**2. Materials and Methods**

#### *2.1. 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.

#### *2.2. 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).



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].

#### *2.3. Subcellular Localization Analysis of MbERF11 Protein*

The *MbERF11* ORF was cloned into the *Sac*I and *Bam*HI sites of the pSAT6-GFP-N1 vector. This vector contains a modified red-shifted green fluorescent protein (GFP) at *Sac*I–*Bam*HI 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.

#### *2.4. A. Thaliana Transformation*

To construct an expression vector for the transformation of *A. thaliana*, restriction enzyme cut sites of *Sac*I and *Bam*HI 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 *Sac*I and *Bam*HI, 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.

#### *2.5. 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].

#### *2.7. 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.

#### **3. Results**

#### *3.1. 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.


**Figure 1.** Nucleotide and deduced amino acid sequences of *MbERF11* gene. Underlines indicate conserved amino acid sequences. Black boxes indicate specific amino acids of the AP2/ERF domain. Blue boxes indicate conserved elements.
