**Deguo Han, Jiaxin Han, Guohui Yang, Shuang Wang, Tianlong Xu and Wenhui Li \***

Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, College of Horticulture & Landscape Architecture, Northeast Agricultural University, Harbin 150030, China; deguohan\_neau@126.com (D.H.); A02140301@163.com (J.H.); yangguohui\_neau@126.com (G.Y.); ws18045312966@163.com (S.W.); 18846829610@163.com (T.X.) **\*** Correspondence: lwh\_neau@126.com; Tel.: +86-4515-5190-781

Received: 2 April 2020; Accepted: 29 April 2020; Published: 2 May 2020

**Abstract:** 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).

**Keywords:** *Malus baccata*; *MbERF11*; cold stress; salt stress; transgenic plant

## **1. 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 [4–7]. 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].

The APETALA2/ethylene responsive factor (AP2/ERF) TFs are widely distributed in different types of plants [10]. About 145 AP2/ERF TF genes have been isolated from *A. thaliana* [11], and 167 AP2/ERF TFs were also found in Oryza sativa [12]. The AP2/ERF TF family may have evolved from HNHAP2 endonucleases in bacteria and viruses [13]. The AP2/ERF genes were widely isolated and studied from different plants, such as *APETALA2*, *AINTEGUMENTA* and *AtCBF1* from *Arabidopsis* [14–16], *ThCRF1* from *Tamarix hispida* [17], and *CsERF025* from cucumber [18]. The AP2/ERF TFs were found to participate in almost every process of plant growth and development, especially in response to biotic or abiotic stresses in plants [19].

As a class of TFs, AP2/ERF TFs usually contain an AP2/ERF domain consisting of about 60 amino acid residues [20]. This domain was firstly discovered from APETALA2 homologues of *A. thaliana* [21], a similar sequence was also found in tobacco [22]. According to the different numbers of AP2/ERF domains contained in AP2/ERF, the AP2/ERF TFs were divided into two subfamilies. Among them, one subfamily is called ERF containing one AP2/ERF domain. The other subfamily is called the AP2 subfamily containing two AP2/ERF domains. The ERF subfamily can be further divided into DREB (dehydration responsive element binding protein), ERF and other three categories [23]. In the AP2/ERF domain of DREB subfamily, the 14th and 19th amino acids are valine (V) and glutamic acid (E), respectively, while they are alanine and aspartate in the AP2/ERF domain of ERF subfamily [24].

ERF TFs are widely involved in biotic and abiotic stress responses, which play important roles in drought, high salt and low temperature tolerance, as well as plant development, hormone response and other regulatory networks [24,25]. Previous researches found ERF TFs were also involved in organ development, cell division, differentiation, flower development and fruit maturation in plants [26,27]. Overexpression of *Sl-ERF2* gene in tomato could activate *Sl-Man2*, a mannanase-encoding gene, and result in the premature germination of tomato immature seeds [28]. The *Sub1A*/*C* gene in *O. sativa* participates in plant growth and metabolism [29]. The *MdERF1*/*2* genes in *Malus domestica* are associated with fruit ripening [30]. However, these studies mostly focused on model plants or crops, and the roles of the ERF TFs genes in *Malus* plant stress responses were less well known.

*M. baccata* is widely used as an apple rootstock in northern China, and also as a source of forestry wood or greening tree species. Due to its high resistance to low temperature and drought, M. baccata is also used as a breeding material for cold and drought resistance [31]. From the transcriptome analysis of *M. baccata* seedlings under cold and/or drought stresses (results not presented here), 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]. To better understand the role of ERF TFs genes involved in low temperature and salt stress, and to provide potentially genetic resources for the improvement of the drought resistance of *Malus* plant, a new ERF TFs gene was isolated from *M. baccata* and designated as *MbERF11*. Moreover, it was found that the tolerance of transgenic *A. thaliana* to cold and salt stress was increased because of the overexpression of *MbERF11*.
