Plant seeds including wild-type (WT) A. thaliana (Columbia ecotype glabrous1), G. max (L.) Merr. cultivars Jackson (salt-sensitive), Lee68 (salt-tolerant) and N23674 (salt-tolerant), Escherichia coli DH5α, Agrobacterium tumefaciens strain GV3101, Agrobacterium rhizogenes strain K599, binary vector for plant transformation pCAMBIA1300, the yeast Δgef1 mutant (derived from Saccharomyces cerevisiae BY4741) and yeast expression plasmid pYES2 were used in this study.

The seeds of G. max N23674 cultivar were surface-sterilized with 1 g dm^-3 HgCl₂ for 5 min, then fully rinsed in distilled water, soaked in distilled water for 6 h, and finally germinated at 25°C in the dark. The germinated seeds were grown on vermiculite irrigated with 1/2 Hoagland solution in a greenhouse with temperature at 25 ± 2°C and humidity ranging from 60 to 70%. Total RNA was extracted from 10-days-old seedlings using the Trizol reagent (Invitrogen, USA). First-strand cDNAs were synthesized with 2 μg total RNAs using a PrimeScript^TM II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian) according to the manufacturer’s protocol. The full-length coding region of GmCLC1 was amplified from the cDNA using the following PCR protocol: 94°C 3 min; 30 cycles of 94°C 30 s, 55°C 30 s, and 72°C 2 min 30 s; and 72°C 5 min in a 25 μL reaction mixture [2 μL of first strand cDNA, 0.15 mM MgCl₂, 0.2 mM dNTPs, 0.4 μM of each primer, 0.25 U Taq DNA polymerase (TaKaRa, Dalian) and 10× PCR buffer]. Primers used: 5′-ATGGGTGAGGAATCCAGTTT-3′ and 5′-CTTCCTCTTTGATTTTGCCAG-3′. The PCR products were then cloned into pMD19-T vector (TaKaRa, Dalian) for sequencing.

Subsequently, the open reading frame of GmCLC1 was amplified from cDNA by PCR (95°C 3 min, 28 cycles of 95°C for 30 s, 55°C for 30 s, and 68°C for 2.5 min) with KOD-Plus (TOYOBO, Japan), and ligated into the plasmid pCAMBIA1300 or pYES2 to obtain the recombinant plasmid pCAMBIA1300-GmCLC1 (primers used: 5′-GGGGTACCATGGGTGAGGAATCCAGTTT-3′ and 5′-GGACTAGTCTTCCTCTTTGATTTTGCCAG-3′) or pYES2-GmCLC1 (primers used: 5′-CCGGAATTCATGGGTGAGGAATCCAG-3′ and 5′-CCGCTCGAGTCACTTCCTCTTTGATTTTG-3′). After sequence verification, the recombinant plasmid pCAMBIA1300-GmCLC1 was transformed into A. tumefaciens GV3101 or A. rhizogenes K599, respectively.

The binary vector pCambia1300-GmCLC1 was transformed into WT Arabidopsis plants using the floral dip method mediated by A. tumefaciens (Clough and Bent, 1998). Transformants were selected by germination on Murashige and Skoog (MS) agar medium supplemented with 40 mg L^-1 hygromycin B. Homozygous lines (L₁–L₅) of T₂ plants were identified by PCR. The seeds of A. thaliana WT and a homozygous GmCLC1-transgenic line (L₁) were sterilized and sown evenly on MS agar medium (pH = 5.8; Nie et al., 2015) containing 0, 150, and 200 mM NaCl, respectively. The agar plates were put in an illuminated growth chamber under a 14 h light/10 h dark cycle at 20 ± 2°C, with 60–70% relative humidity. The seed germination percentage (%) was recorded after 7 days. For measuring the root elongation of A. thaliana under salt stress, seeds of WT and GmCLC1-transgenic plants were cultured on MS agar medium (pH = 5.8) without NaCl for 5 days. Then seedlings with similar root lengths were selected and transferred onto MS agar medium (pH = 5.8) containing 0, 150, and 200 mM NaCl. After placing the agar plates vertically for 5 days, the seedlings of WT and GmCLC1-transgenics were photographed and the root lengths were measured.

Seeds of A. thaliana WT and GmCLC1-transgenic line (L₁) were sterilized and sown on MS agar medium (pH = 5.8), and then placed in a growth chamber under a 14 h light/10 h dark cycle at 20 ± 2°C, with 60–70% relative humidity. After 8 days, the seedlings were then transferred to pots containing a sterilized peat moss and vermiculite mixture and grown for 5 days. Then the seedlings were treated with increasing concentrations of NaCl (50 mM NaCl for 2 days, 100 mM NaCl for the next 2 days, 150 mM NaCl solution for another 7 days). Finally, leaf relative water content (RWC) was measured according to the method described by Hu et al. (2016). Leaf chlorophyll fluorescence (Fv/Fm) was measured at room temperature with a plant efficiency analyzer (Handy PEA Fluorometer, Hansatech Instruments, UK; (Tian et al., 2014). Root or leaf relative electrolyte leakage (REL) was assayed using the method described by Hu et al. (2016) with a digital conductivity meter (DDS-307, Shanghai, China).

Seeds of WT and GmCLC1-transgenic A. thaliana were sterilized and sown in pots containing a sterilized peat moss and vermiculite mixture and grown in a growth chamber under a 14 h light/10 h dark cycle at 20 ± 2°C, with 60–70% relative humidity. After 30 days, plants were treated with 1/2 X Hoagland solution containing 150 mM NaCl for 0, 4, 12, 24 h, 2, 4, and 6 days, respectively. Then the roots and shoots of Arabidopsis seedlings were sampled and Cl^- contents were measured with the method described (Zhou and Yu, 2009).

Hairy root transformation was performed according to Ali et al. (2012). A. rhizogenes strain K599 containing the recombinant binary vector pCAMBIA1300-GmCLC1 was grown in yeast extract peptone (YEP) medium containing 50 mg/L ampicillin (Amp) and 200 μM acetosyringone at 28°C for 16 h. Then the bacterial culture was centrifuged, and the pellet was resuspended gently in 10 mM MgSO₄ solution followed by two washings, and adjusted to OD₆₀₀ ≈ 0.5.

The cotyledons of soybean (Jackson and Lee68 cultivars) were scored with a scalpel and the wounds were infected with the pCAMBIA-containing A. rhizogenes infection solution for 1 h in dark at room temperature, and then transferred to moist filter paper and incubated in the dark for 5 days (at 25 ± 2°C). After that, the infected cotyledons were transferred to a growth chamber under a 12 h light/12 h dark cycle at 25 ± 2°C. New hair roots that sprout from the infected cotyledons that were free of Agrobacterium as screened by PCR and at similar lengths were selected and transferred into 1/2 X Hoagland solution (pH = 6.5) containing 0, 100, and 150 mM NaCl, respectively Hairy roots infected with A. rhizogenes strain K599 containing the empty vector pCAMBIA1300 served as the control. After 5 days treatment, hairy root growth (maximum root length and root fresh weight) was photographed and measured (Qi et al., 2014).

For whole-plant transformation, surface-sterilized soybean seeds (Jackson and Lee68 cultivars) were sown in pots containing a sterilized peat moss and vermiculite mixture. When the first pair of true leaves had fully expanded, the cotyledon nodes of the soybean seedlings were infected with the A. rhizogenes strain K599 infection solution for 1 h at room temperature by injection. Then the wounds were covered with moist vermiculite and incubated for 5 days at 28°C under a 12 h light/12 h dark cycle. After 7 days, hairy root lines that were free of Agrobacterium as screened by PCR were selected and the original roots removed from the seedlings. These seedlings were then cultured in 1/2 X Hoagland solution. Seedlings infected with A. rhizogenes strain K599 containing the empty vector pCAMBIA1300 served as the negative control. After 10 days, Jackson and Lee68 seedlings with similar-length hairy roots were selected and treated with 1/2 X Hoagland solution containing 120 mM NaCl for 3 and 5 days, respectively, and seedlings grown in Hoagland solution without NaCl served as the untreated control. All the solutions listed above were replaced every 3 days throughout the experiments. REL and Cl^- contents in roots, stems, first true leaves, and the first and second trifoliates were measured accordingly.

The yeast expression vector pYES2-GmCLC1 was transformed into Δgef1 mutant yeast (S. cerevisiae) using the PEG/LiAc procedure and transformants were screened by PCR (Gietz, 2014). Ten-fold serial dilutions (starting at OD₅₅₀ ≈ 0.5) of each yeast culture were plated on agar supplemented with YPD medium (1% yeast extract/2% peptone/2% dextrose), YPG medium (1% yeast extract/2% peptone/2% galactose), or YPG medium supplemented with 1 M NaCl, 1 M KCl, or 3 mM MnCl₂. Plates were then incubated at 30°C and photographs were taken after 60 h. For Cl^- content measurements, the above-mentioned yeast cells were grown in the liquid YPG medium plus 1.0 M NaCl, and collected during the exponential growth phase (OD₅₅₀ ≈ 0.2), and their Cl^- contents were assayed by AgNO₃ titration as described by Zhou and Yu (2009).

Data were expressed as mean ± SD of three independent experiments and were analyzed using one-way analysis of variance by SPSS 19.0, and pairwise comparisons were performed using Duncan’s test.

The cDNA of GmCLC1 was cloned from G. max cultivar N23674. The encoded protein product has the same sequence as the annotated product of Glyma05g14760. We have constructed five independent transgenic lines expressing GmCLC1. All transgenic lines exhibited NaCl tolerance (Supplementary Figure S1) and hence we selected one typical line for detailed analysis. The A. thaliana plant was successfully transformed with GmCLC1 as shown with genotyping by PCR (Figure 1A). When grown on plate containing 150 or 200 mM NaCl, the seed germination rates of both WT and GmCLC1-transgenic line (L₁) declined as the NaCl concentration increased. However, under 150 or 200 mM NaCl treatments, the transgenic line exhibited a higher germination rate than WT (Figure 1B). Also, under 200 mM NaCl treatment, the seedling growth of WT and GmCLC1-transgenic line in both roots and shoot were inhibited compared to the untreated control (Figure 1C). However, the root elongation of the GmCLC1-transgenic line was significantly higher than those of WT under either 150 or 200 mM NaCl stress (p < 0.01; Figure 1D). This indicates that ectopic expression of GmCLC1 in A. thaliana could enhance seed germination and seedling growth under salt stress.

When WT and GmCLC1-transgenic seedlings were continuously exposed to NaCl stress with increasing concentrations (50 mM NaCl solution for 2 days, and 100 mM NaCl solution for next 2 days, followed by 150 mM NaCl solution for 7 days), the transgenic plants appeared to be healthier than WT (Figure 2A), and RWC and Fv/Fm were also significantly higher than those of WT (p < 0.01; Figures 2B,C), while the REL values were significantly lower than those of WT (p < 0.01 for root, p < 0.05 for leaf; Figure 2D). This further shows that the GmCLC1-transgenic A. thaliana plants suffered less leaf water loss, exhibited more stable photosynthetic capacity and had reduced salt injuries compared to untransformed WT when under salt stress.

During the 6-days treatment with 150 mM NaCl, the Cl^- contents in the roots of both WT and GmCLC1-transgenic seedlings increased significantly within the first 4 h and then leveled off (Figure 3A). However, the Cl^- contents in the shoots of both WT and transgenic seedlings increased steadily throughout the duration of the experiment, with the increase in the shoots of WT being significantly higher than in the transgenic plants (p < 0.01; Figure 3B), indicating that GmCLC1 reduces salt stress partly by reducing the Cl^- accumulation in shoots.

The transformation of soybean cotyledon hairy roots with GmCLC1 was shown to be successful through genotyping by PCR (Figure 4A). Under normal growing condition without additional NaCl, both empty vector- and GmCLC1-transformed hairy roots grew well, with no obvious difference (Figure 4B). However, under 100 mM NaCl stress, the growth of the hairy roots of both GmCLC1-transformed and empty vector-transformed lines were significantly inhibited, and the numbers of root branching were also reduced as compared to the untreated control. The maximum root lengths and fresh weights of empty vector-transformed hairy roots decreased more significantly than the GmCLC1-transformed ones (p < 0.01). At the NaCl concentration of 150 mM, the growth of the hairy roots of both GmCLC1- and empty vector-transformed cotyledons were inhibited, but there was no significant difference between the two (Figures 4C,D).

Building on the results we obtained using the GmCLC1-transgenic soybean cotyledon hairy root system, we further tested the response of whole GmCLC1-transgenic soybean hairy root composite plants to salt stress. First, the presence of the transgene in the soybean plants was confirmed by PCR (Figure 5A). Without any additional NaCl in the culture medium, there was no obvious phenotypic difference between the GmCLC1-transgenic soybean (including both Jackson and Lee68 cultivars) hairy root composite plants and the empty vector-transformed plants (Figure 5B). There was also no significant difference in the REL values of roots, first true leaves, the first and second trifoliate leaves among all the genotypes when grown in NaCl-free medium (Figure 5C).

When Jackson or Lee68 GmCLC1-transgenic soybean hairy root composite plants and the corresponding empty vector-transformed plants were exposed to 150 mM NaCl solution, the relatively salt-sensitive Jackson cultivar (both vector-only and GmCLC1) displayed obvious salt injury symptoms (with severely withered leaves) on the 3rd day, while the salt-tolerant Lee68 cultivar (both vector-only and GmCLC1) showed only mildly withered leaves up to the 5th day (Figure 5B). Furthermore, the hairy root composite plants of both cultivars that were transformed with GmCLC1 showed better salt adaptation than their empty vector-transformed counterparts, especially for the more salt-tolerant Lee68 cultivar (Figure 5B). When examining the REL values in separate parts of the composite soybean plants, we found the REL values of roots, first true leaves, the first and second trifoliate leaves of GmCLC1 were all significantly lower than their empty vector-transformed counterparts from the Lee68 cultivar (p < 0.01). The REL values of the second trifoliates of the GmCLC1-transgenic Lee68 cultivar, especially, were comparable to those of the water control. The REL values of the roots and second trifoliate leaves of GmCLC1 of the Jackson cultivar were also significantly lower than their empty vector-transformed counterparts (p < 0.01 and 0.05, respectively; Figure 5C).

In addition, under salt stress, the Cl^- contents in roots, stems, first true leaves and the first trifoliate leaves of both Jackson and Lee68 hairy root composite plants (including both GmCLC1 and vector-only) were dramatically increased compared to the water control. It is clear that the GmCLC1-transgenic plants of the Lee68 cultivar, when compared to their empty vector-transformed counterparts, had significantly lower Cl^- accumulation in their stems, first true leaves and the first trifoliate leaves (p < 0.01), and GmCLC1-transgenic plants of the Jackson cultivar also had significantly lower Cl^- accumulation in the first trifoliates (p < 0.01; Figure 5D). This indicates that the ectopic expression of GmCLC1 in Jackson or Lee68 hairy root composite plants can reduce the Cl^- transportation and accumulation in the aerial parts of the plant, especially for the salt-tolerant Lee68 cultivar.

All the yeast strains, including the mutant Δgef1 (a GEF1-deficient mutant), and Δgef1 transformed with empty vector, GEF1, and GmCLC1, grew well on both YPD and YPG media under normal growing conditions. When cultured on YPG medium supplemented with 3 mM MnCl₂, the mutant Δgef1 and Δgef1/Vector were unable to grow, but the mutants transformed with yeast GEF1 or soybean GmCLC1 could. Similarly, mutants transformed with yeast GEF1 or soybean GmCLC1 grew better on YPG medium supplemented with 1.0 M NaCl or 1.0 M KCl (Figure 6A). The Cl^- contents in the cells of Δgef1/GEF1 and Δgef1/GmCLC1 grown in liquid YPG medium plus 1.0 M NaCl were significantly higher than in the control cells without intracellular vesicle-localized GEF1 transporter activity (Δgef1 and Δgef1/Vector; p < 0.01; Figure 6B). Thus GmCLC1 may have similar functions much like the yeast GEF1.