Introduction

Heavy metal toxicity, such as aluminum (Al) toxicity, is a major limiting factor for crop production worldwide [1]. When the pH of the soil is lower than 5.0, aluminum is present in an ionic form, i.e., Al3+, which strongly inhibits root growth and function, reducing crop yields [2]. Plant species and varieties vary widely in their ability to tolerate aluminum toxicity. Some plant species or varieties have evolved high levels of tolerance mechanisms to survive in acidic soils. Wild soybean has been growing in acidic soils in South China for a long time, and as such, there resources available that can provide tolerance, which plays an important role in improving the stress resistance of soybean [3]. Transcription factors are involved in stress responses; transcription factors from the WRKY, bZIP and NAC families have been shown to participate in the aluminum stress response and to regulate the aluminum tolerance of plants [4,5,6]. However, the involvement of the ERF transcription factor family in the aluminum stress response has not been reported. Plants employ a complex regulatory network to cope with a variety of stresses during growth and development. A variety of plant hormones play important roles from the beginning of sensing stress signals to the response of plants to stress. Under normal circumstances, the ethylene content in plants is maintained at a low level. However, plant ethylene content changes in response to biological stress or abiotic stress. The response of ethylene production after stress stimulation is transmitted through corresponding signal transduction pathways, which can regulate downstream genes, causing a series of reactions in plant cells and an associated response to stress [7,8,9]. Previous studies have shown that when plants are subjected heavy metal toxicity, the general response involves increased production of ethylene. For example, plants increase their production of ethylene under toxic levels of cadmium (Cd), copper (Cu), iron (Fe), nickel (Ni) and zinc (Zn). Moreover, it has been found that the change in ethylene under heavy metal stress is due to the increased expression of ethylene-related biosynthesis-related genes and/or changes in the expression of ethylene-responsive genes. Regarding these changes, it has been found that the increase in ethylene during stress can have negative effects on plants. However, ethylene can alleviate the inhibition of the photosynthetic capacity of mustard under cadmium stress. These findings suggest that ethylene involves a complex two-way regulatory function under stress, which depends on its endogenous level [10,11,12,13].

ERF transcription factors (ethylene response factors) constitute a subfamily of the AP2/ERF superfamily and can be divided into three categories according to the number of AP2/ERF domains: AP2, ERF and RAV [14]. The ERF family protein members contain an AP2/ERF domain consisting of 58–60 highly conserved amino acids, which constitutes the main functional region of ERF family proteins [15]. Ethylene response factors (ERFs) not only play important roles in plant growth and development but also play very important roles in the plant response to stress [15]. Previous studies have shown that ERF family genes are involved in plant growth and development in rice, Arabidopsis and other plant species. For example, OsERF1 is constitutively expressed in different organs of rice and is upregulated by ethylene. Overexpression of OsERF1 significantly affects the growth and development of transgenic Arabidopsis by promoting the expression of the ethylene-responsive genes PDF1.2 and β-chitinase [16]. AtERF71/HRE2 can activate the expression of downstream genes by binding the motifs of GCC boxes and DRE/CRT elements, regulate the expansion of root cells and play important roles in root development [17]. Julien Pirrello found that overexpression of the Sl-ERF2 gene in transgenic tomato lines can lead to early seed germination and enhanced hypocotyl formation in dark-grown seedlings. Recently, the transcription factor ERF139 was found in poplar to regulate the expansion of xylem cells and the deposition of secondary cell walls [18].

In recent years, an increasing number of ERF family genes have been found to function in stress tolerance in plants. Under drought stress, overexpression of the rice genes OsERF71, OsERF101 and OsERF48 was shown enhance the drought resistance of rice [19,20,21,22]. Heterologous overexpression of the soybean gene GmERF3 can enhance tobacco drought resistance [23], and overexpression of AtERF019 can enhance drought resistance in Arabidopsis [24]. Overexpression of GmERF135 can enhance the salt tolerance of Arabidopsis plants under salt stress. Moreover, GmERF135 can promote the growth of transgenic hairy roots under salt stress [25]. In wheat, overexpression of ERF1-V can enhance the salt tolerance of wheat, and heterologous overexpression of GmERF7 can enhance the salt tolerance of tobacco [26, 56], and the primers used for qRT PCR are listed in Supplementary Table S1.

GsERF1 gene isolation and sequence analysis

The GsERF1 gene was isolated from wild soybean line BW69. The full sequence of GsERF1 was amplified via PCR in conjunction with the following primer pair: 5′ – GGATCACGCCTCAAGTT − 3′ and 5′- CGAACCCTAAATCATCAG − 3′. The PCR products were inserted into the multiple cloning site of a pLB vector (Tiangen Biotech, Bei**g, China), and the positive clones were sent for sequencing. Multiple sequence alignment analysis was performed using DNAMAN software. Homology analysis of GsERF1 and the other 44 reference ERF superfamily genes was performed using MEGA 6.0 software through a neighbor-joining method. The amino acid sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and Phytozome (http://phytozome.jgi.doe.gov/pz/portal.html).

Subcellular localization analysis

To analyze the subcellular localization of the GsERF1 protein, full-length GsERF1 was inserted into the NcoI/SpeI sites of a pCAMBIA1302 vector to generate a GsERF1-eGFP construct. The pCAMBIA1302-GsERF1-eGFP fusion construct was subsequently transformed into tobacco epidermal cells. After 2–3 days, the green fluorescence signals in tobacco epidermal cells were observed under a confocal laser-scanning microscope (Olympus FluoView FV1000, Japan). The excitation wavelengths used were 488 nm for eGFP and 580 nm for RFP, and the resolution was 600 dpi [57].

In vitro transcriptional activation assays

For transactivation assays, the full-length GsERF1 gene was inserted into the EcoRI/BamHI sites of a pGBKT7 vector. The pGBKT7-GsERF1 construct was then transformed into yeast strain Y2H gold, and the transformants were grown on SD/−Trp media (Clontech) at 30 °C for 3 days. After selection of the yeast transformants carrying the GsERF1 gene on SD (−Trp) media, they were transferred to SD (−Trp, X-α-Gal) media to evaluate their transcriptional activation. An empty plasmid was used as a negative control.

Arabidopsis transformation and soybean hairy root transformation

Arabidopsis ecotype Col-0 was used for transformation. The full coding region of GsERF1 driven by the CaMV 35S promotor was inserted into the plant expression binary vector pTF101.1, yielding pTF101.1-GsERF1. The construct was subsequently transformed into Agrobacterium tumefaciens strain GV3101, and then the target gene was transferred into Arabidopsis plants by the floral-dip method [58].

Five-day-old seedlings with unfolded cotyledons were used for soybean hairy root production. For the RNAi construct, 233 bp of the GsERF1 coding region was cloned and inserted into a pMU103 vector. The overexpression vector and RNAi interference vector were then transferred into A. rhizogenes strain K599, after which the plants were transformed with the cells by hypocotyl injection [59]. An empty pTF101.1 plant expression binary vector was used as a control.

Hematoxylin staining

The expression of the GsERF1 gene in the hairy root lines was analyzed, and appropriate hairy root lines were selected for subsequent experiments. The hairy roots were treated with 0 or 25 μM AlCl3 (0.5 mM CaCl2, pH 4.5) for 6 h. After AlCl3 treatment, the hairy roots were washed three times with sterilized water and then stained with hematoxylin. The dyed roots were subsequently washed in sterile water for 30 minutes, after which they were observed and imaged through a Leica S8APO stereomicroscope (Leica, Germany) [46].

Phenotypic analysis of Arabidopsis tolerance to aluminum stress

To analyze the phenotypes of GsERF1-overexpressing (OX) and wild-type (WT) Arabidopsis under aluminum stress, seeds of T3GsERF1-overexpressing and WT plants were used. Among them, three transgenic lines with high expression levels were selected. The seed surfaces were sterilized with 10% sodium hypochlorite for 10 minutes and subsequently washed with deionized water. The sterilized seeds were grown on 1/2-strength MS agar plates in darkness for 4 days at 4 °C. Then, the plates were oriented upright and placed in a growth chamber at 22–24 °C, a 60% relative humidity, a 100 μmol photons m-2 s-1 light intensity, and 16 h light/8 h darkness photoperiod. Seedlings with a root length of 1 cm were selected and transferred to 1/2-strength MS agar media (pH 4.5) with different AlCl3 concentrations. After 10 days, the length from the base of the rosette leaf to the tip of the taproot was measured with a ruler, and images were taken with a Canon EOS 750d camera [60].

Physiological index assays

GsERF1 overexpression and WT lines were treated with or without aluminum for 10 days, and whole plants were selected as samples. The free proline content was measured as described in detail previously [23]. The ethylene precursor (ACC) and abscisic acid contents were determined using an enzyme-linked immunosorbent assay (ELISA) [61].

Statistical analysis

All experiments involving each group were performed at least in triplicate. The data are reported as the means ± SDs. All the data were analyzed via t tests by GraphPad Prism 6.01 software to assess significant differences between the means.