An ERF Transcription Factor Gene from Malus baccata (L.) Borkh, MbERF11, Affects Cold and Salt Stress Tolerance in Arabidopsis

Apple, as one of the most important economic forest tree species, is widely grown in the world. Abiotic stress, such as low temperature and high salt, affect apple growth and development. Ethylene response factors (ERFs) are widely involved in the responses of plants to biotic and abiotic stresses. In this study, a new ethylene response factor gene was isolated from Malus baccata (L.) Borkh and designated as MbERF11. The MbERF11 gene encoded a protein of 160 amino acid residues with a theoretical isoelectric point of 9.27 and a predicated molecular mass of 17.97 kDa. Subcellular localization showed that MbERF11 was localized to the nucleus. The expression of MbERF11 was enriched in root and stem, and was highly affected by cold, salt, and ethylene treatments in M. baccata seedlings. When MbERF11 was introduced into Arabidopsis thaliana, it greatly increased the cold and salt tolerance in transgenic plant. Increased expression of MbERF11 in transgenic A. thaliana also resulted in higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), higher contents of proline and chlorophyll, while malondialdehyde (MDA) content was lower, especially in response to cold and salt stress. Therefore, these results suggest that MbERF11 probably plays an important role in the response to cold and salt stress in Arabidopsis by enhancing the scavenging capability for reactive oxygen species (ROS).


Introduction
During growth and development, plants are frequently exposed to various abiotic stresses, such as drought, cold, salt, heat, and nutrient deprivation. Under different environmental stresses, plants have developed different adaptable mechanisms to ensure their normal growth and development [1,2]. The response mechanism of plants to abiotic stresses was regulated by multiple signaling pathways [3]. In these processes, transcription factors (TFs) play a key role in the interaction of these signaling pathways [4][5][6][7]. TFs, also known as trans-acting factors, are a class of DNA-binding proteins that specifically bind to cis-acting elements. Interactions between transcription factors and cis-acting elements or other proteins can be transcriptional activation or inhibition. Studies have clarified that TF genes are abundantly present in plants, and can regulate plant growth and metabolism [8,9].

Plant Material and Growth Conditions
The M. baccata test-tube seedlings were rapidly propagated in MS growth medium (containing 0.6 mg/L IBA + 0.6 mg/L 6-BA) for 30 days. Then they were transplanted to MS + 1.2 mg/L IBA for 45 days for rooting. Finally, the seedlings were transferred to Hoagland solution for 40 days for growth. The solution was changed three times per week. The temperature of the culture chamber was maintained at 20 • C and the relative humidity was maintained at about 85%. When the test-tube seedlings grew to 8-9 leaves (completely expanded), a part of them was placed in Hoagland solution with a NaCl concentration of 200 µM and pH 5.8 for salt stress treatment. The other part of them was placed in Hoagland solution at 4 • C for cold stress treatment. Test-tube seedlings incubated in Hoagland solution at 20 • C were used as control. The unexpanded young leaf, the completely unfolded mature leaf, the phloem at the second and third node stem segments, and the newly emerged root were taken as samples. The samples of all control and treated plants were sealed after treatments of respectively 0, 2,4,8,12,24, and 48 h, immediately frozen in liquid nitrogen, and then stored at −80 • C for RNA extraction.

The qRT-PCR Expression Analysis of MbERF11
Total RNA was respectively extracted from young leaf, mature leaf, new root, and stem using the EasyPure Plant RNA Kit (TransGen, Beijing). Synthesis of cDNA first strands with TransGen's Trans Script ® First-Strand cDNA Synthesis Super Mix (TransGen, Beijing). The whole sequence of MbERF11 was obtained by PCR, with the first strand cDNA of M. baccata as a template. A pair of primers (MbERF11-F and MbERF11-R, Table 1) were designed based on the homologous regions of MdERF011 (MDP0000258562) to amplify the full-length cDNA sequence. The obtained DNA fragments were gel purified and cloned into the pEasy-T1 vector (TransGen) and sequenced (BGI, Beijing). Table 1. Primers used in this study.

Name of Primer
Sequence of Primer (5 →3 ) Purpose The qRT-PCR expression analysis of MbERF11 was performed according to method of Han et al. [33]. The Malus ACTIN gene (AB638619.1) amplified from M. baccata tissues was as control, which was stably expressed under various conditions [34]. We designed the primers (ACTIN-F and ACTIN-R, Table 1) from the sequences and published in the GenBank databases. The primers (MF and MR, Table 1) of MbERF11 were designed from partial sequences cloned in this study for qRT-PCR detection. The thermal cycling program was one initial cycle of 94 • C for 30 s, followed by 40 cycles of 94 • C for 15 s, and 55 • C for 30 s. The relative transcription level data was analyzed by the Pfaffl method [35].

Subcellular Localization Analysis of MbERF11 Protein
The MbERF11 ORF was cloned into the SacI and BamHI sites of the pSAT6-GFP-N1 vector. This vector contains a modified red-shifted green fluorescent protein (GFP) at SacI-BamHI sites. The MbERF11-GFP construct was transformed into onion (Allium cepa) epidermal cells by particle bombardment [36]. The DAPI staining was used as a nucleus marker for nucleus detection. The transient expression of the MbERF11-GFP fusion protein was observed by confocal microscopy.

A. Thaliana Transformation
To construct an expression vector for the transformation of A. thaliana, restriction enzyme cut sites of SacI and BamHI at 5 and 3 ends of the MbERF11 cDNA was respectively added by PCR with the primers (Site-F, Site-R, Table 1). To construct the PCAM3301-MbERF11 vector, PCAM3301 and the products of PCR were digested by SacI and BamHI, and linked together by T4 DNA ligase. The MbERF11 gene driven by the CaMV 35S promoter and the vecror (only PCAM3301) were introduced into A. thaliana by Agrobacterium-mediated LBA4404 transformation [37]. Columbia ecotype A. thaliana plants were transformed using the vacuum infiltration method. Transformants (transgenic lines and vector line) were selected on MS medium containing 6 mg/mL glufosinate. The transgenic lines (roots used as materials) were confirmed by qRT-PCR analysis with wild type (WT) and vacant-vector line (VL) as control. T3 generation plants were used for further analysis.

Determination Survival Rates Under Cold and/or Salt Stress Treatment in Transgenic Arabidopsis
Wild-type A. thaliana (WT), vacant-vector line (VL, the line only transformed with vacant vector) and three MbERF11 transgenic lines (S2, S6, S7) were respectively sown in culture medium, and transferred to nutrient soil for two weeks after 10 days. The WT, VL and T3 transgenic A. thaliana were cultured under control condition, low temperature treatment (−4 • C) for 12 h, and high salinity stress (200 mM NaCl) for 7 d, respectively, after which their survival rates were recorded with 15 nutrition pots.
2.6. Detection of the Contents of Chlorophyll, MDA and Proline and the Activity of SOD, POD and CAT All the materials of different lines above were collected for measurements. The chlorophyll content was determined with method of Li et al. [38]. The proline content was measured according to the spectrophotometric method [39]. The MDA content and the activities of SOD, POD, and CAT were measured according to the protocol described by Shin et al. [40].

Statistical Analysis
DPS 7.05 data processing system software was used for one-way analysis of variance (ANOVA). All experiments were repeated for three times and the standard errors (±SE) were measured, respectively. Statistical differences were referred to as significant * p ≤ 0.05, ** p ≤ 0.01.

Isolation of MbERF11 Gene from M. Baccata
The ProtParam analysis (http://www.expasy.org/tools/protparam.html) showed that the MbERF11 gene encodes 160 amino acids (Figure 1). The theoretical molecular mass of MbERF11 is 17.97 kDa, with theoretical isoelectric point 9.27 and the average hydrophilicity coefficient −0.995. The underlined part of Figure 1 is the conserved sequence of the AP2/ERF family, which contains two conserved elements, namely YRG and RAYD. The 14th and 19th amino acids of the conserved sequence are valine and glutamic respectively, indicating that it belongs to the DREB subfamily in the AP2/ERF family.

Determination Survival Rates Under Cold and/or Salt Stress Treatment in Transgenic Arabidopsis
Wild-type A. thaliana (WT), vacant-vector line (VL, the line only transformed with vacant vector) and three MbERF11 transgenic lines (S2, S6, S7) were respectively sown in culture medium, and transferred to nutrient soil for two weeks after 10 days. The WT, VL and T3 transgenic A. thaliana were cultured under control condition, low temperature treatment (−4 °C) for 12 h, and high salinity stress (200 mM NaCl) for 7 d, respectively, after which their survival rates were recorded with 15 nutrition pots.

Detection of the Contents of Chlorophyll, MDA and Proline and the Activity of SOD, POD and CAT
All the materials of different lines above were collected for measurements. The chlorophyll content was determined with method of Li et al. [38]. The proline content was measured according to the spectrophotometric method [39]. The MDA content and the activities of SOD, POD, and CAT were measured according to the protocol described by Shin et al. [40].

Statistical Analysis
DPS 7.05 data processing system software was used for one-way analysis of variance (ANOVA). All experiments were repeated for three times and the standard errors (±SE) were measured, respectively. Statistical differences were referred to as significant * p ≤ 0.05, ** p ≤ 0.01.

Isolation of MbERF11 Gene from M. Baccata
The ProtParam analysis (http://www.expasy.org/tools/protparam.html) showed that the MbERF11 gene encodes 160 amino acids (Figure 1). The theoretical molecular mass of MbERF11 is 17.97 kDa, with theoretical isoelectric point 9.27 and the average hydrophilicity coefficient −0.995. The underlined part of Figure 1 is the conserved sequence of the AP2/ERF family, which contains two conserved elements, namely YRG and RAYD. The 14th and 19th amino acids of the conserved sequence are valine and glutamic respectively, indicating that it belongs to the DREB subfamily in the AP2/ERF family.

Phylogenetic Relationship of MbERF11 with Other ERF Proteins
To explore the evolutionary relationship among plant ethylene response factors, DNAman was used to compare MbERF11 with other 13 ERF proteins of different species. The phylogenetic tree

Phylogenetic Relationship of MbERF11 with Other ERF Proteins
To explore the evolutionary relationship among plant ethylene response factors, DNAman was used to compare MbERF11 with other 13 ERF proteins of different species. The phylogenetic tree showed that MbERF11 contains an AP2/ERF conserved domain consisting of 58 amino acid residues. This conserved sequence is the characteristic sequence of the ERF TF in plants ( Figure 2A). As shown in Figure 2A, the TF of ERF family has higher homology in the conserved domain. There are also many changes in the non-conserved domain, which is consistent with the characteristics of the TF. showed that MbERF11 contains an AP2/ERF conserved domain consisting of 58 amino acid residues. This conserved sequence is the characteristic sequence of the ERF TF in plants ( Figure 2A). As shown in Figure 2A, the TF of ERF family has higher homology in the conserved domain. There are also many changes in the non-conserved domain, which is consistent with the characteristics of the TF. However, AtERF7 (NC_003074.8) was grouped into another cluster on its own ( Figure 2B). The homologous phylogenetic tree showed that MbERF11, MdERF011 (XP_008378018, Malus domestica), EjERF7 (AKN10300.1, Eriobotrya japonica), PbERF011 (XP_009359912.1, Pyrus bretschneideri) and PpERF011 (XP_007223577.1, Prunus persica) clustered together. NtERF010 (XP_009614221.1, Nicotiana tomentosiformis), StERF010 (XP_006354856.1, Solanum tuberosum), SiERF008 (XP_011074400.1, Sesamum indicum), MnERF2 (XP_010094641.1, Morus notabilis), and BpERF11 (AMD11605.1, from Betula platyphylla) were grouped into the second cluster, followed by CsERF011 (XP_004141277.1, Cucumis sativus), CmERF011 (XP_008452588.1, Cucumis melo), and McERF011 (XP_022143655.1, Momordica charantia). However, AtERF7 (NC_003074.8) was grouped into another cluster on its own ( Figure 2B).

MbERF11 was Localized to the Nucleus
As shown in Figure 3, the MbERF11-GFP fusion protein is targeted to nucleus ( Figure 3E) with 4 , 6-diamidino-2-phenylindole (DAPI) staining ( Figure 3F), whereas the control GFP alone is distributed throughout the cytoplasm ( Figure 3B). These results determined that MbERF11 is a nucleus localized protein. distributed throughout the cytoplasm ( Figure 3B). These results determined that MbERF11 is a nucleus localized protein.

Expression Analysis of MbERF11 in M. Baccata
As shown in Figure 4A, in control condition, the expression level of MbERF11 in M. baccata seedlings was higher in root and stem, while very low in leaf. When dealt with salt (200 mM NaCl), cold (4 °C), and ethephon treatments (500 μL/L, the ratio of ethylene:water is 1:2000), the expression level of MbERF11 in young leaf of M. baccata increased quickly, reached maximum at 12 h, 24 h, and 4 h, respectively, and then decreased ( Figure 4B). The expression level of MbERF11 in root had a similar trend, which reached the maximum at 8 h, 12 h, and 2 h, respectively, then decreased slightly ( Figure 4C).

Expression Analysis of MbERF11 in M. Baccata
As shown in Figure 4A, in control condition, the expression level of MbERF11 in M. baccata seedlings was higher in root and stem, while very low in leaf. When dealt with salt (200 mM NaCl), cold (4 • C), and ethephon treatments (500 µL/L, the ratio of ethylene:water is 1:2000), the expression level of MbERF11 in young leaf of M. baccata increased quickly, reached maximum at 12 h, 24 h, and 4 h, respectively, and then decreased ( Figure 4B). The expression level of MbERF11 in root had a similar trend, which reached the maximum at 8 h, 12 h, and 2 h, respectively, then decreased slightly ( Figure 4C).

Overexpression of MbERF11 in A. Thaliana Contributed to Low Temperature Stress Tolerance
To study the role of MbERF11 in cold and salt stress responses, MbERF11 gene was transformed into A. thaliana under the control of the CaMV 35S promoter. Among 12 transformed lines, seven of them (S1, S2, S4, S6, S7, S9, and S10) were confirmed by qRT-PCR analysis with wild type (WT) and vacant-vector line (VL) as control ( Figure 5A).

Overexpression of MbERF11 in A. Thaliana Contributed to Low Temperature Stress Tolerance
To study the role of MbERF11 in cold and salt stress responses, MbERF11 gene was transformed into A. thaliana under the control of the CaMV 35S promoter. Among 12 transformed lines, seven of them (S1, S2, S4, S6, S7, S9, and S10) were confirmed by qRT-PCR analysis with wild type (WT) and vacant-vector line (VL) as control ( Figure 5A).
As shown in Figure 5B, no significant difference in appearance was found among all the A. thaliana lines (WT, VL, S2, S6, and S7) under control condition (Cold 0h). However, when dealt with low temperature (−4 • C) stress for 12 h (Cold 12 h), the transgenic plants (S2, S6 and S7) look much healthier than WT and VL. There were no differences in appearance between WT and VL under control condition and cold stress.
Under control condition, there was no significant difference in the survival rates among all A. thaliana lines (WT, VL, S2, S6 and S7). However, when dealt with cold stress, the survival rates of WT and VL A. thaliana were only 16.7% and 18.3%, while the transgenic plants of S2, S6, and S7 were 78.7%, 75.1%, and 81.3%, respectively. The survival rates of transgenic plants were significantly higher than those of WT and VL lines under low temperature treatments ( Figure 5C).  Figure 5B, no significant difference in appearance was found among all the A. thaliana lines (WT, VL, S2, S6, and S7) under control condition (Cold 0h). However, when dealt with low temperature (−4 °C) stress for 12 h (Cold 12 h), the transgenic plants (S2, S6 and S7) look much healthier than WT and VL. There were no differences in appearance between WT and VL under control condition and cold stress.

As shown in
Under control condition, there was no significant difference in the survival rates among all A. thaliana lines (WT, VL, S2, S6 and S7). However, when dealt with cold stress, the survival rates of WT and VL A. thaliana were only 16.7% and 18.3%, while the transgenic plants of S2, S6, and S7 were 78.7%, 75.1%, and 81.3%, respectively. The survival rates of transgenic plants were significantly higher than those of WT and VL lines under low temperature treatments ( Figure 5C).
As shown in Figure 6, for the contents of chlorophyll, MDA and proline, as well as the activities of SOD, POD, and CAT, these was no significant difference among all the A. thaliana lines, i.e., S2, S6, S7, WT and VL plants under control conditions (Cold 0 h). However, when dealt with cold treatment (−4 °C) for 12 h (Cold 12 h), the activities of SOD, POD, and CAT, the chlorophyll and proline contents were higher than those in WT and VL. However, the contents of MDA in transgenic A. thaliana (S2, S6, and S7) were significantly lower than those in WT and VL ( Figure 6). As shown in Figure 6, for the contents of chlorophyll, MDA and proline, as well as the activities of SOD, POD, and CAT, these was no significant difference among all the A. thaliana lines, i.e., S2, S6, S7, WT and VL plants under control conditions (Cold 0 h). However, when dealt with cold treatment (−4 • C) for 12 h (Cold 12 h), the activities of SOD, POD, and CAT, the chlorophyll and proline contents were higher than those in WT and VL. However, the contents of MDA in transgenic A. thaliana (S2, S6, and S7) were significantly lower than those in WT and VL ( Figure 6).

Overexpression of MbERF11 in A. Thaliana Contributed to Improved Salt Stress Tolerance
The WT, VL and transgenic lines (S2, S6, and S7) of A. thaliana were treated with 200 mM NaCl solution daily for seven days, and then the phenotype of each line was observed. As shown in Figure 7A, the transgenic lines (S2, S6, and S7), WT and VL plants all grew well under control condition (Salt 0 d). However, when dealt with salt stress for 7 days (Salt 7 d), the transgenic A. thaliana (S2, S6 and S7) had better appearance than WT and VL plants.

Overexpression of MbERF11 in A. Thaliana Contributed to Improved Salt Stress Tolerance
The WT, VL and transgenic lines (S2, S6, and S7) of A. thaliana were treated with 200 mM NaCl solution daily for seven days, and then the phenotype of each line was observed. As shown in Figure  7A, the transgenic lines (S2, S6, and S7), WT and VL plants all grew well under control condition (Salt 0 d). However, when dealt with salt stress for 7 days (Salt 7 d), the transgenic A. thaliana (S2, S6 and S7) had better appearance than WT and VL plants.   Under control condition, there was no significant difference in the survival rates of all A. thaliana lines (WT, VL, S2, S6, and S7). However, when dealt with salt stress for 7 days, the survival rates of WT and VL plants were only 41.9% and 43.2%, while the transgenic lines of S2, S6, and S7 were 89.7%, 85.8% and 87.6%, respectively. The survival rates of the transgenic plants under salt stress were significantly higher than those of WT and VL plants ( Figure 7B).
As shown in Figure 8, when treated with salt stress (Salt 7 d), overexpression of MbERF11 in transgenic A. thaliana resulted in lower MDA contents, higher levels of chlorophyll and proline Under control condition, there was no significant difference in the survival rates of all A. thaliana lines (WT, VL, S2, S6, and S7). However, when dealt with salt stress for 7 days, the survival rates of WT and VL plants were only 41.9% and 43.2%, while the transgenic lines of S2, S6, and S7 were 89.7%, 85.8% and 87.6%, respectively. The survival rates of the transgenic plants under salt stress were significantly higher than those of WT and VL plants ( Figure 7B).
As shown in Figure 8, when treated with salt stress (Salt 7 d), overexpression of MbERF11 in transgenic A. thaliana resulted in lower MDA contents, higher levels of chlorophyll and proline contents, as well as higher activities of SOD, POD, and CAT than those of WT and VL plants. However, for the indices above, these was no significant difference among the entire test lines (WT, VL, S2, S6, and S7) under control condition (Salt 0 d).

Discussion
From the transcriptome analysis of M. baccata seedlings under cold and/or drought stresses, we found the MbERF11 level was significantly up-regulated under both stresses. More importantly, through NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of MbERF11 gene, we found that the closest Arabidopsis ERF gene is AtERF7, which is a famous ERF TF gene involved in drought stress through ABA signal transduction [32]. Sequence homologous analysis showed that MbERF11 is a member of the ERF family ( Figure 2A). All the ERF family includes one conserved ERF domain in the middle region [41]. These results showed that the ERF family genes are highly conserved during the evolution. ERF genes were widely distributed in apple, A. thaliana, pear, jujube, cucumber, tobacco, and rice, and were known to be involved in a variety of processes, including stress [10,12,18,21,32]. Subcellular localization has revealed that the MbERF11 is a nucleus localized protein (Figure 3), which is consistent with other ERF proteins [5,17,28,30,32]. Phylogenetic tree analysis shows that the relationship between MbERF11 and MdERF011 is the closest among 14 species. Among Arabidopsis ERF TFs, AtERF7 has the highest homology to MbERF11 ( Figure 2B).
The expression level of MbERF11 was more enriched in stem and root than in young leaf and mature leaf ( Figure 4A). This expression pattern indicated that MbERF11 may play an important role in organs that related to transport of stress signal. When dealt with cold, salt, and ethephon treatments, the expression level of MbERF11 in M. baccata was markedly affected. It is possible that MbERF11 plays a key role in regulating stress response in M. baccata. Ethylene is considered as a signal of stress in plants [17,19,25,41], and ethylene treatment affected the expression of MbERF11. The changed expression level of MbERF11 induced by cold and salt probably depends on the synthesis of endogenous ethylene.

Discussion
From the transcriptome analysis of M. baccata seedlings under cold and/or drought stresses, we found the MbERF11 level was significantly up-regulated under both stresses. More importantly, through NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of MbERF11 gene, we found that the closest Arabidopsis ERF gene is AtERF7, which is a famous ERF TF gene involved in drought stress through ABA signal transduction [32]. Sequence homologous analysis showed that MbERF11 is a member of the ERF family ( Figure 2A). All the ERF family includes one conserved ERF domain in the middle region [41]. These results showed that the ERF family genes are highly conserved during the evolution. ERF genes were widely distributed in apple, A. thaliana, pear, jujube, cucumber, tobacco, and rice, and were known to be involved in a variety of processes, including stress [10,12,18,21,32]. Subcellular localization has revealed that the MbERF11 is a nucleus localized protein (Figure 3), which is consistent with other ERF proteins [5,17,28,30,32]. Phylogenetic tree analysis shows that the relationship between MbERF11 and MdERF011 is the closest among 14 species. Among Arabidopsis ERF TFs, AtERF7 has the highest homology to MbERF11 ( Figure 2B).
The expression level of MbERF11 was more enriched in stem and root than in young leaf and mature leaf ( Figure 4A). This expression pattern indicated that MbERF11 may play an important role in organs that related to transport of stress signal. When dealt with cold, salt, and ethephon treatments, the expression level of MbERF11 in M. baccata was markedly affected. It is possible that MbERF11 plays a key role in regulating stress response in M. baccata. Ethylene is considered as a signal of stress in plants [17,19,25,41], and ethylene treatment affected the expression of MbERF11. The changed expression level of MbERF11 induced by cold and salt probably depends on the synthesis of endogenous ethylene.
When dealt with salt, cold, and ethephon treatments, the MbERF11 expression level in root of M. baccata reached the highest level at 8 h, 12 h, and 2 h, respectively ( Figure 4C), while in leaf, which got the maximum at 12 h, 24 h, and 4 h, respectively ( Figure 4B). The results showed that the response rate to stresses, such as low temperature, salt stress and ethephon in root is faster than in leaf, indicating that the expression of MbERF11 in root is more sensitive than that in leaf. This expression profile indicated that MbERF11 may play a key role in the plants' stress signal transportation. Ethylene is a gaseous plant hormone that regulates many aspects of the plant life cycle, including seed germination, leaf senescence, fruit ripening, abscission, as well as biotic and abiotic stress responses [42][43][44]. Consequently, a higher concentration of ethylene in plants may be a signal to regulate plant stress response. This conclusion was consistent with the cold resistance mechanism of VaERF057 [45]. Ethylene had been proposed to protect mitochondrial activity in A. thaliana under cold stress [46]. GmERF7 had been confirmed to regulate the expression of stress-related genes through regulating the content of ethylene, thereby improving the salt tolerance [47]. Therefore, the increased expression level of MbERF11 induced by cold and salt stresses may depend on the biosynthesis of ethylene.
The expression of AtERF7 can be induced by ethylene, ABA and JA [32]. Abiotic stresses could induce the expression of AtERF71, AtERF73, RAP2.1, RAP2.2, and RAP2.3 [48][49][50]. These results were consistent with our research (MbERF11 expression level can be induced by ethylene, salt, and cold treatments). The expression level of GmERF3 increased when dealt with abiotic stresses such as drought and high salinity. External factors such as ethylene and other hormone treatments could also change the expression level of GmERF3. However, low temperature stress had little effect on GmERF3 expression [51]. The expression level of MbERF11 gene in young leaf and new root was also significantly affected by cold, salt, and ethephon treatments ( Figure 4B,C). Low temperature and salt stress could also increase the ethylene levels and trigger cold and salt stress responses in plants [30,46]. Based on the previous studies and theories, we reckon that ethephon treatments induce stress responses, such as the increased expression of MbERF11 in the above parts.
Overexpression of MbERF11 enhanced the tolerance to both cold and salt stresses in transgenic A. thaliana. The levels of chlorophyll, proline, MDA and antioxidant enzymes can be used to indicate the damage extent from stress [52,53]. The higher MDA content indicates higher degree of membranous peroxidation of the plant cells and the more serious damage to the cell membrane [2]. The proline content in WT A. thaliana increased after exposure to low temperature stress. Low temperature stress caused the destruction of chloroplast and the yellowing of plants. Hence, chlorophyll content is one of the important indicators of whether the plant is subjected to adverse stress [54]. The antioxidant enzyme system in plant plays an important role in resisting external environmental stress. They can inhibit the accumulation of free radicals, thereby reducing the occurrence of oxidative damage and lethal effects. The above effects allowed a variety of biochemical metabolic activities in cells to proceed normally. Overexpression of MbERF11 enhanced the tolerance to cold and salt stresses in transgenic A. thaliana ( Figures 5B and 7A), also led to increased activities of SOD, POD, and CAT, contents of proline and chlorophyll, decreased MDA content, especially when dealt with stresses (Figures 6 and 8). It is possible that MbERF11 could increase cold and salt tolerance through changing these physiological indicators in transgenic A. thaliana under stress.
These results indicate that the MbERF11 may be an upstream regulatory gene for stress resistance, and the overexpression of MbERF11 gene may enhance the cold and salt tolerance of A. thaliana. More works need to be done to further verify the function of MbERF11 through heterologous expression in Arabidopsis mutants (AtERF7 gene deletion). Clarifying the role of the different domains of MbERF11 in stress response will be helpful in breeding stress-resistant Malus by gene transfer. Further experiments are required to identify other functions of MbERF11 gene.

Conclusions
In the present study, a new ERF gene was isolated from M. baccata and named as MbERF11. Subcellular localization showed that MbERF11 protein was located to the nucleus. When MbERF11 was introduced into A. thaliana, it increased the levels of proline and chlorophyll, and improved the activities of SOD, POD, and CAT, but decreased MDA content, especially under cold and salt treatments. Taken together, our results suggest that MbERF11 plays an important role in response to cold and salt stress by enhancing the capability of scavenging ROS.

Conflicts of Interest:
The authors declare no conflicts of interest.