate was then observed after 10-14 days. Seeds of overexpressing T3 homozygous and control Arabidopsis lines were then sown on fresh 1/2 MS medium and observed for five days for germination. They were then transferred to 1/2 MS medium plates with or without (control) 100 mM NaCl and cultured at 22°C with a photoperiod of 16 h light and a photon flux of 80-120 µmol·m −2 ·s −1 . After 7-day continuous cultivation, fresh weights were then measured. Seedlings of 4-week-old T3 transgenic and WT Arabidopsis lines grown in pots were also treated with 100 mM NaCl solution for 10 days. Well-watered seedlings were used as a control. The planting method, nutrient substrate and cultivation environment were as described in Section 4.5. The phenotypes of the seedlings were then photographed with Canon EOS 7D MARKII, and contents of chlorophyll and proline in the leaves were determined. Chlorophyll contents was determined according to the description of Lightenthaler (1987) [47]. The samples were fully ground with liquid nitrogen and treated with acetone. The optical densities of supernate at wavelengths of 663 nm, 645 nm, 652 nm and 470 nm were measured. For proline assay, the ground samples were assayed by a colorimetric assay kit based on the ninhydrin reaction (Jiancheng Bio. Co., Nanjing, China) and the reaction product was quantified by its absorbance at 520 nm, calibrated with proline standards).

Histochemical Detection of Reactive Oxygen Species (ROS)
Leaves of 4-week-old T3 transgenic and WT Arabidopsis lines were sampled at 0, 3, 6 and 12 h after salt treatment. They were then stained with NBT (Nitrotetrazolium Blue chloride) for detection of H 2 O 2 and with DAB (Diaminobenzene) for detection of superoxide (O 2˙− ) [48].

Analysis of Antioxidant Enzyme Activity
Leaves of 4-week-old T3 transgenic and WT Arabidopsis lines were sampled after 12 h salt treatment. Stress-treated samples were collected for analysis of antioxidant enzyme activity. Superoxide dismutase (SOD) and peroxidase (POD) activities were determined using corresponding kits based on the manufacturers' instructions (Nanjing Jiancheng, Nanjing, China). Samples were obtained from at least nine seedlings per line, and all experiments were repeated three times.

Statistical Analyses
Statistical analyses were carried out using SPSS software version 20.0. Mean ± standard deviations (SD) were obtained from the average of three biological replicates. Data were compared using Student's t test, and differences were considered significant at p < 0.05.

Conclusions
The DHN gene PtrDHN-3 was successfully cloned from P. trichocarpa. Expression of PtrDHN-3 was subsequently found to be induced by NaCl, suggesting a role in the response to salt stress. PtrDHN-3 also improved tolerance to NaCl stress in transgenic yeast, as well as improving SOD and POD activity and reducing the accumulation of H 2 O 2 and O 2 − in transgenic Arabidopsis, preventing ROS oxidative harm and promoting osmotic regulation via accumulation of proline. Taken together, these findings suggest that PtrDHN-3 plays an important physiological role in resistance to salt stress in plants, providing a foundation for future research into the functions of DHN in stress resistance.
Author Contributions: M.Z. wrote the manuscript and performed some of the assays. N.P. provided the materials. C.Y. provided the gene (PtrDHN-3). C.W. provided funds for the current study, designed the study and revised the manuscript. All authors have read and agreed to the published version of the manuscript.