Introduction

Plants frequently face adverse environmental conditions, such as salt, disease and chilling injury, which may have serious effects on their growth and the crop yields. As the main grain and oil crop, soybean [Glycine max (L.) Merr.] often suffers from cold damage in many cold growing areas, which seriously affects the crop productivity and quality. Therefore, it is very important to increase the crop yield and keep stable yields in cold conditions in order to ensure food supplies for an increasing global population. In recent years, many typical, cold related transcription factors (bZIP, MYB, WRKY and C₂H₂) have been cloned and characterized (Luo et al. 2012; Yu et al. 2014), to confirm a critical relationship with the response of plants to cold stress via transcriptional regulation (Ahuja et al. 2010; Xu et al. 2011).

Zinc finger proteins are a kind of transcription factors with finger shape domain (Tian et al. 2010) and were first discovered in Xenopus by Miller et al. (1985). They are divided into nine categories: C₄, C₆, C₈, CCCH, C₂HC, C₂HC₅, C₂H₂, C₃HC₄ and C₄HC₃ (Michael and Chrisopher 2003). The C₂H₂zinc finger proteins were studied most and are composed of about thirty amino acids. The conservative sequence is: CX_2~4CX₃PX₅LX₂HX₃H (Pabo et al. 2001). The zinc finger proteins in plants are associated with the processes of morphological changes during growth, pollen and embryo development. And also plays a key part in the regulation of abiotic stress (Luo et al. 2012; Zhai et al. 2013).

The C₂H₂ zinc finger proteins which contained special conserved motifs which was QALGGH located in the special α helix portion of each protein, are common transcription factors which were characterized (Isernia et al. 2010; Fedotova et al. 2017). Sugano et al. (2010) found that zpt2-3 gene transferred into petunia was also induced by desiccation stress. However, there are relatively few studies on soybean zinc finger proteins; only SCOF-1 has been studied in depth (Kim et al. 2010). Over expression of SCOF-1 can increase the expression of COR gene, which enhances the cold resistance of transgenic tobacco and Arabidopsis. Besides, Luo et al. (2012) confirmed that GsZFP1 enhanced the cold and drought tolerance significantly.

This study was conducted to clone a novel GmC₂H₂4-like gene and characterize its functions in growth, development and its responses to cold stress. The technology of soybean genetic transformation is quite difficult. Therefore, we verified the functions of GmC₂H₂4-like gene in Arabidopsis thaliana, a model plant with easy genetic transformation. This will provide the basis for the further functional study of GmC₂H₂4-like gene in soybean and the cultivation of high-quality transgenic materials in the future.

Materials and Methods

Plant materials

Total RNA of Jilin32 soybean seedlings was extracted for GmC₂H₂4-like gene cloning and the total RNA of various Jilin32 soybean tissues (root, stem, leaf and seed) at 24°C and 4°C was extracted for expression analyses of GmC₂H₂4-like. This Jilin32 soybean variety has strong tolerance to stress and is disease resistant. We constructed the library expression profile of immature Jilin32 embryos for further research. Five hundred plants were planted under natural conditions. Fifty Jilin32 seeds were sowed in the plant incubator at 25°C under 16 h lightness and 23°C under 8 h darkness with 55% relative humidity. The soybean strains are used for gene expression analyses. 10-day-old seedlings were cultivated under cold treatment at 4°C for 24 h before tissues were sampled for qRT-PCR analysis and further investigations. Tissue samples included roots, stems, leaves and mature seeds.

Arabidopsis (Colombia-0) was the basic model material for the transformation of target genes, the extraction of protoplast cells, the observation of phenotype in seedling stage and the cold resistance analysis of GmC₂H₂4-like. After the sterilization and vernalization of Arabidopsis seeds, the seeds of WT and transgenic Arabidopsis plants were simultaneously planted and cultivated in MS solid medium. After half a month, all the Arabidopsis seeds were transplanted to mixed soil with peat and vermiculite for further studies. The roots were used for investigation of root length and development. After one month of growing, the transgenic Arabidopsis and the control (WT) were simultaneously subjected to cold treatment at 4°C for 24 h. After cold treatment, Arabidopsis leaves were immediately sampled into liquid nitrogen for the next experiments. The leaves were sampled from the same Arabidopsis lines at 22°C and 4°C to make sure the experiment rigorous. The leaves were used for qRT-PCR analyses and determination of various physiological and biochemical indexes. All samples were collected randomly in three repeats and saved at -80°C after freezing in liquid nitrogen.

Bioinformatics analysis and cloning of GmC₂H₂4-like in soybean

The whole sequence of GmC₂H₂4-like was obtained in NCBI Blast database (http://www.ncbi.nlm.nih.gov/). The single zinc protein conserved domain was predicted in NCBI with the accession number NM_001255238. Alignment of the cDNA sequence was demonstrated in Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) and Soybase database (https://www.soybase.org/sbt/). The theoretical pI and molecular mass were calculated on the ExPASy (https://web.expasy.org/protparam/) SIB Bioinformatics Resource Portal. The subcellular location of GmC₂H₂4-like protein was preliminarily predicted using Cell-PLoc online software (http://www.csbio. sjtu.edu.cn/bioinf/Cell-PLoc-2/). The tertiary structure were predicted using Phyre² online software (http://www.sbg.bio.ic.ac.uk/servers/phyre2/html/page.).

Total RNA of Jilin32 soybean seedlings were extracted and the cDNA was synthesized using cDNA Synthesis Kit (TaKaRa, Beijing, China). The whole cDNA sequence was obtained with RT-PCR according to forward primers (5′-TAGCTTGAAAACTTAGCACAG-3′) and reverse primers(5′-TAACAGCACATACAGAGCAAA-3′). The RT-PCR experiment system included 1 μL cDNA, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 2.5 μL 10x Ex-Taq Reaction buffer (20 mM), 2 μL dNTP (2.5 mM), 0.5 μL extaq DNA polymerase (5 U/μL) and 17 μL distilled water. The PCR product was recycled using Gel Recycling Kit (Takara, China), and sequenced by Biological sequence Company (Sangon Biotech, China). The nine genes which were most similar to GmC₂H₂4-like and containing one single conserved C₂H₂ domain were analyzed by the online SMART software (Letunic et al. 2006). The accession numbers of nine genes are ATZFP11, NP_181770; Hypothetical petunia hybrid, BAD11142; RABBIT EARS A. thaliana, BAC98433; SUPERMAN, Q38895; LSIF petunia hybrid, BAB58897; AtZFP10, NP_181310; KNUCKLES A. thaliana, AAT27472; Cys2-His2 GmZFP1, NP_001341929 and ATZFP1, NP_178188.

Expression analysis by qRT-PCR

Total RNA of different parts including roots, stems, leaves and seeds in soybean was extracted using the same method as cloning experiments before. The qRT-PCR was performed using TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) and analyzed using ABI PRISM 7900 software according to the QIAGEN Supplementary Protocal (QIAGEN, Germany). Each hole up to 20 μL in the 96 holes testboard was composed of 10 μL 2 x TransStart Tip Green qPCR SuperMix, 1 μL Template (cDNA, 1 μg), 0.4 μL Passive Reference Dye (50x), 0.4 μL forward primer, 0.4 μL reverse primer and 6.8 μL DNAase free water. All the primers of GmC₂H₂4-like, β-Tubulin (GMU12286), soybean and Arabidopsis Actin (J01298 and NM_112764, respectively) were showed in Table 1. These two kinds of internal genes were used to standardize the experiment data (Jian et al. 2008; Hu et al. 2009; Le et al. 2012). The qRT-PCR systems were divided into two steps: 94°C for 30 s, 55°C for 5 s (45 cycles) and 60°C for 30 s. The ΔCT value method was used as a standard to validate the gene expression (Riedel et al. 2014).

Subcellular localization of GmC₂H₂4-like gene

The stop codon of GmC₂H₂4-like was got rid from coding region and the sequence was inserted in the pBI121 vector with XbaI restriction enzymes. The recombinant construct was then introduced into Agrobacterium tumefaciens strain EHA105 and transformed to the protoplast of Arabidopsis cells. Specific experimental methods referred to the paper of Confraria and Baena-González (2016) and Su et al. (2014). Finally, the GFP fluorescence was found using a confocal microscope (Olympus, Tokyo). The nucleus was dyed with DAPI. The location was preliminarily predicted using Cell-PLoc online software according to http://www.csbio. sjtu.edu.cn/bioinf/Cell-PLoc-2/ (Chou and Shen 2010a, b).

Transactivation analysis in yeast

To study the transactivation activity in yeast, the full-length open reading frame of GmC₂H₂4-like was inserted into the GAL4 BD binding domain of pGBKT7 vector (Invitrogen, Carlsbad, CA, USA), which had been digested in advance with the EcoRI and SalI restriction enzymes. The pGBKT7-GmC₂H₂4-like was then transformed into the AH109 yeast as the positive control (Zhao et al. 2017). The transformant yeast was cultivated on medium without Trp at 35°C for 3 days and subsequently, the yeast was shifted to mediun without Trp, His and Ade including 3-amino-1, 2, 4-triazole. A β-galactosidase assay was also conducted to examine the transactivation ability within 10 h. Specific β-galactosidase assay method referred to Zhao et al. (2017). Finally, the result showed according to color reaction which was monitored and photographed.

Arabidopsis transformation

Gateway technology was used to clone and construct an expression vector (Su et al. 2014). The GmC₂H₂4-like gene was inserted into the pDONR221 vector. Through two steps BP and LR recombination reaction, the resultant pCB35S-GFP-GmC₂H₂4-like was obtained and then transformed into A. tumefaciens EHA105, the transgenic Arabidopsis was obtained using the floral dip method. The transgenic Arabidopsis was screened with 5mg/L glufosinate-ammonium.

Detection and screening of transgenic plants

The GmC₂H₂4-like transgenic Arabidopsis were identified using a series of detection methods. The positivity of the transgenic plants for the target gene was examined by RT-PCR. The forward primer (5′- ACCCACGTCATGCCAGTT-3′ and reverse primer 5′- CTAGGGGGATCTACCATG-3′) were used to amplify the 501 bp bar gene. The forward primer pairs (5′-TAGCTTGAAAACTTAGCACAG-3′ and the reverse primer pairs (5′-TAACAGCACATACAGAGCAAA-3′) were used to amplify the 982 bp target gene. The bar strip (A07-13-413, Beijing) is an easy and accurate way to detect the PAT protein in transgenic plants.

Measurement of the cold resistance-related indicators in WT and transgenic Arabidopsis

Fresh Arabidopsis leaves of WT and four transgenic Arabidopsis lines were sampled in 3 biological replicates at the end of the 4 °C treatment for a day for further biochemical analyses. In general, under the stress of adversity, the permeability of plant cell membrane will change. Malondialdehyde (MDA) is used commonly as index of cell membrane peroxidation, which can reflect the degree of membrane peroxidation and the response to stress. Malondialdehyde (MDA) content was calculated by the chromogenic reaction of thiobarbituric acid and MDA under acidic conditions. After 4°C cold treatment, Arabidopsis leaves (1g) were grinded with quartz sand and 10% trichloroacetic acid. The homogenate was centrifuged at 5000 rpm for 10 min and the supernatant was the extract of malondialdehyde. Then the samples were measured the absorbance values in 532 nm, 450 nm and 600 nm wavelength. The results were calculated referring to the formula in the paper of Tirani and Haghjou (2019). Peroxidase (POD) can measure the resistance of plants to adversity. The stronger the resistance was, the higher the POD value was (Weng et al. 2015; Shekaari et al. 2019). Peroxidase (POD) activity was measured by the guaiacol method (Wang et al. 2019). The enzyme of Arabidopsis leaves (0.1 g) were extracted, then the enzyme activity was determined in

Fig. 1: Amino acid sequence analyses of GmC₂H₂4-like (A) Alignment of amino acids of GmC₂H₂4-like. Partial sequences of GmC₂H₂4-like with other nine single zinc finger proteins in plants. Sequences were aligned using ClustalW. Positions containing identical residues are marked with an asterisk (*). The thin-lined red frame contains a special zinc-finger domain. QALGGH is a conserved motif. The GenBank accession numbers are: ATZFP11, NP_181770; Hypothetical petunia hybrid, BAD11142; RABBIT EARS A. thaliana, BAC98433; SUPERMAN, Q38895; LSIF petunia hybrid, BAB58897; AtZFP10, NP_181310; KNUCKLES A. thaliana, AAT27472; Cys2-His2 GmZFP1, NP_001341929; ATZFP1, NP_178188. (B) Phylogenetic tree of GmC₂H₂4-like and other TFIIIA single zinc finger proteins. The tree was constructed using the neighbor-joining method with the program MEGA 5.1 (Tamura et al. 2011). Branch numbers represent bootstrap values from 1000 sampling replicates and branch lengths are proportional

Table 1: Gene specific primers for qRT-PCR

Gene name

Forward primer (5'-3')

Reverse primer (5'-3')

GmC2H24-like

β-Tubulin

soybean Actin

Arabidopsis Actin

5′-AAAGAACAATAGCGAAGAG-3′

5′-GGAAGGCTTTCTTGCATTGGTA-3′

5′-GTCCTTTCAGGAGGTACAACC-3′

5′-CCTTGAAGTATCCTATTGAGC-3′

5′-GAGGGAACCTGATGGTAG-3′

5′-AGTGGCATCCTGGTACTGC-3′

5′-CCACATCTGCTGGAAGGTGC-3′

5′-GGTCTTTGAGGTTTCCATCT-3′

spectrophotometer in OD₄₇₀. The results were recorded each 30 s. The relative electric conductivity of the leaves was measured with a conductometer (Chen and Han 2010; Zhang et al. 2018). The soluble sugar in the leaves (0.5–1.0 g) was tested by the modified phenol sulfuric acid method. The samples were measured the absorbance values in 485 nm wavelength. The results were calculated by the standard curve (Klotke et al. 2010). The proline content in WT and transgenic Arabidopsis (0.5–1 g) was determined by the sulfosalicylic acid method. The samples were measured the absorbance values in 520 nm wavelength. The results were still calculated referring to the formula in the paper of Xu et al. (2013).

Results

Isolation and characterization of GmC₂H₂4-like

According to the expression of GmC₂H₂4-like in different immature pods, we found four unknown cDNA clones which showed much homology with other differentially expressed C₂H₂ genes in plants. The four predicted translation products of the clones contained one or two C₂H₂ domains. QRT-PCR experiments were conducted to analyze the expression of GmC₂H₂s in response to the growth and development of the plant and their tolerance to cold. One gene designated GmC₂H₂4-like showed significant changes compared to the WT in four candidate genes. Based on this discovery, GmC₂H₂4-like was selected for further functional analyses. The open reading frame of GmC₂H₂4-like was 756 bp and 251 amino acids were encoded in the protein with a calculated mass of 27.64 kDa and a pI of 6.30. According to the results of alignment of the cDNA sequences in the Soybase and Phytozome database, GmC₂H₂4-like contained one intron and was located on No.18 Chromosome. The GmC₂H₂4-like protein contained one single conserved C₂H₂ domain, including a conserved QALGGH motif according to the SMART analyses (Fig. 1A). Phylogenetic analysis revealed that the novel single zinc finger gene GmC2H24-like from soybean clustered with Arabidopsis AtZFP1, which belongs to the C₂H₂ protein family (Fig. 1B). GmC₂H₂4-like protein was preliminarily predicted to be localized in the nucleus. Besides, the results of secondary structure and tertiary structure of GmC₂H₂4-like in Phrye² software; the results of gene and protein BLASTs in NCBI were shown in Fig. S1.

Fig. 2: GmC₂H₂4-like expressions in different soybean tissues and different temperatures. Expression analysis of GmC₂H₂4-like in different temperatures (24°C and 4°C) and tissues (root, stem, leaf, and seed) of soybean Jilin32 is determined by qRT-PCR. The data represent the average of three independent experiments ± SD. Results were normalized against for β-tubulin and soybean Actin. Statistical significance was determined by independent-sample t-test (*P < 0.05, **P < 0.01)

Fig. 3: Subcellular localization of the GmC₂H₂4-like protein in the protoplast of Arabidopsis thaliana cells. (A) Images expressing the GFP control (p35S-GFP); (B) Images of expression in the GmC₂H₂4-like-GFP fusion protein. The cells were visualized mainly localized in the nucleus in bright and fluorescent light fields. The cell nucleus was dyed with DAPI, and the pictures were merged. Scale bars = 10 μm

Expression analyses of GmC₂H₂4-like in different soybean tissues at 24°C and 4°C

The expression levels of GmC₂H₂4-like in roots, stems, leaves and mature seeds of soybean (Jilin 32) at normal and cold temperatures were examined by qRT-PCR (Fig. 2). The expression of GmC₂H₂4-like was significantly higher in the roots and seeds, however, relatively lower in the stems and leaves at different temperatures. Data showed that the expression of GmC₂H₂4-like in different tissues after cold treatment was even higher compared to normal temperature. The results were consistent with the predictions obtained using online software.

Subcellular localization of the GmC₂H₂4-like protein

The online prediction tool Cell-PLoc, predicted that the GmC₂H₂4-like protein was localized in the cell nucleus. To further identify localization of GmC₂H₂4-like, the stop codon of GmC₂H₂4-like was deleted and the full length was fused to the GFP reporter gene with the CaMV 35S promoter. The results showed that the GmC₂H₂4-like: GFP fusion protein was distributed throughout the plant cell, mainly in the nucleus (Fig. 3B). 35S-GFP was a control that showed a distributed fluorescence through all the protoplast cells (Fig. 3A). The result was consistent with the predictions obtained using Cell-PLoc online software.

Transactivation activity of GmC₂H₂4-like in yeast

The transactivation activity of GmC₂H₂4-like was detected in the pGBKT7 vector which expresses special proteins fused to the GAL4 domain from Alcohol Dehydrogenase1 promoter in the yeast system. C₂H₂ proteins usually function as

Fig. 4: Transcription activation analysis of the GmC₂H₂4-like protein. (A) Yeasts containing pGAL4, pGBKT7 and pGBKT7-GmC2H24-like grown on SD solid medium without Trp. (B) Yeasts containing pGAL4, pGBKT7, and pGBKT7-GmC₂H₂4-like grown on SD solid medium SD without Trp, His and Ade with 3-amino-1, 2, 4-triazole. (C) β-galactosidase activity assay

transcriptional activators or repressors. The results showed that all transformants grew normally on SD without Trp medium (Fig. 4A). The transformant of pGAL4 served as positive control. As a result, transformants of pGAL4 and pGBKT7 fused with GmC₂H₂4-like could grow on the selective SD medium without Trp, Ade and His. They exhibited β-galactosidase activity in the filter paper with X-gal (Fig. 4B–C). These results showed that GmC₂H₂4-like gene was a transcriptional activator.

Generation and screening of GmC₂H₂4-like transgenic Arabidopsis

The 756 bp GmC₂H₂4-like open reading frame was cloned into the pCB35S-GFP vector. The recombinant plasmid pCB35S-GFP-GmC₂H₂4-like was transformed into the A. tumefaciens EHA105. All T₀ transgenic Arabidopsis seeds were sowed and cultivated at 23°C in MS solid medium that contained 5 mg/L glufosinate-ammonium (Fig. 5A). Finally, four regenerated T3 transgenic Arabidopsis were confirmed positive by PCR and bar strip analysis (Fig. 5B–D). The different tissues (roots, stems, and leaves) of transgenic Arabidopsis and CK (WT) were sampled before and after the 4°C treatment and used in qRT-PCR (Fig. 5E–F) to verify the heterologous expression of GmC₂H₂4-like. GmC₂H₂4-like transgenic Arabidopsis was more tolerant to cold stress than WT.

The key role of GmC₂H₂4-like in plant development

Compared to the WT, the roots of GmC₂H₂4-like transgenic Arabidopsis were much longer (Fig. 6A). Moreover, after being transplanted into soil, transgenic plants grew faster than that of the WT (Fig. 6B). This kind of growth and development state was mainly obvious in the early stage of growth. 30 roots from each GmC₂H₂4-like transgenic Arabidopsis line and the WT, totally 120 roots from four transgenic lines were sampled for statistical analyses. The root lengths of transgenic Arabidopsis were significantly longer than the WT (Fig. 6C).

The heterologous expression of GmC₂H₂4-like enhances the cold tolerance

After the 4°C treatment for 24 h, five physiological and biochemical indicators, namely, electrical conductivity, POD activity and MDA, soluble sugar and proline contents were measured. The WT and four different transgenic Arabidopsis lines presented significant differences. Under normal temperature conditions, there was no much difference between the leaves of transgenic and WT Arabidopsis in relative electrical conductivity. After the 4°C cold treatment, the relative conductivity of the transformed Arabidopsis was significantly lower than the WT (Table 2), indicating that cell membrane of the transgenic lines was less damaged at low temperatures. At 22°C, POD activity in transgenic plants was not significantly different compared to the WT plants. After the cold treatment, the POD activity of transgenic Arabidopsis was significantly higher compared to
Table 2: Physiological and biochemical indexes (relative conductivity, POD activity, and MDA, soluble sugar, and proline content) of WT and four different transgenic lines (1 to 4) at 22°C and 4°C

	Relative conductivity		POD activity (ΔA470/g. min)		Malondialdehyde (MDA) (μmol•g^-1)		Soluble sugar (mmol•g^-1)		Proline content (ng•mg^-1)
	22°C	4°C	22°C	4°C	22°C	4°C	22°C	4°C	22°C	4°C
WT	88.86 ±0.11^a	95.31±0.22^a	1.83 ± 0.22^a	15.66 ± 0.12^b	0.013 ± 0.24^a	0.073 ± 0.21^a	0.5387±0.25^a	0.7413±0.33^c	128.54±0.25^b	232.57±0.26^c
1	89.38 ±0.14^a	92.03±0.16^b	1.83 ± 0.24^a	20.15 ± 0.18^a	0.028 ± 0.28^a	0.059 ± 0.29^b	0.6210±0.31^a	0.8061±0.28^a	141.05±0.31^a	291.5±0.21^b
2	90.39 ±0.21^a	92.78±0.12^b	1.76 ± 0.15^a	20.05 ± 0.26^a	0.019 ± 0.21^a	0.048 ± 0.31^c	0.5944±0.24^a	0.7804±0.27^b	139.28±0.28^a	282.6±0.18^b
3	90.77 ±0.17^a	91.68±0.17^b	1.85 ± 0.21^a	21.48 ± 0.24^a	0.025 ± 0.17^a	0.044 ± 0.28^c	0.6306±0.22^a	0.8422±0.19^a	144.71±0.28^a	309.7±0.19^a
4	90.42 ±0.15^a	92.18±0.21^b	1.88 ± 0.12^a	22.13 ± 0.19^a	0.022 ± 0.23^a	0.041 ± 0.17^c	0.6022±0.27^a	0.8140±0.33^a	140.53±0.31^a	290.6±0.23^b

Data are the average values of three biological replicates at 22°C and 4°C. Significantly different results are indicated by different letters (a, b, c)

Fig. 5: Molecular characteristics of GmC₂H₂4-like transgenic plants. (A) T₀ transgenic Arabidopsis seeds were sowed in MS solid medium containing 5 mg/L glufosinate-ammonium. (B) Electrophoresis images of four regenerated T3 transgenic lines were confirmed positive by PCR. (C) Bar strip detection of four transgenic Arabidopsis lines. (D) Electrophoresis images of four regenerated T3 transgenic lines were confirmed positive by RT-PCR. (E, F) Expression analysis of GmC₂H₂4-like in different tissues of WT and transgenic Arabidopsis at 22°C and 4°C by qRT-PCR. The data represent the average of three independent experiments ± SD. Values were normalized of β-tubulin and Arabidopsis Actin. Two asterisks (**) indicates that the differences between the transgenic lines and WT are highly significant (P < 0.01).

Fig. 6: Comparison of WT and transgenic Arabidopsis at various developmental periods. (A) Comparison of length of 30 roots of the WT and transgenic Arabidopsis. (B) Growth and development of the WT and transgenic Arabidopsis in soil. (C) The roots length of ten-day-old Arabidopsis lines at 22°C. WT means wild type Arabidopsis; GmC₂H₂4-like -1, 2, 4 and 5 are four transgenic lines. Scale bars = 1.0 cm in A and B

the WT, (Table 2), indicating that the reactive oxygen species (ROS) scavenging capacity of the antioxidant systems of transgenic Arabidopsis was much higher than the non-transgenic ones. At 22°C, the content of MDA showed no significant difference. After 4°C treatment, the content of MDA in the transgenic Arabidopsis was significantly lower than the WT, indicating that transgenic leaves suffered less adverse effects (Table 2). Besides, the contents of soluble sugar and proline in transgenic Arabidopsis after the 4 °C treatment improved significantly compared to that of WT Arabidopsis (Table 2), indicating that the content of soluble sugars facilitated osmoregulation. All these experiments implied that the heterologous expression of GmC₂H₂4-like enhanced the cold tolerance of the transgenic Arabidopsis plants.

Discussion

It is reported that C₂H₂ zinc finger proteins are involved with plant growth and various adaptive responses to all kinds of stress (Zhang et al. 2016; Wang et al. 2018). Although C₂H₂zinc finger proteins are reported that they were connected with various stress responses and growth and developmental processes, the detailed functions of single zinc finger proteins involved in cold response of soybean are rarely reported (Luo et al. 2012).

In our study, a typical single zinc finger protein was cloned from soybean and transferred into Arabidopsis. We observed the phenotype including root length, plant size and development speed of the WT and transgenic Arabidopsis lines at various stages of growth and development, especially in the early stage. We found that heterologous expression of GmC₂H₂4-like can promote the plant development which was consistent with reported papers (Pomeranz et al. 2011). Sendon et al. (2014) found the Arabidopsis thaliana dwarf1 (Atdwa1) mutant displayed severe dwarfism and loss of apical dominance, as well as other pleiotropic defects, such as earlier flowering, fewer leaves, and shorter sliliques than those of the wild-type plant. They indicated that the zinc finger proteins may play a role in regulation of plant growth and development. Therefore, we speculated that the GmC₂H₂4-like gene affected plant growth and development in transgenic Arabidopsis, consistent with its reported functions (Xu et al. 2020).

There were not many effects that have been proposed or reported for soybean single zinc finger proteins on cold tolerance. The double zinc finger protein plays an essential role in resistance to many stresses, and the single zinc finger protein controls plant development. Zhang et al. (2016) found that GmZFP3 in soybean belonging to C₂H₂ zinc finger protein contained a special conserved motif, and negatively regulates drought responses by transgenic Arabidopsis. Yu et al. (2014) reported that GmZF1 in soybean enhances cold tolerance in transgenic Arabidopsis because of the cold gene regulated. Therefore, the expression and many physiological and biochemical indicators of WT and transgenic GmC₂H₂4-like Arabidopsis were measured under normal and cold conditions to verify its functions. Transgenic Arabidopsis showed higher cold resistance than the WT. This corroborates previously reported functions of double zinc finger proteins. Kielbowicz-Matuk (2012) conducted a detailed study and analyzed the transcription factors of a double C₂H₂ protein involved in stress responses. Heterologous expression of GmC₂H₂4-like improves much of the proline and soluble sugar contents in transgenic Arabidopsis in cold stress, indicating that GmC₂H₂4-like transgenic Arabidopsis have adaptive physiological mechanisms to cold. The research of Yu et al. (2014) showed that over-expression of GmZF1 increases the expression level of cold-regulated cor6.6. GmZF1 could interact with cold regulation genes to improve cold tolerance. A double zinc finger protein, ZAT12 was identified as a negative regulator downregulating the expression of the CBF genes. Compared to WT, ZAT12 over-expressing plants exhibit cold tolerance under freezing stress. The results of transcriptome profiling and mutagenesis experiments indicated that additional cold response pathways exist and may have important roles in life at low temperature (Vogel et al. 2010). Besides, Yang et al. (2016) identified 118 members of the tobacco C₂H₂ zinc finger protein transcription factor family from the N. tabacum genome database by using Pfam, SMART and Blastp. The analyses of phylogenetic tree, physical and chemical properties, chromosomal mapping, gene structures, protein three-dimensional structures and tissue expression patterns were performed. Therefore, there were still many difficult problems that need to be studied in depth In our study, qRT-PCR analyses showed that GmC₂H₂4-like expression was connected with cold stress. Under cold treatments, the physiological and biochemical indexes showed that transgenic Arabidopsis were more tolerant to cold stress compared to the WT. Furthermore, we speculated that the GmC₂H₂4-like transcription factor with single zinc finger protein positively regulated the cold stress response in Arabidopsis. However, the function of this GmC₂H₂4-like gene in soybean has not been confirmed. Gene editing and silencing are possible methods to verify the functions in soybean. Further research on regulatory mechanism in our study needs a more step.

Recent studies have shown that the zinc finger proteins can interact with themselves and similar kinds of zinc finger proteins, as well as with some other types of proteins, to regulate their corresponding expressions. The interaction between different zinc finger proteins can allow the recognition of different DNAs or prevent the zinc finger proteins from binding with the corresponding DNA to regulate gene transcription and expression. Therefore, the interaction of GmC₂H₂4-like zinc finger protein and other key proteins needs to be further studied.

In our study, a new single zinc finger gene, GmC₂H₂4-like, was cloned in soybean and transfected into Arabidopsis. GmC₂H₂4-like encodes a protein localized in the nucleus and has a transcription activation. The results of our phenotypic observations and physiological and biochemical analyses suggested that transgenic Arabidopsis was superior to the WT in growth development, and cold tolerance. However, the specific mechanism of cold resistance needs additional clarification to ascertain its functional importance.

Conclusion

In conclusion, our study reported a new C₂H₂gene, GmC₂H₂4-like, which contains a single C₂H₂ domain and belongs to the zinc finger proteins. GmC₂H₂4-like protein is mainly localized in the nucleus and activates the transcription of the reporter genes. Heterologous expression of GmC₂H₂4-like in Arabidopsis can improve the cold tolerance and promote the plant growth and development.

Acknowledgments

Thanks to the Project of the National Natural Science Foundation of China (No.32001572), the Major Science and Technology Sponsored Program for Transgenic Biological Breeding (No. 2016ZX08004-003) and the National Key Research and Development Program of China (No. 2017YFD0101304).

Author Contributions

YX and FY conducted the experiments; YL, YW and QW and conceived the idea; YX, FY analyzed the data and results. YX, FY and QW designed and conducted the study. YX and FY finished the manuscript. QW, JL, YL and FY critically commented on the manuscript.

Conflict of Interest

There are no conflicts of interest.

Data Availability

Primary and supplementary data reported in this article are available with the corresponding authors

Ethics Approval

Not applicable

Funding Source

This study was supported by the National Key Research and Development Program of China (No. 2017YFD0101304), the Major Science and Technology Sponsored Program for Transgenic Biological Breeding (No. 2016ZX08004-003) and the National Natural Science Foundation of China (No. 32001572).

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