*3.4. 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).

**Figure 4.** Time-course expression patterns of *MbERF11* in *Malus baccata* using qRT-PCR methord. (**A**) Expression patterns of *MbERF11* in young leaf (partly expanded), mature leaf (fully expanded), root and stem in normal condition (room temperature and normal nutrient solution). The expression level of young leaf was as control. (**B**,**C**) Expression patterns of *MbERF11* in control, salt (200 μM), cold (4 ◦C) and ethephon (500 μL/L) in young leaf (**B**), and root (**C**) at the following time points: 0 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. The reference genes *MdACTIN* and *MdGAPDH* were used as controls in this study. The error bars represent standard deviation. Asterisks above the error bars indicate a significant difference between the treatment and control (0 h) using Student's *t* test (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01).

#### *3.5. 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 5.** Overexpression of *MbERF11* in *Arabidopsis*improved cold tolerance. (**A**) Transgenic *Arabidopsis* qRT-PCR validation. (**B**) Phenotypic map of *Arabidopsis* under control condition (Cold 0 h), cold stress (Cold 12 h) and recover. All the test lines, including the T3 transgenic *A. thaliana* (S2, S6, and S7), WT and VL were dealt with low temperature stress (−4 ◦C) for 12 h, and then with control temperature (20 ◦C) 1 h for recover. Scale bar corresponds to 3 cm. (**C**) The survival rates of transgenic and WT *Arabidopsis* under control condition and low temperature stress. Extremely significant differences between transgenic *Arabidopsis* (S2, S6, and S7), vacant-vector line (VL) and WT line were shown by the *t* test, \*\* *p* ≤ 0.01.

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).

#### *3.6. 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.

**Figure 6.** The levels of malondialdehyde content ((**A**) MDA); superoxide dismutase activity ((**B**), SOD); peroxidase activity ((**C**), POD); catalase activity ((**D**), CAT); chlorophyll content ((**E**), Chl) and proline content ((**F**), Pro) Proline in WT, VL (vacant-vector line) and *MbERF11*-OE (S2, S6, and S7) *Arabidopsis* under control condition (Cold 0 h) and cold stresses (Cold 12 h). Asterisks above the error bars indicate an extremely significant difference between the transgenic and WT plants with Student's *t* test (\*\* *p* ≤ 0.01). The level of each index in WT was as control.

**Figure 7.** Overexpression of *MbERF11* in *Arabidopsis* improved salt tolerance in transgenic plants. (**A**) Phenotypic map of *Arabidopsis* under control condition (Salt 0 d), salt stress (Salt 7 d) and recover. All the test lines, including the T3 transgenic *A. thaliana* (S2, S6, and S7), WT and VL were dealt with salt stress (200 mM NaCl) for 7 d, and then with control water management 3 d for recover. Scale bar corresponds to 3 cm. (**B**) The survival rate of *Arabidopsis* under control condition and salt stress. Extremely significant differences between transgenic *Arabidopsis* (S2, S6, and S7), VL and WT line were shown by the *t* test, \*\* *p* ≤ 0.01.

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).

**Figure 8.** The levels of malondialdehyde content ((**A**), MDA); superoxide dismutase activity ((**B**), SOD); peroxidase activity ((**C**), POD); catalase activity ((**D**), CAT); chlorophyll content ((**E**), Chl) and proline content ((**F**), Pro) Proline in WT, VL (vacant-vector line) and *MbERF11*-OE (S2, S6, and S7) under control condition (Salt 0 d) and salt stresses (Salt 7 d). Asterisks above the error bars indicate an extremely significant difference between the transgenic and WT *Arabidopsis* with Student's *t* test (\*\* *p* ≤ 0.01). The level of each index in WT was as control.

#### **4. 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 [42–44]. 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* [48–50]. 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.

## **5. 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.

**Author Contributions:** D.H. and W.L. designed the experiments; D.H., J.H., G.Y., and S.W. performed the experiments; T.X. and J.H. analyzed the data; D.H. and W.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by by University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (UNPYSCT-2017004), National Natural Science Foundation of China (31301757), Postdoctoral Scientific Research Development Fund of Heilongjiang Province, China (LBH-Q16020, LBH-Z15019), the Natural Science Fund Joint Guidance Project of Heilongjiang Province (LH2019C031) and the Open Project of Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs (neauhc201803).

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