Isolation, sequencing, and expression analysis of 30 AP2/ERF transcription factors in apple

Background AP2/ERF transcription factors are involved in the regulation of plant growth, development, and stress responses. Our research objective was to characterize novel apple (Malus × domestica Borkh.) genes encoding AP2/ERF transcription factors involved in regulation of plant growth, development, and stress response. The transcriptional level of apple AP2/ERF genes in different tissues and under various biotic and abiotic stress was determined to provide valuable insights into the function of AP2/ERF transcription factors in apple. Methods Thirty full-length cDNA sequences of apple AP2/ERF genes were isolated from ‘Zihong Fuji’ apple (Malus × domestica cv. Zihong Fuji) via homologous comparison and RT-PCR confirmation, and the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. Expression levels of apple AP2/ERF genes were detected in 16 different tissues using a known array. Expression patterns of apple AP2/ERF genes were detected in response to Alternaria alternata apple pathotype (AAAP) infection using RNA-seq with existing data, and the expression of apple AP2/ERF genes was analyzed under NaCl and mannitol treatments using qRT-PCR. Results The sequencing results produced 30 cDNAs (designated as MdERF3-8, MdERF11, MdERF16-19, MdERF22-28, MdERF31-35, MdERF39, MdAP2D60, MdAP2D62-65, and MdRAV2). Phylogenetic analysis revealed that MdERF11/16, MdERF33/35, MdERF34/39, and MdERF18/23 belonged to groups A-2, A-4, A-5, and A-6 of the DREB subfamily, respectively; MdERF31, MdERF19, MdERF4/25/28/32, MdERF24, MdERF5/6/27, and MdERF3/7/8/17/22/26 belonged to groups B-1, B-2, B-3, B-4, B-5, and B-6 of the ERF subfamily, respectively; MdAP2D60 and MdAP2D62/63/64/65 belonged to the AP2 subfamily; and MdRAV2 belonged to the RAV subfamily. Array results indicated that 30 apple AP2/ERF genes were expressed in all examined tissues to different degrees. RNA-seq results using previously reported data showed that many members of the apple ERF and DREB subfamilies were induced by Alternaria alternate apple pathotype (AAAP) infection. Under salt treatment, many members in the apple ERF and DREB subfamilies were transcriptionally up or down-regulated. Under mannitol treatment, many members of the apple ERF, DREB, and AP2 subfamilies were induced at the transcriptional level. Taken together, the results indicated that the cloned apple AP2/ERF genes were expressed in all examined tissues. These genes were up-regulated or down-regulated in response to AAAP infection and to salt or mannitol treatment, which suggested they may be involved in regulating growth, development, and stress response in apple.

Apple (Malus × domestica Borkh.) is one of the most important tree fruits in the world. However, progress on the ERF transcription factors in apple is more limited than that in model plants like Arabidopsis thaliana, and most researches about apple are focused on fruit ripening and softening (Wang et al., 2007;Tacken et al., 2010;Li et al., 2016;An et al., 2017;Li et al., 2017;Han et al., 2018a;Han et al., 2018b). In this study, we obtained the AP2/ERF transcription factor in apple based on previous results (Girardi et al., 2013) and from the Plant Transcription Factor Database (http://planttfdb.cbi.pku.edu.cn/). When the 60 known transcription factors were excluded by sequence alignment in the GenBank database (Tacken et al., 2010), the other genes were cloned and analyzed. In total, 30 genes in the AP2/ERF family were obtained. Furthermore, we analyzed the phylogenetic relationships, subcellular locations, and expression levels in different tissues under different biotic and abiotic stresses for the 30 AP2/ERF genes. The results are helpful for further studying roles of AP2/ERF transcription factors played in growth, development, and biotic and abiotic stress in apple.

Plant materials
The apple cultivar 'Gala' (Malus × domestica cv. Gala) was used as material under stress conditions. In vitro seedlings of 'Gala' were cultivated on basic subculture medium (MS medium + 0.2 mg L −1 indole-3-acetic acid (IAA) + 0.8 mg L −1 6-benzylaminopurine (6-BA) + 30 g L −1 sucrose + 7 g L −1 agrose) that was changed every 30 d. The cultivation conditions were under 14-h light/10-h dark and a temperature of 24 ± 2 • C. On the 20th day on the basic subculture medium, some relatively uniform seedlings were selected and transplanted to different media. The basic subculture medium was used as the control. We added 150 mmol L −1 NaCl or 300 mmol L −1 mannitol to the basic subculture medium to create different treatments (Li et al., 2019).

Gene cloning and sequence analysis
RNA was extracted in the fully expanded leaves of 'Zihong Fuji' apple (Malus × domestica cv. Zihong Fuji) by the CTAB method, then cDNA was synthesized using a PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Based on the nucleotide sequence of 259 identified members in the apple AP2/ERF gene family and the 60 known transcription factors in the GenBank database (Tacken et al., 2010;Velasco et al., 2010;Girardi et al., 2013), we designed primers for PCR amplification and 30 apple AP2/ERF genes were finally cloned (Table S1). The PCR reaction conditions were 94 • C for 5 min, then 35 cycles for 94 • C for 1 min 20 s, 56-60 • C for 1 min, 72 • C for 2 min, and a final extension at 72 • C for 10 min. PCR products were purified and cloned into pMD19-T vector to construct recombinant plasmids. The recombinant plasmids were transformed into the competent cells of Escherichia coli DH5α, and then the positive clones were selected.

Subcellular localization analysis
The full-length cDNA without the stop codon of MdERF28 was introduced into the pCAMBIA2300-GFP vector. The fusion vectors were then introduced into Agrobacterium tumefaciens strain EHA105 and then infiltrated into tobacco leaves. Those infected tissues were analyzed 72 h after infiltration, under a fluorescence microscope (BX63; Olmypus, Tokyo, Japan).
The RNA was extracted from the treated tissues of 'Gala' using a RNeasy Plant Mini Kit (QIAGEN, China, Item No. 74903), and the cDNA was synthesized using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The qRT-PCR primers (Table S1) were designed based on the 3 -or 5 -UTR of AP2/ERF genes, and then qRT-PCR was conducted using a 3-step method by BIO-RAD IQ5 (USA) with MdMDH RNA as the internal reference gene (Perini et al., 2014). Three independent biological replicates were used for calculations. Each 20 µL qRT-PCR reaction mixture consisted of SYBR Green Master I 10 µL, 5 µmol L −1 forward prime 1 µL, 5 µmol L −1 reverse prime 1 µL, template 1 µL, and ddH 2 O 7 µL. qRT-PCR conditions were 95 • C for 3 min, then 40 cycles for 95 • C for 10 s, 58.5 • C for 30 s, 72 • C for 15 sand, after annealing to 55 • C, the temperature was increased 0.5 • C every 7 s till 95 • C, with 81 cycles in total. The 2 − CT method was used to analyze the data (Livak & Schmittgen, 2001).

Cloned genes in the AP2/ERF family in apple
Based on the nucleotide sequence of 259 identified members in the apple AP2/ERF gene family and the 60 known transcription factors in the GenBank database (Tacken et al., 2010;Velasco et al., 2010;Girardi et al., 2013), the other primers for PCR amplification were designed, and a total of 30 genes in the apple AP2/ERF family were cloned (Table 1). Homology alignment for the amino acid information showed that all the MdAP2/ERF proteins contained an AP2 conserved domain (Fig. 1). Both MdAP2D60 and MdAP2D62-65 had two AP2 conserved domains, and MdRAV2 had one B3 conserved domain (Fig. 1).

Subcellular locations of AP2/ERF proteins in apple
Subcellular localization of AP2/ERF proteins was performed by SoftBerry ProtComp 9.0, CELLO, and PORST using their protein sequences. All prediction results indicated that MdERF3-8, MdERF11, MdERF16-19, MdERF22-28, MdERF33-35, MdERF39, MdAP2D60, MdAP2D62-65, and MdRAV2 were target to nuclear (Table 2). To further verification of these subcellular locations revealed by the online software, the MdERF28-GFP fusion protein was performed to detect the subcellular location of MdERF28 protein and a transient transfection assay into tobacco leaves. The GFP control was ubiquitously distributed throughout the cell, whereas MdERF28-GFP fusion protein was predominantly detected in the nucleus (Fig. 3), indicating that MdERF28 was localized in the nucleus.

Expression analysis of 30 AP2/ERF gene family in apple
The array (GSE42873) in 16 different apple tissues in GEO (https://www.ncbi.nlm.nih. gov/geo/) was used to evaluate the expression level of the AP2/ERF gene family in different tissues (Fig. 4). The 30 AP2/ERF genes exhibited diverse expression patterns among the various tissues (Fig. 4).
Further, we detected the expression level of the response of the AP2/ERF gene family to AAAP infection using RNA-seq with existing data (>two-fold and FDR<0.001) (Zhu et al., 2017). MdERF16 in A2, MdERF35 in A4, MdERF23 in A6, MdERF25/28/32 in B3, MdERF6/27 in B5, and MdERF8 in B6 were all up-regulated in the response of apples' AAAP infection ( Fig. 5 and File S3). Particularly, the expression level of B3 in MdERF32 was increased significantly, which was 12.6-folds by 18 h post inoculation (HPI). Expression levels of MdERF23 in A6, MdERF25 in B3, MdERF28 in B3, and MdERF27 in B5 were all increased, which were 18.2, 8.4, 16.2, and 8.7-fold by 72 HPI, respectively. During the early (12 HPI) and intermediate (18 and 36 HPI) phase of infection, expression levels of  MdERF4 in B3 and MdERF5 in B5 were increased at the beginning and then decreased later, and expression of MdERF4 was 4.6-fold by 18 HPI. Expression levels of MdERF22 in B6 and MdAP2D65 was down-regulated on 72 HPI ( Fig. 5 and File S3). The relative expression level of other genes did not change significantly ( Fig. 5 and File S3). The AP2/ERF gene family expression in 'Gala' seedlings under mannitol and NaCl stress was analyzed by qRT-PCR. Under NaCl stress, eight members in the AP2/ERF family were up-regulated, which included MdERF16 in A2, MdERF23 in A6, MdERF25/28/32 in B3, MdERF24 in B4, MdERF17 in B6, and MdRAV2 (Fig. 6). Among them, the expression level of MdERF23, MdERF25, and MdERF28 were increased more than 10 times when treated for 48 h compared with that of the control. MdERF11 in A2, MdERF33 in A4, MdERF34 in A5, MdERF18 in A6, MdERF31 in B1, MdERF4 in B3, MdERF5 in B5, and MdERF22/26 in B6 were down-regulated. Expression levels of MdERF5 and MdERF39 were only 0.04 and  0.23 times that of the control, respectively, when treated with NaCl for 24 h, but expression level of MdERF39 was increased to 3.11 times that of the control when treated for 48 h. The other AP2/ERF genes under NaCl stress had almost the same expression level compared with that of the control (Fig. 6). Under mannitol treatment condition, the relative expression levels of MdERF11 (A2), MdERF33 (A4), MdERF39 (A5), MdERF31 (B1), MdERF19 (B2), MdERF28/32 (B3), MdERF5/6/27 (B5), MdERF7 (B6), and MdAP2D60/62/64/65 were increased compared with the control, MdERF39 (A5) reached 5.96 times that of the control at 24 h, and MdERF11 (A2) and MdAP2D65 reached 7.99 and 10.7 times that of the control, respectively, when treated for 48 h (Fig. 6).The relative expression level of MdERF25 (B3) and MdRAV2 were inhibited compared with that of the control, but the relative expression level of other AP2/ERF genes did not change significantly under mannitol treatment (Fig. 6).  et al., 2010;Girardi et al., 2013). In this study, we cloned 30 apple AP2/ERF genes, which belonged to the AP2, ERF, DREB, and RAV subfamilies of AP2/ERF, and their changes in expression level in different tissues were analyzed under AAAP infection, and NaCl and mannitol stresses. ERF is one the largest transcription factor families in plants. The A. thaliana genome contained 147 AP2/ERF proteins, which were divided into the AP2, ERF (ERF and DREB), and RAV sub-families based on their similarity in amino acid sequences and domain number (Nakano et al., 2006;Mizoi, Shinozaki & Yamaguchi-Shinozaki, 2012). The 30 genes cloned in this study were divided into four subfamilies; 8, 16, 5, and 1 gene belonged to the subfamilies DREB, ERF, AP2, and RAV, respectively (Fig. 2). AAAP infection, NaCl stress, and mannitol stress all affected the expression of MdERF4/25/28/32 in the B3 group at transcriptional level, except for MdERF4 under mannitol stress. In A. thaliana, AtERF1, AtERF2, AtERF5, and AtERF6 in the B3 group, which could be induced by osmotic stress (Moffat et al., 2012), were responded to Saprophytic bacteria by up-regulating the downstream resistance genes PDF1.2 and b-CHI, resulting in enhanced resistance to S. bacteria infection (Fujimoto et al., 2000;Berrocal-Lobo, Molina & Solano, 2002;Lorenzo et al., 2003;Moffat et al., 2012). Alfafa exhibited increased resistance from MtERF1-1 in the B3 group that up-regulated the resistance downstream gene PDF1.2 (Anderson et al.,  2010). In wheat, TaPIEP1 in the B3 group was up-regulated by Bipolaris sorokiniana, which boosting disease resistance (Dong et al., 2010). Transgenic tobacco plants had enhanced resistance to Tobacco Mosaic Virus and brown spot through overexpression of NtERF5 and GbERF2 (Fischer & Droge-Laser, 2004;Zuo et al., 2007). The transgenic A. thaliana with SpERF1, which was the ERF member of the B3 group in Stipa purpurea, had increased drought tolerance when SpERF1 was up-regulated (Yang et al., 2016). In this study, MdERF4/25/28/32 was clustered into the B3 group of ERF and was up-regulated significantly under AAAP infection and NaCl stress; also, mannitol stress had some effects on MdERF4/25/28/32 expression (Figs. 5 and 6). These results indicated that MdERF4/25/28/32 may play important roles in response to various biotic and abiotic stress.
Several studies have proved that the DREB transcription factor subfamily was important for abiotic stress (Nakano et al., 2006). For example, A. thaliana showed increased tolerance to high-salt and drought by overexpression of certain DREB transcription factors that included DREB2A and DREB2B in the A2 group, HARDY in the A4 group, and RAP2.4 in the A6 group. DREB2C, DREB2D, and DREB2F in A. thaliana played an important role in high-salt stress (Nakano et al., 2006;Sakuma et al., 2006;Karaba et al., 2007;Qin et al., 2007;Lin, Park & Wang, 2008). Drought tolerance in maize was enhanced by DREB2A overexpression in the A2 group (Qin et al., 2007). Overexpression of the PsAP2 gene in the A6 group of Papaver somniferum enhanced the resistance of transgenic tobacco to pathogenic bacteria, salt, and mannitol stresses (Mishra et al., 2015). In this study, under mannitol stress, MdERF11 in the A2 group, MdERF33 in the A4 group, and MdERF39 in the A5 group were up-regulated at transcriptional level. Seven genes were induced by NaCl at transcriptional level. Three of them, MdERF16 in A4, MdERF39 in A5, and MdERF23 in A6, were up-regulated at transcriptional level under NaCl stress, and four genes, which included MdERF11 in A2, MdERF33 in A4, MdERF34 in A5, and MdERF18 in A6, were down-regulated. In addition, there were four genes, which included MdERF16 in A2, MdERF35 in A4, and MdERF23 in A6, were up-regulated at transcriptional level by AAAP infection (Figs. 5 and 6). These results showed that the DREB transcription factors cloned in this study might be important for responding to abiotic stress, and some members might play a role in response to biotic stress. The AP2 subfamily may be important for plant growth and development (Maes et al., 2001;Jofuku et al., 2005;Chung et al., 2010;Horstman et al., 2014), but also be critical for defending against biotic and abiotic stress (Park et al., 2001;Yi et al., 2004). For example, the overexpression of the Tsi1 gene improved tobacco's tolerance to pathogenic bacteria and osmotic stress (Park et al., 2001), and the CaPF1 gene in Capsicum annuum cv. Bukang responded to ethylene (ET), jasmonic acid (JA), and cold stress, and its overexpression Figure 6 Expression heatmap of apple AP2/ERF genes under normal growth, mannitol and salt treatments. The expression data of apple AP2/ERF genes under normal growth, mannitol and salt treatments were obtained from qRT-PCR. The heat map of apple AP2/ERF genes was generated by TIGR MeV v4.8.1 software.
Full-size DOI: 10.7717/peerj.8391/ fig-6 improved A. thaliana resistance to low temperature and to infection by Pseudomonas syringae pv. tomato DC3000 (Yi et al., 2004). In this study, MdAP2D65 in AP2 responded to AAAP infection only at transcriptional level, but it did not respond to NaCl stress, and MdAP2D60/62/64/65 were up-regulated by mannitol stress (Figs. 5 and 6). These results indicated that MdAP2D60/62/64/65 had some effect on osmotic stress, and MdAP2D65 might be involved in responding to biotic stress.

CONCLUSIONS
Thirty novel AP2/ERF genes have been successfully isolated from Malus domestica, which belong to DREB, ERF, AP2, and RAV subfamily. Results of a known array and RNA-seq analysis using existing data as well as qRT-PCR-based transcription profiling indicated that 30 apple AP2/ERF genes were expressed in all examined tissues at different expression levels, and responded differentially to various stresses, suggesting that these genes may be involved in the regulation of growth, development, and stress responses in apple. These results serve as the theoretical basis for understanding the biological function and regulation of AP2/ERF transcription factors in apple.
• Kun Ran conceived and designed the experiments, performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences: The MdERF3