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. 2024 Sep 3;73:187–198. doi: 10.1016/j.jare.2024.09.004

GmBSK1-GmGSK1-GmBES1.5 regulatory module controls heat tolerance in soybean

Ze-Hao Hou a,1, Yuan Gao a,1, Jia-Cheng Zheng b,1, Meng-Jie Zhao a, Ying Liu a, Xiao-Yu Cui c, Zhi-Yong Li d, Ji-Tong Wei a, Tai-Fei Yu a, Lei Zheng a, Yuan-Chen Jiao a, Shu-Hui Yang a, Jia-Min Hao a, Jun Chen a, Yong-Bin Zhou a, Ming Chen a, Lijuan Qiu a, You-Zhi Ma a,e, Zhao-Shi Xu a,e,⁎
PMCID: PMC12225950  PMID: 39236976

Graphical abstract

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Keywords: Soybean, BR signaling pathway, Heat stress, ROS, Regulation mechanism

Highlights

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    GmBSK1 improves heat stress tolerance by enhancing antioxidant ability.

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    GmBES1.5 was involved in the GmBSK1-mediated heat stress response pathway in soybean.

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    GmBES1.5 directly binds to the E-box cis-element and regulates the transcript level of abiotic stress-related genes.

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    GmBSK1 antagonized the interaction between GmGSK1 and GmBES1.5, and released the transcriptional activity of GmBES1.5.

Abstract

Introduction

Heat stress poses a severe threat to the growth and production of soybean (Glycine max). Brassinosteroids (BRs) actively participate in plant responses to abiotic stresses, however, the role of BR signaling pathway genes in response to heat stress in soybean remains poorly understood.

Objectives

In this study, we investigate the regulatory mechanisms of GmBSK1 and GmBES1.5 in response to heat stress and the physiological characteristics and yield performance under heat stress conditions.

Methods

Transgenic technology and CRISPR/Cas9 technology were used to generated GmBSK1-OE, GmBES1.5-OE and gmbsk1 transgenic soybean plants, and transcriptome analysis, LUC activity assay and EMSA assay were carried out to elucidate the potential molecular mechanism underlying GmBSK1-GmBES1.5-mediated heat stress tolerance in soybean.

Results

CRISPR/Cas9-generated gmbsk1 knockout mutants exhibited increased sensitivity to heat stress due to a reduction in their ability to scavenge reactive oxygen species (ROS). The expression of GmBES1.5 was up-regulated in GmBSK1-OE plants under heat stress conditions, and it directly binds to the E-box motif present in the promoters of abiotic stress-related genes, thereby enhancing heat stress tolerance in soybean plants. Furthermore, we identified an interaction between GmGSK1 and GmBES1.5, while GmGSK1 inhibits the transcriptional activity of GmBES1.5. Interestingly, the interaction between GmBSK1 and GmGSK1 promotes the localization of GmGSK1 to the plasma membrane and releases the transcriptional activity of GmBES1.5.

Conclusion

Our findings suggest that both GmBSK1 and GmBES1.5 play crucial roles in conferring heat stress tolerance, highlighting a potential strategy for breeding heat-tolerant soybean crops involving the regulatory module consisting of GmBSK1-GmGSK1-GmBES1.5.

Introduction

Heat stress, a detrimental environmental alteration, significantly constrains plant development process and yield formation [1]. The generation of ROS represents the primary characteristic of plant responses to external environmental stresses [2], [3]. Under heat stress, excessive accumulation of ROS can induce cellular and chloroplast structural damage, leading to lipid peroxidation [4], [5]. Plants have evolved a highly efficient antioxidant system to scavenge ROS and mitigate the detrimental effects of oxidative stress [6], [7]. Previous studies have demonstrated that antioxidative enzyme genes, including CAT, POD, and GST, confer plant abiotic stress resistance by augmenting the scavenging capacity of ROS [8], [9], [10], [11]. Furthermore, heat shock transcription factors (TFs), HSFs, are another pivotal regulator that enhance the transcription of heat shock proteins (HSPs) to promptly respond to thermal stress conditions [7], [12], [13]. As molecular chaperones, HSPs confer tolerance to heat stress by positively regulating the antioxidative enzyme system for detoxifying reactive oxygen species (ROS) and safeguarding cellular membrane stability [5], [12].

Recently conducted studies have revealed that brassinosteroids (BRs) actively participate in abiotic stress response through various mechanisms, including precise modulation of stress-responsive gene expression patterns, activation of antioxidant defense machinery, and facilitation of the osmoprotection system [13], [14], [15]. BR-INSENSITIVE 2 (BIN2), a member of the glycogen synthase kinase-3 like kinase (GSK3) family, phosphorylates and stabilizes the stress-responsive transcription factor RD26 to enhance drought stress response efficiency [16]. The maize brassinosteroid-signaling kinase 1 (ZmBSK1) enhances water deficit and salt stress tolerance in plants [17], [18]. TaBZR2, a member of the BZR1/BES1 TFs families, positively activates TaGST1 and augments the ROS scavenging system to confer drought stress resistance in wheat [8], while AtBES1 interacts with AtHSFA1 to positively regulate heat stress tolerance in Arabidopsis [19].

Despite the characterization of the BR signaling pathway in various plant species, our understanding of the response of BR signaling pathway genes to heat stress in soybean remains limited. Soybean, being a crucial oilseed crop globally, faces significant growth and production constraints due to abiotic factors including water deprivation, high temperature, and salinity [12]. Molecular techniques serve as potent tools for conferring abiotic stress resistance in plants [20]. Previous studies have demonstrated the function of BSKs in plant responses to abiotic stress [17], [18], [21]. Here, we present the induction of a soybean GmBSK1 gene by BR and heat stress treatment. Overexpression of GmBSK1 enhances heat stress tolerance in soybean by activating ROS scavenging system. We identified GmGSK1 as an interacting protein of GmBSK1 and GmBES1.5, while GmBSK1 antagonizes the interaction between GmGSK1 and GmBES1.5, thereby rescuing the transcriptional activation of inhibited GmBES1.5 by GmGSK1. Our findings elucidate the regulatory mechanism through which GmBSK1 enhances heat stress tolerance in soybean.

Materials and methods

Plant materials and growth conditions

The full-length cDNA of GmBSK1 and GmBSK1-G2A were cloned into pBI121 and introduced into GV3101 Agrobacterium tumefaciens strain for transgenic Arabidopsis lines. And transgenic Arabidopsis plants were selected on 1/2 MS agar plates with kanamycin. The generation of transgenic soybean lines, including 35S::GmBSK1 and 35S::GmBES1.5, as well as CRISPR/Cas9-generated gmbsk1 knockout mutants (Williams 82), was previously described by Chen et al. [22]. To evaluate the heat tolerance of soybean at both seedling and flowering stages, mature seeds were germinated and sown in standardized flowerpots within controlled growth chambers under a 16 h (light)/8h (dark) with 25 ℃ and 50 % humidity [23]. Two-week-old seedlings were then subjected to heat stress for assessment. For evaluation during the flowering stage, 5 days old soybean seedlings were planted in standardized planters measuring approximately thirty cm in diameter and allowed to grow until reaching maturity within a controlled greenhouse environment before being exposed to heat stress for fifteen days. Finally, agronomic traits and yield characteristics of 10 plants per genotype were determined following harvest as previously described [23]. For gene expression pattern analysis, 5-day-old seedlings cultivated hydroponically were exposed to a temperature of 45 ℃ and treated with 1 μmol/L BR, following previous studies [4], [24]. Subsequently, the leaves of soybean seedlings were harvested at different time points (0, 1, 2, 3, 8, 12, 24, and 48 h) and rapidly frozen in liquid nitrogen before storage at −80 ℃ until used.

Heat stress treatment, phenotypic and physiological measurement

For short-term heat treatment of soybean plants at the seedling stage, two-week-old seedlings were subjected to 42 ℃ for 8 h in controlled growth chambers. For long-term heat treatment of soybean plants during the flowering stage, the plants were transferred to a controlled greenhouse with a thermal cycle of 42/28 ℃ following a light–dark regime of 16/8 h, as described in a previous study [4]. To analyze physiological characteristics, leaves from soybean seedlings at the seedling stage were harvested at 0 and 8 h after heat treatment, while leaves from soybean plants at the flowering stage were harvested four days after treatment according to previously established protocols [4]. The chlorophyll content, MDA content, POD content, O2•- content, H2O2 content, CAT activity, and POD activity were analyzed using the Comin physiological characteristics assay kit (China). Relative water content (RWC) was calculated as previously described [25]. For histochemical staining of detached soybean leaves, 0.5 mg/mL NBT solution (pH 7.8; Coolaber, China) and 1 mg/mL DAB solution (pH3.8, Coolaber,) were employed as previously reported [26].

RNA isolation, RT-qPCR and transcriptome analysis

Total RNA was isolated using the RNAprep Pure Plant Plus Kit (Tiangen, China) following the protocol and reverse transcribed into cDNA using cDNA Synthesis SuperMix (Transgen). RT-qPCR was conducted with Talent qPCR PreMix Kit (SYBR Green) on a Bioer Quant Gene 9600 System (Bioer, China). The primers utilized in this study are listed in Table S1. The relative transcription level was determined by employing the 2-ΔΔCT method as described by Yu et al [23]. For transcriptome analysis, ten leaves per sample were gathered for total RNA extraction, and three biological duplications were conducted. Transcriptome analysis were performed by Tiangen biotech, and different expression genes (DEGs) were identified by DESeq2 with pvalue < 0.05. The raw reads were submitted to the NCBI SRA with a Bioproject ID: PRJNA1050321 and PRJNA1055254.

BiFC and LCI assay via transiently expressed fusion proteins

For the BiFC assay, we cloned the open reading frames (ORFs) of GmBSK1, GmGSK1, and GmBES1.5 into the pXY104-cYFP and pXY106-nYFP vectors following the protocol described by Yu et al [27]. The co-injection of vectors (pXY104-GmBSK1-cYFP, pXY104-GmBES1.5-cYFP, and pXY106-GmGSK1-nYFP) was performed in Nicotiana leaves using Agrobacterium-mediated transformation with GV3101 strain. YFP fluorescence was visualized using a Zeiss confocal laser scanning microscope (LSM 700, Germany). For LCI assay, we cloned the ORFs of GmBSK1, GmGSK1, and GmBES1.5 into either pCAMBIA1300-nLUC or pCAMBIA1300-cLUC vectors and co-injected them into Nicotiana leaves using Agrobacterium-mediated transformation with GV3101 strain as previously described [23]. The luciferase activity was visualized using the Rocel plant living imaging system equipped with a low-light cooled CCD imaging apparatus (Beijing Rong Jing Science and Technology Development Co. Ltd, China).

LUC activity analysis

To assess transcriptional activity, the promoters of the target gene were cloned into pGreenⅡ0800 vector and transformed into GV3101 (pSoup and p19) chemically competent cells (Zoman, China). The 35S::GmBSK1-3 × FLAG, 35S::GmBES1.5-GFP and 35S::GmGSK1-3 × HA vectors were used as effectors. The expression of effector proteins was confirmed by western blot (WB) assay using anti-FLAG-Tag antibody, anti-HA-Tag antibody and anti-GFP-Tag antibody ABclonal (China). Visualization was achieved using a Chemiluminescent Imaging System (Tanon-5200). LUC signal was visualized using Rocel plant living imaging system (Beijing Rong Jing Science and Technology Development Co. Ltd, China), and relative LUC/REN ratio was calculated using Dual Luciferase Reporter Gene Assay Kit (Yeasen, China).

Chromatin immunoprecipitation (ChIP) assay

The 3 g of the twice and third expanded leaves of GmBES1.5-OE plants were fixed in 1 % formaldehyde solution for ChIP-qPCR assay. ChIP-qPCR assay was performed according to previously described [28]. The chromatin from the GmBES1.5-OE plant cells was purified with an anti-GFP antibody, and the purified DNA samples were tested by RT-qPCR assay, and GmEFL1B was used as a negative control [28]. Primers used in this assay are listed in Table S1.

In vitro EMSA assay and pulldown assay

The coding sequence (CDS) of GmBES1.5 was cloned into the pGEX-4 T-1 vector and then transformed into Transetta (DE3) chemically competent cells (Transgen) to express GST-tagged GmBES1.5 protein. The EMSA assay was performed as previously study [23], using the LightShift™ Chemiluminescent EMSA Kit (Thermo), and visualized using a Tanon-5200 System (China). The CDS of GmBSK1 and GmGSK1 were cloned into the pClod-TF and pMAL-c2x vectors, respectively, and transformed into Transetta (DE3) cells (Transgen) for repressive expression of GmBSK1-His and GmGSK1-MBP proteins. The pulldown assay was carried out according the previously described protocol [23], using the One Step Western Kit HRP (Mouse) with anti-His-Tag antibody and anti-MBP-Tag antibody from ABclonal (China). Visualization was achieved using a Chemiluminescent Imaging System (Tanon-5200).

Results

GmBSK1, a heat tolerance regulator in soybean

We performed transcriptome sequencing analysis to identify candidate genes associated with heat stress, and observed that GmBSK1, a BR-signaling kinase protein, was upregulated in response to both high temperature and BR treatment (Fig. S1). The analysis of gene expression patterns revealed a significant upregulation of GmBSK1 by more than 5-fold and 2.5-fold in response to treatment with BR and heat stress, respectively (Fig. 1A). Previous studies showed that the overexpression of BSK protein effectively suppresses the dwarf phenotype in atbri mutants, thereby providing evidence for the activation of BR signaling downstream of BRI1 by BSKs [29], [30]. Our results demonstrated that the expression of GmBSK1 suppressed the dwarf phenotype of atbri, and the introduction of GmBSK1-G2A (a BSK1 mutant with a disrupted myristoylation site) failed to restore the phenotype in atbri background (Fig. 2B). These findings were consistent with previous studies by Tang et al [30]. Therefore, our findings suggest that GmBSK1 may act as a crucial regulator in soybean's response to heat stress through the BR signaling pathway. Thus, we generated gmbsk1 mutants using CRISPR/Cas9 technology and overexpression lines for further investigation (Fig. 1C, Fig. S1). Under normal growth conditions, there were no differences between the wild-type (WT) plants and transgenic lines (Fig. 1C). However, the GmBSK1-OE lines exhibited enhanced tolerance to heat stress with less severe wilting compared to WT plants, while the gmbsk1 lines displayed more pronounced stress phenotypes such as wrinkled leaves with reduced relative water content (RWC) and lower survival rates under heat stress conditions (Fig. 1C-E). Furthermore, measurements of chlorophyll content revealed that GmBSK1-OE plant seedlings maintained the highest chlorophyll levels following exposure to heat stress (Fig. 1F-H).

Fig. 1.

Fig. 1

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GmBSK1 is a heat tolerance regulator. (A) Expression patterns of the GmBSK1 gene under heat stress and BR treatments determined by RT-qPCR analysis. (B) Phenotypic analysis WT, atbri, or transgenic atbri overexpressing GmBSK1 and GmBSK1-G2A. (C) Identification of CRISPR/Cas9-gmbsk1 soybean mutant plants, and phenotypic analysis of different plants under heat stress; Bar = 10 cm (D) Survival rate of different lines under heat stress; (E) Relative water content (RWC) of different lines under heat stress. (F-H) Chlorophyll content of different plants under heat stress. Different letters indicate means that were significantly different at p < 0.05.

Fig. 2.

Fig. 2

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Heat stress tolerance of various soybean plants at flowering stage. (A) Phenotypic analysis of the different plants at flowering stage under heat stress. (B, C) MDA and proline content of different plants under heat stress condition. (D, E) CAT and POD activities of different plants under heat stress condition; (F-I) Biomass of plant (F), number of pods per plant (G), the grain weight per plant (H) and the stem base circumference (I) of different plants at adult stage under heat stress condition; (J-K) Phenotypic analysis of seeds under heat stress condition at the adult stage and the relative proportions of different seed types.

GmBSK1 improved physiological characteristics and yield performance under heat stress

We further assessed the tolerance to heat stress in gmbsk1 and GmBSK1-OE plants during the flowering stage in a greenhouse. Physiological index analyses revealed that GmBSK1-OE plants exhibited reduced levels of MDA and increased proline content following heat stress treatment (Fig. 2A-C). Conversely, gmbsk1 plants displayed decreased activities of CAT and POD compare with WT plants (Fig. 2D, E). Subsequently, we evaluated plant agronomic traits during the maturation period and observed that GmBSK1-OE plants demonstrated enhanced performance under heat stress conditions (Fig. 2F-I, Fig. S2), including higher plant biomass and yield per plant. However, there were no significant differences in stem base circumference among gmbsk1, WT, and GmBSK1-OE plants. Based on plumpness (Fig. 2J), seed traits were divided into four groups (Ⅰ to Ⅳ), and under heat stress conditions, GmBSK1-OE plants exhibited a larger number of groups Ⅰ seeds than WT plants, and gmbsk1 transgenic lines showed more groups Ⅱ and Ⅲ seeds (Fig. 2 K, Table S2). These results indicated that GmBSK1 plays a positive regulator in soybean's response to heat stress.

GmBSK1 reduced ROS accumulation and modulated the BR signaling pathway genes

Transcriptomic analysis was conducted on wild-type (WT) and transgenic plants (GmBSK1-OE5 and gmbsk1-KO6) to elucidate the molecular mechanism underlying GmBSK1-mediated heat stress tolerance, and the differentially expressed genes (DEGs) was listed in Table S3. Principal component analysis (PCA) was performed, and the results showed that the samples of the control treatment were clearly separated from the heat stress treatment in various soybean plants (Fig. S3). GO enrichment analysis showed that DEGs which response to abiotic stimulus, photosynthesis, and hormone response were significantly up-regulated in GmBSK1-OE plants under heat stress conditions (Fig. 3A, Fig. S3, Table S4). Conversely, DEGs related to oxidative stress and ROS were significantly down-regulated in GmBSK1-OE plants but markedly up-regulated in gmbsk1 plants compared to WT controls (Fig. 3B). Furthermore, the ROS scavenging-related genes, including glutaredoxins (GRXs), thioredoxins (TRXs), glutathione peroxidases (GPXs), CAT4, and POD17, were up-regulated in GmBSK1-OE lines but down-regulated in gmbsk1 transgenic lines compared to the wild type (WT) under heat stress conditions. NBT staining and DAB staining results demonstrated that GmBSK1-OE plants exhibited reduced O2•- and H2O2 production compared to WT under heat stress condition, while there were no significantly different between WT and transgenic lines under normal growth conditions (Fig. 3C, Fig. S4). Conversely, determination of O2•- and H2O2 concentrations revealed that gmbsk1 lines accumulated higher levels of O2•- and H2O2 under heat stress conditions, with no significant difference observed between WT and transgenic lines under normal growth conditions (Fig. 3D, E). The expression levels of abiotic stress-responsive genes, including GmGST, GmCAT4, GmPOD17, and GmGPX6, were assessed using RT-qPCR (Fig. 3F-I). Additionally, we observed an upregulation of GmBES1/2 (GmBES1.5 and GmBEH2) in GmBSK1-OE lines under heat stress conditions. These results suggested that GmBSK1 may play a crucial role in enhancing the antioxidant defense system and modulating the BR signaling pathway to respond to heat stress.

Fig. 3.

Fig. 3

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GmBSK1 enhances ROS scavenging ability and modulating the expression of BES/BZR genes in soybean plants. (A) GO enrichment analysis of DEGs under heat stress. (B) Heat map of DEGs involved in ROS scavenging based on transcriptome analysis of GmBSK1-OE, Gmbsk1-KO and WT plants subjected to heat stress. (C) NBT and DAB staining of GmBSK1-OE, Gmbsk1-KO and WT plants under heat stress condition. (D, E) O2•- and H2O2 content of GmBSK1-OE, Gmbsk1-KO and WT plants under heat stress condition. (F-K) RT-qPCR of the stress-related genes and BES/BZR genes identified from RNA-seq data under heat stress conditions. * indicates a significant difference at p < 0.05 level.

GmBES1.5 confers heat tolerance by alleviates oxidative damage in soybean

The expression level of GmBES1.5 was upregulated in GmBSK1-overexpressing plants under heat stress, suggesting that GmBES1.5 may also act as a regulator in the GmBSK1-mediated heat stress response pathway. Amino acid analysis alignment revealed the presence of conserved BES1 domains in the N-terminal of the GmBES1.5 protein, and subcellular localization analysis demonstrated specific nuclear localization of GmBES1.5 (Fig. S5). The transcript level of GmBES1.5 increased by more than 2.5-fold and 3-fold after BR and heat stress treatment in seedling leaves, respectively (Fig. S5). To demonstrated the potential role of GmBES1.5 in soybean response to heat stress, we generated GmBES1.5-OE plants (Fig. S4), and two independent lines (GmBES1.5-OE1 and GmBES1.5-OE2) were exposed to heat stress. Under high temperature conditions, the GmBES1.5-OE lines exhibited greater tolerance with less severe wilting, higher RWC, and chlorophyll content compared to WT plants (Fig. 4 A-D). NBT staining and DAB staining showed that the GmBES1.5-OE plants produced less ROS than WT plants under heat stress (Fig. 4F, Fig. S6). In contrast, under heat stress conditions, the GmBES1.5-OE lines showed lower levels of O2•-, H2O2 and MDA compared to WT plants (Fig. 4G, H). These results suggest that GmBES1.5 enhances soybean's resistance to heat stress by alleviating oxidative damage.

Fig. 4.

Fig. 4

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Overexpression of GmBES1.5 in soybean improves heat stress tolerance. (A) Phenotypic analysis of the different lines under heat stress condition. (B) RWC of leaves of GmBES1.5-OE and WT plants under heat stress condition. (C, D) Chlorophyll content of GmBES1.5-OE lines and WT under heat stress. (E) MDA content in the leaves of GmBES1.5-OE lines and WT under heat stress. (F) NBT and DAB staining of leaves of GmBES1.5-OE and WT plants under heat stress condition. (G) O2•- content and (H) H2O2 content in the leaves of GmBES1.5-OE lines and WT under heat stress. (I) Volcano plots depicting the differential expression of genes between GmBES1.5-OE lines and WT under heat stress conditions in the transcriptome analysis. (J) Heat map of DEGs involved in abiotic stress responsive based on transcriptome analysis of GmBES1.5-OE lines and WT. (K-R) RT-qPCR of the abiotic stress responsive genes Identified from RNA-seq data under heat stress conditions. * indicates a significant difference at p < 0.05 level.

We performed transcriptome analysis of GmBES1.5-OE and WT plants to illustrate the molecular mechanism behind GmBES1.5-mediated heat stress tolerance. We found that 95 DEGs, including a variety of abiotic stress-related genes, were up-regulated in both GmBSK1-OE and GmBES1.5-OE plants (Fig. 4I, J, Fig. S7). Furthermore, RT-qPCR analysis revealed that eight stress-related genes including GmNHL13, GmCYP707A2, GmCIPK11, GmCML41, GmPP2C27, GmPLDα1, GmPAP17 and GmELP1 were induced by heat stress treatment in GmBES1.5-OE plants (Fig. 4 K-R).

GmBES1.5 directly binds to the E-box and activities abiotic stress response genes

The promoter sequences of these abiotic stress-related genes were found to contain E-box cis-acting elements (Fig. S8), indicating that these genes are potential regulatory targets of GmBES1.5. Therefore, the promoters of four genes were selected and cloning into the pGreen II0800-LUC vector, and transient luciferase transcriptional activity assay was performed to assess their activation by GmBES1.5. The results showed a significant enhance in transcript levels of the LUC reporter gene driven by these four promoters upon overexpression of GmBES1.5, with enhanced activation observed under heat stress and BR treatment conditions (Fig. 5 A, B). Furthermore, in order to further determine the specificity binding site of GmBES1.5, the Chip-qPCR was performed using the anti-GFP antibody to pull down GFP-tagged GmBES1.5 proteins from GmBES1.5-OE lines. And our results showed that the enrichment of GmBES1.5 at the fragments containing E-box cis-elements (Fig. 5C-F). Additionally, electrophoretic mobility shift assays (EMSA) using biotin-labeled probes synthesized from the promoter sequences revealed that the GmBES1.5 protein can bind to the E-box cis-element in vitro (Fig. 5 G). Collectively, these findings indicate direct binding capability of GmBES1.5 to the E-box cis-element and its role in regulating transcript levels of abiotic stress-responsive genes.

Fig. 5.

Fig. 5

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GmBES1.5 binds to the E-box and activities abiotic stress responsive genes (A, B) GmBES1.5 activates the transcription of abiotic stress responsive genes, as determined by dual-luciferase assays in Nicotiana leaves; (C-F) Chip-qPCR assay showed in vivo binding of GmBES1.5 to the E-box cis-element. * indicates statistically significant differences (p < 0.05) (G) EMSA analysis showing GmBES1.5 bind to the E-box cis-element.

GmGSK1 was identified interacts with both GmBSK1 and GmBES1.5

For further investigate the mechanism of heat stress response mediated by GmBSK1 and GmBES1.5 in soybean, we employed luciferase complementation imaging (LCI) assay as described previously [31] to screen for interacting proteins with GmBSK1 and GmBES1.5. As a result, we identified a protein named GmGSK1 (GLYMA_05G159400), which exhibited high homology with Arabidopsis BIN2 and rice OsGSK1 (Fig. S9), as an interactor with both GmBSK1 and GmBES1.5 in Nicotiana leaves (Fig. 6 A, B). And pulldown assays confirmed that in vitro interactions occur between GmGSK1 with both GmBSK1 and GmBES1.5 proteins; notably, this interaction was antagonized by the presence of recombinant His-tagged-GmBSK protein (Fig. 6C). Furthermore, we performed Bimolecular Fluorescence Complementation (BiFC) assay to demonstrate the interaction between GmGSK1 and either GmBSK1 or GmBES1.5 in Nicotiana leaves. Co-expression of GmBSK1-cYFP and GmGSK1-nYFP resulted in a predominant fluorescence signal localized in the plasma membrane. Conversely, co-expression of GmGSK1-nYFP and GmBES1.5-cYFP exhibited a robust fluorescence signal specifically within the nucleus (Fig. 6D). Intriguingly, simultaneous co-expression of GmBSK1-cYFP, GmBES1.5-cYFP, and GmGSK1-nYFP demonstrated a fluorescence signal primarily at the plasma membrane (Fig. 6D).

Fig. 6.

Fig. 6

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GmGSK1 interacts with GmBSK1 and GmBES1.5. (A) LCI assays identified a GmGSK1 protein interacts with GmBSK1 and GmBES1.5. (B) Luciferase activity measurement. (C) Pulldown assays showing that GmBSK1 antagonized the interaction between GmGSK1 and GmBES1.5 in vitro. (D) BiFC assays showing the interaction of GmGSK1 and GmBSK1 mainly in plasma membrane, and the interaction of GmGSK1 and GmBES1.5 mainly in nucleus. Bar scale = 50 μm.

GmBSK1 releasing transcriptional activity of GmBES1.5 repressed by GmGSK1

It has been previously reported that the transcriptional activity of BZR/BES proteins is regulated by an interaction mediated by a GSK3-like kinase BIN2, and BSKs are known to function upstream of BIN2 in the BR signaling pathway in Arabidopsis [30]. The transient luciferase reporter assay was carried out to determine the influence of GmBSK1 and GmGSK1 on the transcriptional activity of GmBES1.5, and the expression of effector proteins was confirmed by western blot (WB) assay (Fig. S10). The results showed that GmBES1.5 markedly enhanced the transcript level of LUC reporter gene driven by promoters of GmPP2C27, and GmPLDα1 (Fig. 7A-C). Furthermore, the transcriptional activity of GmBES1.5 was suppressed by GmGSK1; however, it could be derepressed by GmBSK1 through antagonizing with GmGSK1 (Fig. 7C). Moreover, Immunodetection of GmBES1.5-GST protein in response to heat stress and BR treatment in GmBSK1-KO, WT and GmBSK1-OE plants was carried out, and our results showed that overexpression of GmBSK1 in soybean significantly preserved the stability of GmBES1.5 protein under heat stress and BR treatment (Fig. 7E). These findings suggest that GmBSK1 acts as an antagonist, disrupting the interaction between GmGSK1 and GmBES1.5, ultimately leading to the liberation of transcriptional activity in GmBES1.5.

Fig. 7.

Fig. 7

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The transcriptional activity of GmBES1.5 was affected by GmGSK1 and GmBSK1. (A-C) LUC activity assay and (D) relative activity assay showing the transcriptional activity of GmBES1.5 was inhibited by GmGSK1 and derepress by GmBSK1. (E) Immunodetection of GmBES1.5-GST protein in response to heat stress and BR treatment in various soybean plants.

Discussion

Global climate warming poses a threat to food security, as it has been reported that each one-degree Celsius increase in global mean temperature would result in a 3.1 % reduction in soybean production [32], [33]. Under heat stress conditions, plants undergo significant shifts in the expression patterns of various stress response genes to adapt morphologically and cope with high temperatures [7], [34]. Exposure of plants to heat stress leads to the accumulation of excessive ROS, which disrupts cellular and metabolic processes [35], [36]. Through retrograde signaling pathways utilizing ROS and chlorophyll biosynthetic intermediates, chloroplasts transmit high-temperature stimuli to the nucleus for regulating the expression pattern of stress-responsive genes [37]. Heat stress has been shown to induce alterations in chloroplast membranes [38], resulting in reduced chlorophyll content and increased ROS production [39]. To mitigate oxidative damage caused by ROS, plants modulate the activities of antioxidant enzymes to maintain ROS homeostasis [40]. In this study, the presence of leaf wrinkling and reduced chlorophyll content in gmbsk1-KO plants (Fig. 1, Fig. 2) indicated that loss of GmBSK1 function rendered them susceptible to heat stress. Moreover, GmBSK1-OE plants exhibited decreased production of active oxygen under heat stress conditions, accompanied by upregulation of genes encoding antioxidant defense enzymes and stress-responsive factors such as GmGST, GmCAT4, GmPOD17, and GmGPX6 (Fig. 3). Previous investigations have reported that ZmBSK1 confers protection against drought and salt stress through enhanced activity of antioxidant enzymes [17], [18]. Our findings indicated that GmBSK1 play a positive role in enhancing soybean's tolerance to heat stress by augmenting ROS scavenging mechanisms. Previous research has shown that ROS can mediate the signaling crosstalk between various hormones, and hormones and ROS-mediated transcriptional regulation play a significant role in modulating the heat stress responses in plants [7], [13], [15]. For instance, plants can develop heat stress tolerance through the interaction of ABA and BRs, which is mediated by ROS [41], [42]. Additionally, it was demonstrated that ROS caused the oxidation of BZR1, which in turn enhanced its ability to interact with phytochrome-interacting factor 4 (PIF4) and auxin response factor 6 (ARF6) to modify stress response [43]. Additionally, BR positively controls the components of the SA pathway, NPR1 and WRKY70, to mediate thermotolerance and defense gene expression [44], and the crosstalk between IAA, BR, and GA regulated the growth of the hypocotyl under heat stress [45]. Our study showed that the hormone response genes were significantly up-regulated in GmBSK1-OE plants under heat stress conditions (Fig. 3, Fig. S3), suggesting that GmBSK1 may function as a regulator on the hormone-signaling component, and further research is required to understand the underlying networks triggered by phytohormones in response to heat stress.

BIN2 exerts a negative role in BR signaling by phosphorylating and destabilizing BES/BZR TFs in plants [16]. It has been reported that BIN2 reduced cold tolerance by destabilizing ICE1 in plants [46] and acts as a molecular switch between salt stress tolerance and plant growth recovery [47]. In this study, we identified GmGSK1, a member of the GSK3-like kinase protein family, which interacts with GmBES1.5 and inhibits its transcriptional activity. Additionally, the interaction between GmBSK1 and GmGSK1 promotes the localization of GmGSK1 to the plasma membrane, thereby releasing the transcriptional activity of GmBES1.5 (Fig. 6, Fig. 7). BES/BZR family TFs play a pivotal role in plant growth and stress tolerance within the BR signaling pathway [48], [49]. Previous studies have reported that the BES/BZR TFs can directly bind to promoters containing E-box elements (ACNNGT), G-box elements (CACGTG), or BRRE elements (CGTGT/CG) to regulate gene expression in plants [50], [51], [52]. In this study, we observed an induction of GmBES1.5 expression in GmBSK1-OE plants under heat stress conditions (Fig. 3). Functional analysis demonstrated that GmBES1.5 can specifically bind to the E-box cis-elements, with its transcriptional activity being enhanced during heat stress exposure (Fig. 5). Previous studies have reported that the TaBZR2 protein positively regulates the expression of TaGST1 by binding to the E-box cis-element in its promoter, thereby conferring drought stress tolerance in wheat [8]. BR signaling promotes plant thermotolerance by releasing the BIN2 suppression of HsfA1d to facilitate its nuclear localization and DNA binding [53], and BES1 enhances heat stress tolerance in Arabidopsis by interacting with HSFA1 and activating HSP genes through binding to heat shock elements (HSEs) [19]. In this study, the transcriptional level of stress-related genes, such as GmPLDα1, GmCML41, GmCIPK11, and GmNHL13, were increased in GmBES1.5-OE plants under heat conditions. Previous reports have demonstrated that overexpression of PLDα1 reduces transpirational water loss in response to abiotic stress [54], [55], while late embryogenesis abundant (LEA), CIPK and CML proteins act as regulators of ROS homeostasis under abiotic stress in plants [26], [56], [57], [58]. These findings suggest that GmBES1.5 confers heat stress tolerance in soybean through increasing the expression of genes involved in the response to abiotic stress and scavenging reactive oxygen species.

Our findings demonstrate that GmBSK1 is induced by the BR and heat stress, GmBSK1 enhances heat stress tolerance by augmenting ROS scavenging capacity and promoting the localization of GmGSK1 to the plasma membrane, thereby derepressing the transcriptional activity of GmBES1.5. Subsequently, GmBES1.5 positively regulates the transcription of abiotic stress-responsive genes, ultimately enhancing soybean's ability to withstand heat stress (Fig. 8). It is noteworthy that the regulatory module involving GmBSK1-GmGSK1-GmBES1.5 holds promising potential for genetic breeding aimed at developing heat-tolerant soybean cultivars.

Fig. 8.

Fig. 8

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Diagram summarizing the GmBSK1-GmGSK1-GmBES1.5 regulatory module in soybean heat stress tolerance.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are grateful to Drs Wensheng Hou and Shi Sun of the Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS) for kindly providing soybean seeds, high-efficiency A. rhizogenes-mediated transformation and valuable reviews, respectively. This research was financially supported by the National Key R & D Program of China (2022YFF1001600), Innovation Program of Chinese Academy of Agricultural Sciences, Hainan Seed Industry Laboratory (B23CJ0208), and Nanfan special project, CAAS (YBXM04).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2024.09.004.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Data 1
mmc1.docx (2.9MB, docx)
Supplementary Data 2
mmc2.xlsx (1.7MB, xlsx)

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