*3.2. 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.

**Figure 2.** Comparison and phylogenetic relationship of MbERF11 with ethylene-responsive transcription factor proteins from other species. (**A**) Comparison completes alignment of MbERF11 with other plant ethylene-responsive transcription factor proteins. Conserved domains are shown in blue boxes. Positions containing identical residues are shaded in navy blue, while conservative residues are shown in green. (**B**) Phylogenetic tree analysis of MbERF11 and other plant ethylene-responsive transcription factor proteins. The tree was constructed by the neighbour-joining method with MEGA 7 (http://www.megasoftware.net). The gene accession numbers are listed in 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).

#### *3.3. 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.

**Figure 3.** Subcellular localization of MbERF11 protein. The transient vector harboring 35S-GFP and 35S-*MbERF11*-GFP cassettes were transformed into onion epidermal cells by particle bombardment. Transient expressions of green fluorescent protein 35S-GFP (**B**) and 35S-*MbERF11*-GFP (**E**) translational product were visualized in onion epidermal cells by fluorescence microscopy. Onion epidermal cells of 35S-GFP (**C**) and 35S-*MbERF11*-GFP (**F**) stained with DAPI for 24 h. (**A**,**D**) were taken in the bright light. Scale bar corresponds to 5 μm.
