Overexpression of a Malus baccata NAC Transcription Factor Gene MbNAC25 Increases Cold and Salinity Tolerance in Arabidopsis

NAC (no apical meristem (NAM), Arabidopsis thaliana transcription activation factor (ATAF1/2) and cup shaped cotyledon (CUC2)) transcription factors play crucial roles in plant development and stress responses. Nevertheless, to date, only a few reports regarding stress-related NAC genes are available in Malus baccata (L.) Borkh. In this study, the transcription factor MbNAC25 in M. baccata was isolated as a member of the plant-specific NAC family that regulates stress responses. Expression of MbNAC25 was induced by abiotic stresses such as drought, cold, high salinity and heat. The ORF of MbNAC25 is 1122 bp, encodes 373 amino acids and subcellular localization showed that MbNAC25 protein was localized in the nucleus. In addition, MbNAC25 was highly expressed in new leaves and stems using real-time PCR. To analyze the function of MbNAC25 in plants, we generated transgenic Arabidopsis plants that overexpressed MbNAC25. Under low-temperature stress (4 °C) and high-salt stress (200 mM NaCl), plants overexpressing MbNAC25 enhanced tolerance against cold and drought salinity conferring a higher survival rate than that of wild-type (WT). Correspondingly, the chlorophyll content, proline content, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly increased, while malondialdehyde (MDA) content was lower. These results indicated that the overexpression of MbNAC25 in Arabidopsis plants improved the tolerance to cold and salinity stress via enhanced scavenging capability of reactive oxygen species (ROS).


Introduction
Plants play a very important role in our life, but various stresses in the environment affect the normal growth and development of plants seriously. The stress signal is perceived and transduced, ultimately resulting in the expression of functional proteins that protect the plant. Stress-responsive genes are mainly regulated by transcription factors, which are particularly important in the tolerance of plants to changeable environmental conditions [1]. NAC genes play important roles in plant resistance including abiotic and biotic stress responses. Previous studies have shown that there are a large number of transcription factors (TFs) exist in plants, which affect plants' response to stress [2]. NAC TFs (NAM, ATAF1/2 and CUC2) are widely distributed in plants. So far, 117 NAC family TFs have  The evolutionary relationship between MbNAC25 protein and other NAC proteins from different species was analyzed by DNAMAN (v5.0) ( Figure 1B). The sequences in the red frame are the conserved amino acid sequences of the MbNAC25 gene, which consist of 143 amino acids. They are also conservative sequences in NAC TFs of other species. In addition, the phylogenetic tree shows that MbNAC25 protein has the highest homology with MdNAC25-like (NP_001280970.1, from Malus domestica).

Subcellular Localization of MbNAC25 Protein
In order to determine the specific location of MbNAC25 protein, a fusion expression vector of green fluorescent protein (GFP) with MbNAC25 gene was constructed. As a control, the fluorescence of GFP was distributed in the plasma, membrane and nucleus ( Figure 2B), while the MbNAC25-GFP was only distributed in the nucleus with 4 , 6-diamidino-2-phenylindole (DAPI) staining ( Figure 2E). It can be concluded that the MbNAC25 protein was expressed in the nucleus.

Subcellular Localization of MbNAC25 Protein
In order to determine the specific location of MbNAC25 protein, a fusion expression vector of green fluorescent protein (GFP) with MbNAC25 gene was constructed. As a control, the fluorescence of GFP was distributed in the plasma, membrane and nucleus ( Figure 2B), while the MbNAC25-GFP was only distributed in the nucleus with 4′, 6-diamidino-2-phenylindole (DAPI) staining ( Figure 2E). It can be concluded that the MbNAC25 protein was expressed in the nucleus.

Expression Analysis of MbNAC25 in M. baccata
In the control condition, the expression of the MbNAC25 gene was higher in new leaves and stems than old leaves and roots ( Figure 3A). In cold, the expression of MbNAC25 increased rapidly, reached the maximum at 3 h in the new leaf and then decreased. Under high salinity and hightemperature stress, the expression level of MbNAC25 increased for 12 h, then decreased gradually. For drought stress, the expression levels of MbNAC25 increased after 12 h to 24 h of dehydration treatment then decreased ( Figure 3B). Furthermore, the expression level of MbNAC25 in the root peaked at 12 h, 6 h, 12 h and 24 h under cold, high-salt, drought stress and high-temperature treatments, and then showed a downward trend ( Figure 3C). The results showed that the expression of the MbNAC25 gene in the new leaf and root was induced by cold, high salt, drought and high temperature.

Expression Analysis of MbNAC25 in M. baccata
In the control condition, the expression of the MbNAC25 gene was higher in new leaves and stems than old leaves and roots ( Figure 3A). In cold, the expression of MbNAC25 increased rapidly, reached the maximum at 3 h in the new leaf and then decreased. Under high salinity and high-temperature stress, the expression level of MbNAC25 increased for 12 h, then decreased gradually. For drought stress, the expression levels of MbNAC25 increased after 12 h to 24 h of dehydration treatment then decreased ( Figure 3B). Furthermore, the expression level of MbNAC25 in the root peaked at 12 h, 6 h, 12 h and 24 h under cold, high-salt, drought stress and high-temperature treatments, and then showed a downward trend ( Figure 3C). The results showed that the expression of the MbNAC25 gene in the new leaf and root was induced by cold, high salt, drought and high temperature.

Overexpression of MbNAC25 in Arabidopsis Enhances Cold Tolerance
To investigate the role of MbNAC25 in response to cold stress in plants, transgenic Arabidopsis lines with overexpression of MbNAC25 under the control of the CaMV 35S promoter were generated. Among all the T2 generation transformed lines, the target fragments could be detected in six transformed lines (S2, S3, S4, S6, S9, S10) by RT-PCR with a wild-type (WT, Columbia-0) Arabidopsis line as a negative control ( Figure 4A). When the T3 generation of transgenic lines (S3, S6, S10, randomly selected) and WT plants were cultured under control conditions, both transgenic plants and WT plants grew well, and there was no significant difference in phenotype. However, at low temperature (−4 °C), improved cold tolerance in the transgenic (S3, S6, S10) lines were observed ( Figure 4B), and the survival rate of transgenic Arabidopsis lines were significantly higher than the WT line at 88.43% for S3, 85.52% for S6 and 87.49% for S10, compared to WT at only 24.73% ( Figure  4C). When restored to control conditions, most of the transgenic plants treated by low temperature could resume growth.

Overexpression of MbNAC25 in Arabidopsis Enhances Cold Tolerance
To investigate the role of MbNAC25 in response to cold stress in plants, transgenic Arabidopsis lines with overexpression of MbNAC25 under the control of the CaMV 35S promoter were generated. Among all the T 2 generation transformed lines, the target fragments could be detected in six transformed lines (S2, S3, S4, S6, S9, S10) by RT-PCR with a wild-type (WT, Columbia-0) Arabidopsis line as a negative control ( Figure 4A). When the T 3 generation of transgenic lines (S3, S6, S10, randomly selected) and WT plants were cultured under control conditions, both transgenic plants and WT plants grew well, and there was no significant difference in phenotype. However, at low temperature (−4 • C), improved cold tolerance in the transgenic (S3, S6, S10) lines were observed ( Figure 4B), and the survival rate of transgenic Arabidopsis lines were significantly higher than the WT line at 88.43% for S3, 85.52% for S6 and 87.49% for S10, compared to WT at only 24.73% ( Figure 4C). When restored to control conditions, most of the transgenic plants treated by low temperature could resume growth. In the control condition, there were no significant differences in chlorophyll content, proline content, MDA content, SOD, POD and CAT enzyme activity between transgenic Arabidopsis lines and the WT line. After low-temperature treatment, chlorophyll content, proline content, SOD, POD and CAT activities of MbNAC25 transgenic lines were significantly higher than those of the WT line, while MDA content was significantly lower than that of the WT line ( Figure 5). These results showed that WT plants suffered more serious membrane damage than MbNAC25 transgenic Arabidopsis plants. Hence, our results suggest that overexpression of the MbNAC25 gene could scavenge the intracellular reactive oxygen species (ROS) by increasing the enzyme activities of SOD, POD and CAT. In the control condition, there were no significant differences in chlorophyll content, proline content, MDA content, SOD, POD and CAT enzyme activity between transgenic Arabidopsis lines and the WT line. After low-temperature treatment, chlorophyll content, proline content, SOD, POD and CAT activities of MbNAC25 transgenic lines were significantly higher than those of the WT line, while MDA content was significantly lower than that of the WT line ( Figure 5). These results showed that WT plants suffered more serious membrane damage than MbNAC25 transgenic Arabidopsis plants. Hence, our results suggest that overexpression of the MbNAC25 gene could scavenge the intracellular reactive oxygen species (ROS) by increasing the enzyme activities of SOD, POD and CAT.

Overexpression of MbNAC25 in Transgenic Arabidopsis Increased High Salt Tolerance
To study the role of MbNAC25 in response to high-salt stress in plants, the transgenic Arabidopsis lines (S3, S6, S10) and the WT line were watered by 200 mM NaCl for seven days ( Figure 6A). The more yellowing leaves were observed in the WT plant than transgenic plants. When restored to normal irrigation conditions, most of the transgenic plants treated by salt stress could resume growth and the survival rate of transgenic plants was 85.62%, 90.74% and 85.75%, respectively, while that of the WT line was only 37.52% ( Figure 6B).

Overexpression of MbNAC25 in Transgenic Arabidopsis Increased High Salt Tolerance
To study the role of MbNAC25 in response to high-salt stress in plants, the transgenic Arabidopsis lines (S3, S6, S10) and the WT line were watered by 200 mM NaCl for seven days ( Figure 6A). The more yellowing leaves were observed in the WT plant than transgenic plants. When restored to normal irrigation conditions, most of the transgenic plants treated by salt stress could resume growth and the survival rate of transgenic plants was 85.62%, 90.74% and 85.75%, respectively, while that of the WT line was only 37.52% ( Figure 6B).
In order to study the reasons why the transgenic lines had better appearances under high-salt stress, the chlorophyll content, proline content, MDA content, SOD, POD and CAT activities in both transgenic Arabidopsis lines (S3, S6, S10) and the WT line were measured before and after salt stress. These physiological indexes of MbNAC25 transgenic Arabidopsis lines and the WT line under salt stress were basically the same as those of low temperature. In addition to MDA, transgenic Arabidopsis have higher levels of other indicators than wild-type Arabidopsis (Figure 7). This indicated that MbNAC25 transgenic plants had higher ROS scavenging enzyme activities to remove more ROS and protect the membrane. In order to study the reasons why the transgenic lines had better appearances under high-salt stress, the chlorophyll content, proline content, MDA content, SOD, POD and CAT activities in both transgenic Arabidopsis lines (S3, S6, S10) and the WT line were measured before and after salt stress. These physiological indexes of MbNAC25 transgenic Arabidopsis lines and the WT line under salt stress were basically the same as those of low temperature. In addition to MDA, transgenic Arabidopsis have higher levels of other indicators than wild-type Arabidopsis (Figure 7). This indicated that MbNAC25 transgenic plants had higher ROS scavenging enzyme activities to remove more ROS and protect the membrane.

Discussion
Previous reports have confirmed that NAC TFs are involved in many plant growth and development processes such as apical meristem development, flower morphogenesis and lateral root development. In addition, NAC proteins are also involved in plant defenses' response to abiotic and

Discussion
Previous reports have confirmed that NAC TFs are involved in many plant growth and development processes such as apical meristem development, flower morphogenesis and lateral root development. In addition, NAC proteins are also involved in plant defenses' response to abiotic and biotic stresses [22]. In this study, a NAC gene, named MbNAC25, was isolated by a homologous cloning method from the M. baccata. The protein encoded by the MbNAC25 gene has the typical structural characteristics of the NAC family. It was found that the N-terminus of the MbNAC25 protein contains a highly conserved NAC domain, but the transcriptional regulatory regions at the C-terminus are diverse. The conserved sequence of MbNAC25 protein contains 157 amino acids. In addition, the average hydrophilic coefficient of MbNAC25 protein is -0.684, which indicated that MbNAC25 protein was a hydrophilic protein. According to the analysis of protein sequence, the contents of leucine (Leu), lysine (Lys), proline (Pro), serine (Ser) and threonine (Thr) were relatively high, while methionine (Met) and tryptophan (Trp) were relatively deficient. Evolutionary tree analysis showed that MbNAC25 protein had the highest homology with Malus domestica NAC25 protein (Figure 1). The results of subcellular localization showed that MbNAC25 protein was localized in the nucleus ( Figure 2).
Increasing evidence indicates that NAC TFs are in important regulatory roles in plants in response to stresses such as drought, low temperature and high salt. Ohnishi et al. found that under control condition, it is difficult to detect OsNAC6 in rice, but after induction by drought, high salt, low temperature and ABA, the expression of OsNAC6 was significantly increased; moreover, overexpression of OsNAC6 could induce the expression of many abiotic stress-related genes [26]. Similarly, drought, high salt and ABA could induce the expression of ANAC019, ANAC055 and ANAC072 in Arabidopsis and overexpression of these three genes could also improve the drought tolerance of Arabidopsis [27]. In addition, a dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway [28]. Tran et al. found that nine GmNAC genes in soybean were up-regulated in shoots and roots induced by drought stress [29]. The results of this study are similar to the findings above. According to the results of real-time quantitative PCR, the highest expression of the MbNAC25 gene in new leaves under low-temperature, high-salt, drought and high-temperature stresses were 3 h, 12 h, 24 h and 12 h ( Figure 3B), while the highest expression of the MbNAC25 gene in the roots under same abiotic stresses were 12 h, 6 h, 12 h and 24 h ( Figure 3C). It could be due to the fact that the response of roots to high salinity and drought stresses is faster than that of new leaves, which is due to the fact that stresses always start from roots in high salinity and drought treatment. However, the response rate of new leaves to low-temperature and high-temperature stresses is faster than that of roots, which indicated that stress starting from new leaves in low-temperature and high-temperature treatments. Therefore, the expression of the MbNAC25 gene is induced by low-temperature, high salinity, drought and high-temperature stresses.
A large amount of ROS is produced in the cells when plants are subjected to stress. H 2 O 2 and O 2·were detected after drought and salt stresses, and a lower amount of H 2 O 2 and O 2was observed in the roots of transgenic plants than in the WT plant [30]. Plants have gradually formed complex and delicate mechanisms to cope with oxidative stress in the process of evolution such as active oxygen scavenger enzyme system, including peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), to remove excess reactive oxygen species and free radicals in plants. NAC proteins are involved in mediating the antioxidative system under adversity stress [31]. An elevated malondialdehyde (MDA, a product of lipid peroxidation) level is used frequently as an indicator of reactive oxygen species (ROS) and associated cell membrane degradation or dysfunction. The MDA content of bluegrass leaves gradually increased with decreasing temperature. The higher the content of MDA, the weaker the plant's frost resistance [32]. The overexpression of GmNAC085 enhanced antioxidant capacity in the transgenic plants to reduce drought-induced oxidative damage via reducing MDA content accompanied by increased activities of superoxide dismutase, catalase and ascorbate peroxidase [33]. Our study discussed the role of MbNAC25 in plants under cold stress. The results indicated that wild-type Arabidopsis showed obvious wilting, while the transgenic Arabidopsis were normal; that is, the overexpression of the MbNAC25 gene could significantly improve the resistance of transgenic plants under low-temperature stress (4 • C) (Figure 4). The chlorophyll content of wild-type and transgenic Arabidopsis decreased under low-temperature stress, but the former decreased faster. At the same time, the physiological indexes such as proline content, MDA content, SOD, POD and CAT activity of transgenic and wild Arabidopsis increased after low-temperature stress, which indicated that both transgenic and wild Arabidopsis were damaged under low-temperature stress. Compared with wild Arabidopsis, these physiological indexes except MDA of transgenic Arabidopsis increased more, and the rising of MDA content was lower ( Figure 5). This indicated that the transgenic Arabidopsis suffered less damage under low-temperature stress. The MbNAC25 gene significantly enhanced the tolerance of Arabidopsis to low-temperature stress.
Due to the high salt concentration in the plant rhizosphere, the soil water potential is lower than the water potential in the plant tissue, which makes it difficult for the plant to absorb water. The high salt concentration can also cause water infiltration in the plant, cause water loss and even cause plant death [34]. Similarly, the wild-type after salt stress treatment showed more obvious yellowing than the transgenic Arabidopsis (Figure 6), and the changes of chlorophyll content, proline content, MDA content, SOD, POD and CAT activity under salt stress were similar to those under low-temperature stress, but the change ranges were different (Figure 7). The MbNAC25 gene significantly enhanced the tolerance of Arabidopsis to salt stress.
In a word, our results suggest MbNAC25 was induced by low-temperature, high-salt, high-temperature and drought stress and the overexpression of MbNAC25 in transgenic Arabidopsis plants enhances tolerance to cold and salinity stress.

Plant Material and Treatments
The tissue culture plantlets of M. baccata were planted in Murashige and Skoog (MS) growth medium (MS + 0.6 mg·L −1 6-BA + 0.6 mg·L −1 IBA). After a month, the tissue culture plantlets were transplanted into rooting medium (MS + 1.2 mg·L −1 IBA) until white rootstocks emerged. Then, the tissue culture seedlings should be transferred to Hoagland nutrient solution with 80% relative humidity. Moreover, the nutrient solution should be replaced every 3-4 days. When the leaves were fully unfolded, seedlings were divided into four parts for salt stress (watered with 200 mM NaCl solution), low-temperature stress (placed in 4 • C growth incubator), drought stress (watered with 15% PEG solution) and high-temperature stress (placed in 38 • C growth incubator). The seedlings cultured under normal Hoagland nutrient solution were as control. After 0, 1, 3, 6, 12, 24 and 48 h for stress treatment, the plant materials were obtained and frozen with liquid nitrogen immediately, then stored in −80 • C for RNA extraction [35].

Isolation and Cloning of MbNAC25
An OminiPlant RNA Kit (Kangweishiji, Beijing, China) was used to extract RNA from new leaves, old leaves, root and shoot tips of hydroponic seedlings. TransScript ® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) was used to synthesize the cDNA First Strands. RNA and cDNA were assessed by 1.0% agarose gel electrophoresis. The whole sequence of MbNAC25 was obtained by polymerase chain reaction (PCR) with primers MbNAC25F and MbNAC25R, using the first-strand cDNA of M. baccata as a template. The primers used in this study are shown in Table 1. The obtained DNA fragments were purified and cloned into objective vectors using the pEASY ® -T1 Cloning Kit (TransGen Biotech, Beijing, China) and sequenced [36][37][38][39].

Subcellular Localization Analysis of the MbNAC25 Protein
The DNA fragmentsof MbNAC25 was cloned into the BamH I and Xba Isites of pSAT6-GFP-N1 vector. Gold powder containing MbNAC25-GFP plasmid was injected into onion epidermal cells by gene gun method [40], and subcellular localization was carried out [41]. The fluorescence of MbNAC25-GFP was observed under a confocal microscope [42].

Quantitative Real-Time PCR Analysis
The expression of MbNAC25 gene under different abiotic stress was analyzed by RT-PCR. The specific primers were shown in Table 1. The expression of the MbNAC25 gene was detected by the Tli RNaseH Plus kit (TaKaRa, Beijing, China) according to the manufacturer's protocol. The expression data were analyzed using the 2 −∆∆CT method [43]. The Actin gene (NC_024251.1, M. domestica) was used as a reference gene.

Vector Construction and Agrobacterium-Mediated Arabidopsis Transformation
The primers with restriction enzyme sites BamH I and Xba I were designed, and the target fragment was amplified by PCR.The PBI121 vector and the target fragment were simultaneously digested with Xba I and BamH I enzyme, then the target fragment was connected to the vector PBI121 to construct the PBI121-MbNAC25 super expression vector. The PBI121-MbNAC25 vector was transfected into Arabidopsis ecotype Columbia-0 by Agrobacterium tumefaciens GV3101 transformation. The seeds of transformants were seeded in 1/2MS medium (containing 50 mg·L −1 kanamycin) [44] to selectthe transformed lines until the T 2 generations. Seeds of the T 2 generation of transgenic plants (S3, S6, S10, randomly selected) were sown and germinated in a nutrient soil to vermiculite ratio of 4:1 in flowerpots (diameter 10 cm) with normal management in a growth chamber at 25 ± 1 • C under a 16 hlight (50 µmol m −2 s −1 ) and 8 h dark regime in parallel with the wild-type (WT) seeds. Twenty-five seedlings were grown for 3 weeks with regular irrigation prior to salt stress. Salt stress experiments were conducted by watering 200 mM NaCl solution for 7 days. Then, the 25 plants of each line (S3, S6, S10 and WT) were rewatered for 6 d to calculate the survivalrate. The experiments were performed three times foreach treatment at each time point. During the wholegrowth process, all Arabidopsis seedlings were observedand recorded by photographing salt stress for 0 days, 7 days and rewatered for 6 days (recover). Forlow-temperature stress, 3-week seedlings were grown in the 4 • C growth incubator parallel withwild-type (WT). The experiments were also performed three times foreach treatment at each time point. During the wholegrowth process, all Arabidopsis seedlings were observedand recorded by photographing cold stress for 0 h, 12 h and recovered at room temperature for 24 h (Recover).

Determination of Related Physiological Indexes
The seeds of transgenic Arabidopsis lines (S3, S6, S10) and a WT line were sown in a screening medium (1/2 MS + 50 mg·L −1 kanamycin) and transferred to nutrient soil for two weeks. Twenty-five plants of each transgenic lines and the WT line were placed in low temperature (4 • C for 12 h), high salt (200 mM NaCl for 7 days) and normal conditions as control. Chlorophyll content was measured according to the method of Xu et al. [45]. Proline content was measured according to the method of