CBL-Interacting Protein Kinase 2 Improves Salt Tolerance in Soybean (Glycine max L.)

Abstract

Salt stress severely limits soybean production worldwide. Calcineurin B-like protein-interacting protein kinases (CIPKs) play a pivotal role in a plant’s adaption to salt stress. However, their biological roles in soybean adaption to salt stress remain poorly understood. Here, the GmCIPK2 expression was increased by NaCl and hydrogen peroxide (H₂O₂). GmCIPK2-overexpression Arabidopsis and soybean hairy roots displayed improved salt tolerance, whereas the RNA interference of hairy roots exhibited enhanced salt sensitivity. Further analyses demonstrated that, upon salt stress, GmCIPK2 enhanced the proline content and antioxidant enzyme activity and decreased the H₂O₂ content, malondialdehyde (MDA) content, and Na⁺/K⁺ ratios in soybean. Moreover, GmCIPK2 promoted the expression of salt- and antioxidant-related genes in response to salt stress. Moreover, the GmCIPK2-interacting sensor, GmCBL4, increased the salt tolerance of soybean hairy roots. Overall, these results suggest that GmCIPK2 functions positively in soybean adaption to salt stress.

1. Introduction

Plants are sessile organisms that often encounter various environmental changes, including salt, drought, and extreme temperatures [1,2]. During evolution, plants have evolved complex strategies to adapt to unfavorable conditions [2,3]. Calcium (Ca²⁺) is a universal secondary messenger that regulates plant growth, development, and stress responses [4,5]. Environmental stimuli can trigger spatio-temporal changes in cytoplasmic Ca²⁺ concentrations [4,6]. The changes are then detected by Ca²⁺ sensors, such as Ca²⁺-dependent protein kinases, calmodulins, and calcineurin B-like proteins (CBLs) [6,7]. Subsequently, the Ca²⁺ sensors interact with their downstream targets, causing a series of physiological and metabolic alterations in plants [7,8].

CBLs can specifically bind to CBL-interacting protein kinases (CIPKs) to form plant-specific Ca²⁺ signal decoding systems [4,7,9]. CIPKs have been identified as a class of serine/threonine (Ser/Thr) kinases that share a close evolutionary relationship with SNF1 (sucrose non-fermenting-1)-related kinases 3 [7,8]. The catalytic domain of CIPKs is located at the N-terminus and contains an activation loop and an ATP binding site [7,9]. A NAF/FISL motif is found in the C-terminus of CIPKs, adjacent to the junction domain [10]. The NAF/FISL motif is critical for the interaction with CBLs [8,9]. Since the CBL-CIPK pathway was discovered in Arabidopsis (Arabidopsis thaliana L.) [11], CIPK homologs have been found in rice (Oryza sativa L.) [12], pepper (Capsicum annuum L.) [13], maize (Zea mays L.) [14], tomato (Solanum lycopersicum L.) [15], soybean (Glycine max L.) [16], wheat (Triticum aestivum L.) [17], apple (Malus domestica Borkh.) [18], and cotton (Gossypium hirsutum L.) [19].

CIPKs affect ion homeostasis, hormonal signaling, and tolerance to abiotic stresses [4,9]. For example, AtCIPK8 has been demonstrated to positively regulate Arabidopsis’ salt tolerance [20]. AtCIPK24 is a key component of the salt overly sensitive pathway that regulates the salt tolerance of Arabidopsis [21]. Additionally, it has been demonstrated that AtCIPK14 participates in Arabidopsis’ glucose response [22]. Studies have explored the functions of CIPKs in other plant species. For instance, cold stress leads to the increased expression of OsCIPK3 and OsCIPK7, and transgenic plants overexpressing (OE) OsCIPK3 and OsCIPK7 display cold-tolerant phenotypes [23,24]. Furthermore, ZmCIPK16 regulates stress-responsive gene expression to mediate maize adaption to salt stress [25]. TaCIPK27 and TaCIPK23 respond to drought stress by regulating the stomatal movement [26,27]. In addition, CaCIPK13 expression is induced by cold stress, and CaCIPK13-OE tomato plants exhibit cold-resistant phenotypes [28]. SlCIPK24 is found to modulate Na⁺/K⁺ homeostasis in tomato salt responses [15].

Soybean is an important economic crop and a crucial source of edible oil, high-quality protein, and industrial products [29,30]. Salt stress is a major environmental challenge that severely restricts crop quality and yields worldwide [1,31]. It has been established that CIPKs play a crucial role in a plant’s adaption to adverse conditions [4,5]. Nevertheless, whether CIPKs participate in alleviating salt stress in soybean remains largely unknown. Our previous study demonstrated that GmCIPK2 serves as a positive regulator of drought tolerance for soybean [32]. In the present study, the biological functions of GmCIPK2 in the salt response are characterized. Salt stress increases the transcript level of GmCIPK2. Further physiological and molecular assays demonstrate that GmCIPK2 contributes to the salt tolerance of soybean.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Soybean seedlings (Williams 82) were cultured in a growth room under a 16 h light/8 h dark photoperiod, in 70% relative humidity, at a temperature of 25 °C. Fourteen-day-old soybean plants of the same size were transferred to the 1/2 Hoagland’s solution containing 200 mM NaCl and 10 mM H₂O₂ for the expression profile analysis. The soybean leaves that were used for the RNA extraction were harvested at 0, 1, 3, 7, 12, and 24 h. Seeds of Arabidopsis ecotype Columbia-0 were germinated in the 1/2 Murashige and Skoog (MS) medium in an illumination incubator with a photoperiod of 16 h light/8 h dark, in a relative humidity of 70%, at 23 °C. Twenty seeds were sown on each plate and vernalized for three days at 4 °C.

2.2. Transgenic Arabidopsis and Soybean Plant Construction

Arabidopsis with GmCIPK2 overexpression was constructed by a previously described floral dip method [31,32]. Transgenic soybean hFairy roots were constructed by an Agrobacterium rhizogenes-mediated transformation, as described previously [1,30,33]. To obtain the OE transformation vectors, the full-length open reading frames of GmCIPK2 and GmCBL4 were inserted into pCAMBIA3301, driven by cauliflower mosaic virus 35S promoter, respectively. The sense and antisense fragments of GmCIPK2 (28 bp–178 bp) were connected by the intron 6 of the rice zinc finger gene to constitute the specific RNA interference (RNAi) fragment [31,32]. The specific RNAi fragments were ligated into pCAMBIA3301 to generate the pCAMBIA3301-GmCIPK2-RNAi constructs. Subsequently, these vectors were transformed into A. rhizogenes strain K599. The 5-day-old soybean seedling was infected with A. rhizogenes strain K599, harboring the transformation vectors (RNAi, VC, and OE) around the cotyledonary node area with a syringe needle. The infected plants were then covered with plastic cups and kept in the dark at 28 °C. After 24 h, the plastic cups were removed. Meanwhile, the infection sites were covered with wet vermiculite until the hairy roots were generated. Two weeks later, the original roots of the soybean were removed, and then soybean plants were grown with hairy roots forming transgenic soybean hairy root composite plants. The transcript levels of functional genes were detected by qRT-PCR assays. The composite plants were planted in flowerpots (12 cm × 14 cm) containing nutrient soil and vermiculite (1:1). Each pot contained 10 independent composite plants as a sample.

2.3. Quantitative Real-Time-PCR Assay

Total RNA was isolated and extracted using the RNA extraction kit (ZP401, Zomanbio, Beijing, China). The quantitative real-time (qRT) PCR analyses were conducted using an Applied Biosystems real-time PCR system and a TransStart Top Green qPCR SuperMix kit (AQ131, TransGen, Beijing, China) [31]. The relative expression levels of these selected genes were calculated with the 2^-ΔΔCT method, and the Gmtubulin expression was used as an internal reference. The primers used for the qRT-PCR assay are displayed in Table S1.

2.4. Salt Tolerance Assay

The Arabidopsis seedlings were grown in the 1/2 MS medium for seven days. Then, these plants underwent salt treatment (75 mM NaCl). After 10 days, the total root length, proline content, malondialdehyde (MDA) content, and H₂O₂ content were measured. To analyze the soybean salt tolerance, the composite soybean plants were cultured in flowerpots (12 cm × 14 cm) containing nutrient soil and vermiculite (1:1) for one week. For the salt stress treatment, 0.4 L of NaCl solution (200 mM) was added to the bottom tray of each flowerpot once every 3 days. After treatment for 10 days, clearly wilting differences were distinguished between the transgenic (RNAi and OE) and control plants. Each sample contained 10 independent seedlings, and the experiments were repeated three times. For the analysis of the physiological parameters, soybean seedlings underwent the salt treatment for seven days. The transgenic soybean hairy roots were then collected to assay the proline content, MDA content, H₂O₂ content, peroxidase (POD), and glutathione S-transferase (GST) activity using the corresponding detection kit (BC3595, BC0025, BC0095, BC0355, Solarbio, Beijing, China). The contents of Na⁺ and K⁺ were analyzed by an inductively coupled plasma-optical emission spectrometer (ICP-OES, United States), as described previously [1].

2.5. Yeast Two-Hybrid Assay

Using the Matchmaker^TM Two-Hybrid System, empty pGADT7 (AD), empty pGBKT7 (BD), GmCBL4-AD, and GmCIPK2-BD plasmids were transfected into yeast cells (AH109) according to the manufacturer’s protocol. Subsequently, these transformants were plated on the SD/-Ade/-Leu/-Trp/-His medium containing X-α-gal [26,32].

2.6. Pull-Down Assay

To obtain the GmCIPK2-His and GmCBL4-GST recombinant proteins, pCold-GmCIPK2 and pGEX-4T-1-GmCBL4 were constructed and transfected into Escherichia coli (BL21), respectively. The glutathione agarose beads bounded with GmCBL4-GST proteins were then used to combine with the soluble GmCIPK2-His protein. The products were then washed and used for the Western blotting assay, as described previously [2,26].

2.7. Subcellular Localization Assay

The GmCIPK2-GFP and GmCBL4-mCherry plasmids were transformed into Arabidopsis protoplasts using a PEG-mediated transformation system. After incubating in the dark for 12 h, the fluorescence signal in the transfected protoplasts was analyzed by a confocal laser-scanning microscope [3,32].

2.8. Statistical Analysis

All experiments were repeated three times independently. The values are displayed as the mean ± SE of three biological replicates. The differences between the various treatments were analyzed using the one-way analysis of variance (ANOVA) using the SPSS software (SPSS, statistics). Significant differences were determined by a Student’s t-test and labeled as * p < 0.05.

3. Results

3.1. Isolation of Salt Stress-Responsive Gene GmCIPK2

CIPKs function essentially in plant tolerance to environmental stresses, while the functions of soybean CIPKs’ tolerance to salt stress remain largely unknown. The BlASTP and multiple sequence alignment analyses demonstrated that GmCIPK2 has a high sequence identity with OsCIPK2 and AtCIPK2 (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 25 April 2022). The structure analysis result revealed that GmCIPK2 contained typical CIPK domains: the N-terminal Ser/Thr kinase domain and the NAF/FISL domain (Figure S1). Further qRT-PCR assays demonstrated that NaCl-mediated salt stress increased the transcript levels of GmCIPK2, peaking at 3 h (Figure 1A), implying a potential role of GmCIPK2 in salt response. Notably, H₂O₂-mediated oxidative stress led to an enhanced GmCIPK2 expression with a similar expression pattern in response to salt stress (Figure 1B).

3.2. GmCIPK2 Overexpression Confers Transgenic Arabidopsis Tolerance to Salt Stress

To explore the salt resistance associated with GmCIPK2, transgenic Arabidopsis plants with GmCIPK2 overexpression (GmCIPK2-#3, GmCIPK2-#7, and GmCIPK2-#11) were generated (Figure 2B). Under favorable conditions, the OE Arabidopsis plants exhibited a similar phenotype to the wild-type (WT) plants. Nevertheless, the salt treatment resulted in significant differences in physiological traits among the different genotypes. The OE Arabidopsis plants showed salt-tolerant phenotypes with larger biomass accumulation and longer root lengths (Figure 2A,C,D). Proline is a well-characterized osmolyte that enhances plant tolerance to salt stress [3,26]. The salt-treated OE Arabidopsis plants accumulated a significantly larger proline content than the control plants (Figure 2E). Additionally, salt stress promoted malondialdehyde (MDA) synthesis, which was negatively correlated with salt tolerance [3]. The MDA levels of salt-treated OE Arabidopsis plants were lower than that of the control plants (Figure 2F).

3.3. GmCIPK2 Promotes the Salt Tolerance of Soybean Hairy Roots

We generated transgenic hairy roots through the RNAi and OE technologies to verify the salt resistance associated with GmCIPK2 in soybean. GmCIPK2 transcript levels in transgenic hairy roots were examined using qRT-PCR assays (Figure 3B). Before salt stress, the different genotypes have no significant difference (Figure 3A,C). Subsequent salt stress triggers the phenotypic and physiological changes among the different genotypes. Under salt stress, the OE soybean plants exhibited better salt-tolerant phenotypes and higher survival rates than the vector control (VC) plants. In contrast, the RNAi soybean plants had salt-sensitive phenotypes with lower survival rates (Figure 3A,C). Furthermore, upon salt stress, the proline content was higher in the OE hairy roots than in the VC hairy roots. Conversely, the salt-treated RNAi hairy roots contained a lower proline content (Figure 3D). Additionally, the salt-treated OE hairy roots accumulated lower MDA levels than the control ones. However, a larger MDA level accumulated in the RNAi hairy roots than in the VC hairy roots under salt conditions (Figure 3E). Considering that the H₂O₂ treatment led to the increased expression of GmCIPK2, we analyzed the ROS contents in the hairy roots of RNAi, VC, and OE. Under normal conditions, ROS contents in the different genotype hairy roots are comparable (Figure 3F,G). Salt stress promoted ROS synthesis. H₂O₂ is a well-recognized moderately reactive ROS that can induce oxidative stress [1,29]. The DAB staining and quantitative assays showed that the H₂O₂ content was lower in the OE hairy roots than in the VC hairy roots after the salt treatment. By contrast, the salt-treated RNAi hairy roots contained higher H₂O₂ levels (Figure 3F,G). Antioxidant enzymes function essentially in scavenging ROS [1,3,29]. Upon salt stress, compared to the control roots, the OE hairy roots exhibited greater POD activity and GST activity, whereas the activity of the POD and GST enzymes in RNAi hairy roots was lower (Figure 3H,I). Moreover, salt stress usually causes a Na⁺/K⁺ imbalance. The Na⁺/K⁺ ratios are negatively related to the salt tolerance of plants [1,29]. When subjected to salt stress, the OE hairy roots showed a lower Na⁺ content, higher K⁺ content, and lower Na⁺/K⁺ ratios than the control hairy roots. In contrast, the salt-treated RNAi hairy roots displayed a higher Na⁺ content, lower K⁺ content, and larger Na⁺/K⁺ ratios (Figure 3J–L).

3.4. GmCIPK2 Activates the Expression of the Salt Stress- and Antioxidant-Related Genes

To clarify the molecular mechanisms of the GmCIPK2-mediated salt stress adaption in soybean, we examined the transcript levels of several salt- and antioxidant-related genes in the hairy roots of GmCIPK2-RNAi, VC and GmCIPK2-OE under salt treatment. No significant difference was identified among the different genotypes under normal conditions. However, when subjected to salt stress, the GmCIPK2-OE hairy roots showed higher expression levels of salt-responsive genes (GmP5CS, GmMYB118, GmDHN15, GmLEA5, GmSOS1, and GmNHX1) and oxidative-responsive genes (GmPOD21, GmPOD47, GmGST18, and GmGST20) than the control hairy roots. In contrast, the salt-treated GmCIPK2-RNAi hairy roots displayed lower expression levels of these salt- and antioxidant-related genes (Figure 4).

3.5. GmCBL4 Combines with GmCIPK2 at the Plasma Membrane

CIPKs are well-recognized for combining with specific CBLs to modulate plant adaptation to abiotic stress. The candidate CBLs that interact with GmCIPK2 were isolated using a yeast two-hybrid system. The co-expression of GmCBL4-AD with GmCIPK2-BD in the same yeast strain activated the reporter gene expression (Figure 5A). Therefore, GmCBL4 was identified as an interaction sensor of GmCIPK2.

We then performed pull-down assays to confirm the interaction between GmCIPK2 and GmCBL4. In this assay, the soluble GmCIPK2-His recombinant protein was co-purified with GmCBL4-GST but not with the control GST protein (Figure 5B), validating the direct interaction between GmCIPK2 and GmCBL4.

3.6. GmCBL4 Overexpression Imparts Salt Tolerance of Soybean Hairy Roots

To further explore the significance of the combination of GmCIPK2 and GmCBL4, we examined the distribution of both GmCBL4 and GmCIPK2 using the method of PEG-mediated protoplast transformation. PIP-mCherry protein was used as a membrane-localized marker [27]. GFP signals were mainly distributed in the nucleus and cytoplasm when the PIP-mCherry constructs were co-expressed with the GmCIPK2-GFP constructs in the same Arabidopsis protoplasts. However, as the GmCIPK2-GFP and GmCBL4-mCherry constructs were co-expressed in the same Arabidopsis protoplasts, GFP signals were only detected in the plasma membrane. Noteworthily, GmCBL4 encoded a membrane-localized protein (Figure 5C). Therefore, GmCBL4 may bind to GmCIPK2 at the plasma membrane to influence cellular processes.

Considering that GmCBL4 acted as a CIPK2-interacting protein, we generated hairy roots with GmCBL4 overexpression (Figure 6B). Without salt stress, the GmCBL4-OE plants had a similar phenotype as the control plants (Figure 6A). The hairy roots with GmCBL4 overexpression were more tolerant to salt stress and had significantly higher survival rates than the VC plants under salt stress (Figure 6A,C). Further physiological analyses illustrated a significantly lower MDA content and larger proline level that accumulated in the salt-treated GmCBL4-OE hairy roots than in the VC hairy roots in response to the salt stress (Figure 6D,E). More importantly, the results of the DAB staining and quantitative assays showed that salt-treated GmCBL4-OE hairy roots contained a lower content of H₂O₂ than VC hairy roots (Figure 6F,G). Additionally, the GmCBL4-OE hairy roots showed a lower Na⁺ content, higher K⁺ content, and lower Na⁺/K⁺ ratios than the VC hairy roots did under salt stress (Figure 6H–J).

4. Discussion

There is ample evidence to show that CIPKs play a pivotal role in plant tolerance to salt stress [4,5]. Salt stress increases the AcCIPK5 transcripts, and the overexpression of AcCIPK5 confers salt tolerance in transgenic plants [34]. ZmCIPK42 responds to salt stress, and ZmCIPK42 overexpression leads to the improved salt resistance of maize [35]. In recent years, the whole soybean genome has been sequenced [36]; nonetheless, the functions of soybean CIPKs in salt stress responses remain largely unknown. In the assay, a salt-inducible gene, GmCIPK2, was isolated from soybean (Figure 1A). GmCIPK2 overexpression increased the salt tolerance of Arabidopsis plants and soybean hairy roots. Conversely, the downregulation of GmCIPK2 by the RNAi technology resulted in increased salt sensitivity of hairy roots (Figure 2 and Figure 3). Taken together, GmCIPK2 acts as a key regulator in alleviating soybean salt stress.

Salt stress has been documented to trigger the overproduction and accumulation of ROS, which causes oxidative damage to cells, such as membrane damage and enzyme activity disruption, and even cell death [1,29]. To survive, plants have evolved sophisticated antioxidant defense systems to scavenge the redundant ROS [1,17,29]. Previous studies demonstrated that GhCIPK6a and BdCIPK31 increased SOD, POD, CAT, and GST enzyme activity to alleviate salt-induced oxidative stress [19,37]. In this study, the H₂O₂ treatment increased the transcript levels of GmCIPK2 (Figure 1B). Further DAB staining and quantitative assays illustrated that a significantly lower content of H₂O₂ accumulated in the salt-treated GmCIPK2-OE hairy roots than in the control roots. In contrast, the opposite findings were observed in the salt-treated GmCIPK2-RNAi hairy roots (Figure 3F,G). Furthermore, consistent with the role of GmCIPK2 in activating the POD and GST enzymes (Figure 3H,I), GmCIPK2 was found to promote the expression of GmPOD21, GmPOD47, GmGST18, and GmGST20 under salt stress (Figure 4G–J). These results indicate that GmCIPK2 participates in enhancing the antioxidant defense system to respond to salt stress in soybean plants.

Salt treatment increases intracellular Na⁺ concentrations. The excessive accumulation of Na⁺ usually triggers the inhibition of K⁺ absorption, breaking the Na⁺/K⁺ homeostasis [1,29]. CIPKs function crucially in modulating ion transport, especially Na⁺ and K⁺. For example, TaCIPK29 has been shown to enhance the expression of SOS1, NHX2, and NHX4 to reduce the Na⁺/K⁺ ratios, improving the salt tolerance of transgenic plants [38]. NtCIPK9 overexpression promotes the expression of NHX1 and NHX7 to increase the K⁺ content and decrease the Na⁺ content under salt stress [39]. NHXs encode the Na⁺/H⁺ antiporters that function in reducing the intracellular Na⁺ content by regulating Na⁺ extrusion or storing redundant Na⁺ in the vacuole [40]. In this assay, upon salt stress, the overexpression of GmCIPK2 decreased Na⁺ concentrations, increased K⁺ contents, and reduced Na⁺/K⁺ ratios in soybean plants (Figure 3J–L). On the contrary, the salt-treated GmCIPK2-RNAi lines displayed the opposite. Furthermore, GmCIPK2 enhanced the transcript levels of GmNHX1 and GmSOS1 in response to salt stress (Figure 4E,F). Collectively, our findings demonstrate that GmCIPK2 is involved in enhancing Na⁺/K⁺ homeostasis to improve the salt tolerance of soybean.

CIPKs have been reported to modulate salt-responsive gene expression to contribute to salt tolerance in plants. A previous study reported that ZmCIPK21 increases the transcript levels of RD29A, COR15, and DREB to increase salt tolerance in transgenic Arabidopsis [14]. NtCIPK11 was shown to regulate the expression of the proline biosynthesis-related genes to increase the proline content in tobacco under salt stress [41]. In this assay, GmCIPK2 enhanced the transcript levels of the proline biosynthesis gene GmP5CS (Figure 4A), which was consistent with its positive role in increasing the proline content under salt stress (Figure 3D). Moreover, GmCIPK2 was found to increase the transcript levels of GmMYB118, GmLEA5, and GmDHN5 in response to salt stress (Figure 4B–D). MYB transcription factors usually function in mediating stress signal transduction to regulate plant adaption to stress conditions [29,42]. DHN and LEA encode dehydrins that play critical roles in protecting cell membrane stability, regulating ion balance, and controlling ROS homeostasis [1,3]. These findings indicate that GmCIPK2 is associated with increasing the transcript levels of the salt-related gene, contributing to the tolerance of soybean to salt stress.

CIPKs usually combine with specific CBL proteins to regulate plant adaptation to adverse conditions [4,9]. For instance, CBL1/9 has been reported to combine with AtCIPK1 to mediate the Arabidopsis’ adaption to osmotic and salt stresses [43]. According to a subsequent study, the AtCBL1/9-AtCIPK23 complex activates the inward K⁺ channel AKT1 to promote K⁺ absorption [44]. Moreover, CaCBL2 interacts with CaCIPK3 at the plasma membrane to improve the drought tolerance of transgenic tomatoes [45]. In this study, the results of the interaction assays verified that GmCBL4 functions as a GmCIPK2-interacting sensor (Figure 5). Moreover, GmCBL4 overexpression conferred salt tolerance in transgenic hairy roots. Furthermore, compared to the control, the salt-treated GmCBL4-OE hairy roots had a higher proline content, a lower content of MDA and H₂O₂, smaller Na⁺ content and Na⁺/K⁺ ratios, and a higher K⁺ concentration (Figure 6), which is consistent with the function of GmCIPK2 in improving the salt tolerance of soybean (Figure 3). Collectively, these results indicated that the GmCBL4-GmCIPK2 complex contributes to enhancing soybean salt tolerance.

5. Conclusions

GmCIPK2 functions crucially in enhancing soybean tolerance to salt stress. Furthermore, GmCIPK2 alters the antioxidant defense system, Na⁺/K⁺ homeostasis, and salt-related gene expression to respond to salt stress. Moreover, GmCBL4 functions as a GmCIPK2-interacting sensor that improves the salt tolerance of soybean hairy roots. Overall, this study contributed to elucidating the CBL-CIPK mediated salt-responsive mechanism in soybean.