GmTRAB1, a Basic Leucine Zipper Transcription Factor, Positively Regulates Drought Tolerance in Soybean (Glycine max. L)

Abstract

The basic leucine zipper (bZIP) transcription factors play crucial roles in plant resistance to environmental challenges, but the biological functions of soybean bZIP members are still unclear. In this study, a drought-related soybean bZIP gene, GmTRAB1, was analyzed. The transcript of GmTRAB1 was upregulated under drought, ABA, and oxidative stresses. Overexpression of GmTRAB1 improved the osmotic stress tolerance of transgenic Arabidopsis and soybean hairy roots associated with increased proline content and activity of antioxidant enzymes and reduced accumulations of malonaldehyde and reactive oxide species. However, RNA interference silencing of GmTRAB1 in the soybean hairy roots improved drought sensitivity. Furthermore, GmTRAB1 increased the sensitivity of transgenic plants to ABA and participated in modulating ABA-regulated stomatal closure upon drought stress. In addition, GmTRAB1 stimulated the transcript accumulation of drought-, ABA-, and antioxidant-related genes to respond to drought. Collectively, this research will contribute to understanding the molecular mechanisms of bZIP transcription factors in soybean’s resistance to drought.

1. Introduction

Plants usually encounter multiple adverse environments, including extreme temperatures, drought, and salt, and these stresses can greatly affect their growth and development and ultimately destroy their productivity [1]. To survive, plants have evolved a variety of strategies to cope with these environmental challenges. These adaptive responses are highly intricate processes mediated by sophisticated signaling networks [2]. Transcription factors, including those from families including dehydration-responsive element-binding (DREB), myeloblastosis (MYB), NAM, ATAF, and CUC (NAC), WRKY, ethylene-responsive factor (ERF), and basic leucine zipper (bZIP), play critical roles in the signal transduction networks, and they function essentially in transmitting the perceived stress signal to the stress-responsive genes, ultimately leading to physiological and metabolic changes [3,4,5].

The bZIP transcription factors comprise a large and diverse transcription factor family in plants [6]. Members of bZIP proteins have a conserved bZIP domain, which consists of a leucine zipper motif and a conserved base region [7]. The N-X7-R/K motif is composed of approximately 18 amino acids within the bZIP domain and performs critical functions in DNA binding and nuclear localization [8]. The bZIPs usually interact with the promoter fragments harboring an ACGT core cis-element, including C-box (GACGTC), ABRE (CCACGTGG), A-box (TACGTA), and G-box (CACGTG), to influence the expression of downstream targets [9]. The leucine zipper region comprises one or more repeating regions or other hydrophobic amino acids, which is correlated to bZIP recognition and dimerization [10]. The bZIP proteins were first characterized in Arabidopsis, and 75 bZIPs were identified [7]. Subsequent genomics research also found 89 bZIPs in rice [10], 89 bZIPs in barley [11], 55 bZIPs in tomato [12], 247 bZIPs in rapeseed [13], 92 bZIPs in sorghum [14], and 125 bZIPs in maize [15].

The bZIP proteins perform crucial functions in plants’ responses to adverse conditions. For example, in Arabidopsis, AtbZIP1 increased the expression of COR15A, RD17, and RD29A to enhance Arabidopsis’ ability to tolerate salt and osmotic stresses [16]. AtABF3 regulated ABRE-dependent gene expression to influence ABA-mediated drought response [17]. In rice, OsbZIP23, OsbZIP42, and OsbZIP72 were upregulated by ABA and drought stresses and act as key regulators in rendering rice ABA-regulated osmotic stress tolerance [18,19,20]. ZmbZIP4 has been reported to increase ABA synthesis to improve maize adaptation to drought and salt stresses [6]. Under cold conditions, ZmbZIP68 restricts the expression of DREB family proteins to reduce cold tolerance of maize [2]. TabZIP60 is associated with TaCDPK30 to influence the synthesis of ABA and increase the salt tolerance of wheat [5]. TabZIP15 was shown to respond to salt stress, and TabZIP15 overexpression conferred the resistance of transgenic wheat to salt [21]. Overexpression of CsbZIP18 impaired the cold tolerance of transgenic plants through suppressing cold- and ABA-related genes’ expression [22]. Furthermore, in pepper, salt promoted CabZIP25 expression, and CabZIP25-overexpressing (OE) plants displayed salt-tolerant phenotypes [23].

Soybean is a critical cereal and oil crop worldwide [24,25]. Drought acts as the main adverse stimulus to destroy the production of soybean [26]. Studying the mechanism in drought response is an effective way to enhance the adaptability of soybean to drought. Previous studies have reported that there are 160 bZIP genes in the soybean genome [27]. Until now, only a very limited number of bZIP members have been functionally analyzed in soybean. In the present research, we characterized a bZIP family transcription factor, GmTRAB1, and show that its expression is significantly upregulated upon drought, ABA, and oxidative stresses. Further physiology and molecular studies displayed that GmTRAB1 influences the ABA response and antioxidant defense system to enhance soybean tolerance to drought.

2. Results

2.1. Identification and Sequence Analysis of GmTRAB1

Results of bioinformatics analysis have shown that 160 bZIP transcription factors were identified in soybean [27]. GmTRAB1 (Glyma.04G039300) encoded a protein with a conserved bZIP domain and highly shared the sequence with GsABL5 from Glycine soja (99.52%), GmbZIP50 (89.35%), MpABL5 from Mucuna pruriens (87.97%), and VuABL5 from Vigna unguiculata (86.35%) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=JEV01RW2016, accessed on 31 October 2024; Figure 1A). Subsequent phylogenetic analysis demonstrated that GmTRAB1 and its homologous proteins were classified into subgroup A (Figure S1). In the subcellular localization analyses, GmTRAB1-GFP proteins were detected in the nucleus and overlapped with the nuclear marker (OsEHD4-mCherry) [28] (Figure 1B).

2.2. GmTRAB1 Is Induced by Multiple Abiotic Stresses

To assess the function of bZIPs in the modulation of soybean response to environmental challenges, we investigated the expression patterns of GmTRAB1 under drought, ABA, and oxidative stresses using a qRT-PCR system. Drought stimulated the accumulation of the GmTRAB1 transcript, reaching a peak at 6 h (Figure 2A). Moreover, GmTRAB1 transcripts increased under ABA stress and reached their highest level at 24 h (Figure 2B). In addition, oxidative stress also resulted in increased GmTRAB1 expression (Figure 2C).

2.3. Overexpression of GmTRAB1 Enhances Arabidopsis Resistance to Drought

To characterize the role of GmTRAB1 in plants’ resistance to drought, we constructed GmTRAB1-OE Arabidopsis using an Agrobacterium-mediated transformation system. The transcript levels of GmTRAB1 in transgenic plants were detected by semi-qRT-PCR. Three independent homozygous GmTRAB1-OE lines (OE1, OE7, and OE12) with relatively high accumulation of GmTRAB1 transcripts were used for the subsequent functional analysis (Figure 3C,D). Arabidopsis plants with similar growth characteristics were applied for osmotic stress tolerance experiments. There was no remarkable difference in plant morphology and physiological metabolism between GmTRAB1-OE lines and WT plants under sufficient water conditions. Severe drought stress seriously affected plant growth and physiological metabolism, and obvious differences were detected. Upon drought stress, GmTRAB1-OE plants displayed slighter leaf rolling and larger survival rates than the WT plants (Figure 3A,B). Drought usually causes damage to the membranes of plants, restricting their normal growth. The concentration of malonaldehyde (MDA) acted as an indicator to reflect the degree of cell membrane damage. The MDA concentration is proportional to the degree of membrane damage [1]. Proline was an osmoprotectant that functioned crucially in maintaining the stability of the cell membrane and cellular turgor pressure [29,30]. The drought-treated GmTRAB1-OE seedlings showed lower MDA levels and larger proline contents as compared with the WT plants (Figure 3E,F).

2.4. GmTRAB1 Overexpression Leads to Increased ABA Sensitivity in Transgenic Plants

Considering that ABA promotes the increase in GmTRAB1 transcripts, we conducted a seed germination assay to investigate the role of GmTRAB1 in ABA response. The GmTRAB1-OE and WT seeds showed similar germination rates in the absence of ABA (Figure 4A). However, exogenous ABA application inhibited seed germination, but the GmTRAB1-OE seeds presented significantly lower germination rates than those of the control with exposure to ABA treatments (Figure 4B,C).

ABA has been demonstrated to influence stomatal movements. In view of GmTRAB1 overexpression improving ABA sensitivity in transgenic plants, the function of GmTRAB1 in ABA-regulated stomatal movement was investigated. The stomatal closure index was reflected by the stomatal width/length ratio. Without ABA application, the mean of stomatal apertures between GmTRAB1-OE and WT plants is comparable (Figure 4D,E). Nevertheless, with the ABA application, the GmTRAB1-OE plants have a significantly lower mean of stomatal apertures as compared to the control plants (Figure 4D,E). Stomatal movement plays a central role in water loss and is closely related to plant adaptation to drought. Therefore, the water loss rates were also analyzed, and GmTRAB1-OE exhibited lower water loss rates than WT plants (Figure 4F).

2.5. GmTRAB1 Overexpression Confers Osmotic Stress Tolerance in Transgenic Hairy Roots of Soybean

To verify the role of GmTRAB1 in soybean resistance to drought, we generated transgenic hairy roots of soybean with GmTRAB1-RNA interference (RNAi) silencing or overexpressing by Agrobacterium rhizogenes-mediated transformation. The GmTRAB1 expression was detected through qRT-PCR (Figure 5C). There were no significant differences in the growth and physiological characteristics of GmTRAB1 transgenic (RNAi and OE) and empty vector (EV) control soybean seedlings under suitable conditions. However, drought treatment caused remarkable morphological and physiological differences among different genotypes. Upon drought stress, the GmTRAB1-OE seedlings presented a higher survival rate and a lighter leaf-wilting phenotype than the EV seedlings. In contrast, the GmTRAB1-RNAi plants displayed a lower survival rate and a heavier leaf-wilting phenotype (Figure 5A,D). The fresh weights of GmTRAB1-OE hairy roots were larger than the control roots under drought treatment, but the GmTRAB1-RNAi hairy roots displayed lower biomass accumulations (Figure 5B,E). Moreover, the MDA levels in GmTRAB1-OE hairy roots were smaller than in the control roots under drought stress, whereas the GmTRAB1-RNAi hairy roots presented higher MDA accumulations (Figure 5F). In addition, the GmTRAB1-OE hairy roots presented a higher accumulation of proline than the control roots. However, under drought stress, the GmTRAB1-RNAi soybean roots had lower levels of proline (Figure 5G).

2.6. GmTRAB1 Stimulates ROS Scavenging in Response to Drought

Drought caused the excessive production of reactive oxygen species (ROS) that seriously restricted the growth and development of plants. The results of DAB staining and quantitative measurement illustrated that hydrogen peroxide (H₂O₂) accumulation in GmTRAB1 transgenic hairy roots was comparable to that of the EV control roots. With exposure to drought, the H₂O₂ accumulations in GmTRAB1-RNAi, EV, and GmTRAB1-OE hairy roots are increased. Interestingly, significant differences were observed among GmTRAB1-RNAi, EV, and GmTRAB1-OE hairy roots. GmTRAB1-RNAi seedlings accumulated a larger H₂O₂ content in comparison with the EV control roots, but the GmTRAB1-OE hairy roots exhibited a smaller accumulation of H₂O₂ (Figure 6A,B). Antioxidant enzymes play a critical role in eliminating drought-induced ROS. The activity of antioxidant enzymes was measured. After drought treatment, compared with the control, the GmTRAB1-OE roots displayed greater enzyme activity of catalase (CAT) and peroxidase (POD). Nevertheless, the GmTRAB1-RNAi roots had lower activities of CAT and POD enzymes (Figure 6C,D).

2.7. GmTRAB1 Increases Drought-, ABA-, and Antioxidant-Related Gene Expression in Response to Drought

To gain sight into the GmTRAB1-mediated adaptative mechanism in response to drought, the transcripts of drought-, ABA-, and antioxidant-related genes in GmTRAB1-RNAi, EV, and GmTRAB1-OE hairy roots upon drought stress were detected through qRT-PCR. No noticeable difference among GmTRAB1-RNAi, EV, and GmTRAB1-OE hairy roots was detected prior to drought treatment. Drought altered the expression of stress-related genes, and the expression of drought-induced genes (GmDRAB1A, GmWRKY27, and GmCIPK6), ABA-responsive genes (GmP5CS, GmLEA5, and GmNAC6), and antioxidant-related genes (GmPOD3, GmPOD5, and GmCAT4) in drought-treated hairy roots of GmTRAB1-OE were much greater than those of EV hairy roots. In contrast, lower transcript accumulation of the above genes was detected in the GmTRAB1-RNAi hairy roots under drought stress (Figure 7).

3. Discussion

Drought is usually considered the main environmental constraint that restricts crop quality and production [25]. Members of the bZIP transcription factor have been demonstrated to participate in modulating plant adaptation to drought. Nonetheless, the biological roles of bZIP proteins in enhancing soybean drought tolerance are still unclear. In this study, drought led to increased expression of the bZIP gene GmTRAB1 (Figure 2A). Further functional analysis verified that GmTRAB1-OE Arabidopsis seedlings and soybean hairy roots displayed drought-resistant phenotypes. On the contrary, the GmTRAB1-RNAi soybean hairy roots were much more sensitive to drought (Figure 3 and Figure 5). These results indicated that GmTRAB1 positively regulates the osmotic stress tolerance of soybean.

The phytohormone ABA serves as a kind of pivotal signaling molecule that has critical roles in modulating plant response to drought [31]. Drought results in an increase in endogenous ABA, and the increased ABA subsequently alters the transcript accumulation of stress-related genes and stomatal movement in response to drought [32]. It has been shown that members of bZIPs are implicated in ABA-regulated adaptation to drought. For example, OsbZIP42 and OsbZIP71 have been demonstrated to modulate drought adaptation via an ABA-dependent manner [20,33]. In this assay, exogenous ABA induced the accumulation of the GmTRAB1 transcript (Figure 2B). Moreover, with exposure to ABA treatment, the GmTRAB1-OE plants exhibited lower seed germination rate and smaller stomatal aperture as compared with the control plants (Figure 4B–E). Interestingly, GmTRAB1 has a role in reducing the water loss rate (Figure 4F). Additionally, upon drought stress, GmTRAB1 promoted the transcript accumulation of ABA-responsive genes, including GmP5CS, GmLEA5, and GmNAC6 (Figure 7D–F). These results imply that GmTRAB1 is associated with modulating ABA-regulated stomatal closure and stress-responsive gene expression to regulate soybean adaptability to drought.

Drought usually stimulates the generation of ROS, including H₂O₂ and O₂⁻ [34]. The overproduced ROS results in a series of cell toxicity effects, such as membrane damage, nucleic acid and protein degradation, and disruption of enzyme activity [35]. The MDA level is usually used as an important parameter to reflect the degree of cell membrane damage under stress conditions, which is negatively implicated with osmotic stress tolerance in plants [1]. The bZIP proteins have been demonstrated to influence the scavenging of ROS under adverse conditions. For example, HvbZIP21 was implicated in the scavenging of ROS to improve the osmotic stress tolerance in barley [36]. In the assay, oxidative stress induced the expression of GmTRAB1 (Figure 2C). Subsequent quantitative measurements showed that under drought stress, compared to the control, the hairy roots of GmTRAB1-OE accumulated a lower content of MDA and H₂O₂, whereas the MDA and H₂O₂ levels in GmTRAB1-RNAi hairy roots were larger (Figure 3E, Figure 5F, and Figure 6A,B). Proline is a pivotal osmolyte that functions critically in maintaining ROS homeostasis and stabilizing cell membranes under adverse conditions [29,30]. The proline content of GmTRAB1-OE hairy roots under drought stress was larger. Nevertheless, the drought-treated GmTRAB1-RNAi hairy roots demonstrated a lower proline level. Moreover, GmTRAB1 activated the expression of GmP5CS (Figure 7D). Therefore, GmTRAB1 was associated with promoting the generation of proline to reduce drought-induced oxidative damage in soybean.

To mitigate oxidative damage, plants have built complicated antioxidant defense systems to remove the excessive ROS [37]. Antioxidant enzymes, including CAT, POD, and SOD, are essential parts of the antioxidant defense system [34]. The bZIPs have been reported to influence antioxidant enzyme activity to eliminate ROS under adverse conditions. For example, VvbZIP45 participated in increasing the enzyme activity of POD, SOD, and CAT to eliminate ROS under drought stress [38]. IbbZIP1 has been shown to enhance SOD activity to eliminate H₂O₂ under drought and salt stresses [39]. In this research, GmTRAB1 was found to increase the transcript of antioxidant enzyme genes GmPOD3, GmPOD5, and GmCAT4 (Figure 7G–I). Furthermore, compared with the control, the enzyme activity of CAT and POD in GmTRAB1-OE hairy roots under drought stress was larger. Nevertheless, the GmTRAB1-RNAi hairy roots exhibited minor CAT activity and POD activity in response to drought (Figure 6C,D). Collectively, GmTRAB1 has a role in promoting the antioxidant defense system to eliminate ROS and alleviate drought stress in soybean.

The bZIP transcription factors have been shown to alter drought-related gene expression in response to drought. TabZIP156 was found to increase TaDREB1A expression in wheat in response to drought [40]. It has been demonstrated that VlbZIP30 promotes the expression of NAC17, ABF2, PUB19, and PP2C9 to enhance grapevine drought tolerance [41]. In this study, GmTRAB1 functioned in improving the transcript accumulations of GmDREB1A, GmCIPK6, and GmWRKY27 in response to drought (Figure 7A–C). DREB and WRKY transcription factors play crucial functions in increasing plant adaptability to extreme conditions [42,43]. CIPKs have been shown to participate in regulating hormone signaling, ROS scavenging, and the biosynthesis of antioxidants in response to drought [44]. The above results indicated that GmTRAB1 is implicated in stimulating the expression of drought-related genes, thereby promoting the resistance of soybean to drought.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Arabidopsis Columbia-0 and soybean cultivar Williams 82 were used for plant transformation and molecular analysis. The soybean seedlings were cultured in a growth chamber at 70% relative humidity, 25 °C, and a 16 h light/8 h dark photoperiod. To analyze the expression profiles of GmTRAB1 under drought and oxidative stress treatments, the roots of soybean seedlings (2-week-old) were immersed in 15% PEG6000 and 20 µM methyl viologen (MV) solution, respectively. The leaves of seedlings sprayed with 100 µM ABA were used to investigate GmTRAB1 expression profiles upon ABA treatment. The leaves were collected at 0, 1, 3, 6, 12, and 24 h, respectively. Arabidopsis seedlings were growing in an illumination incubator at 70% relative humidity, 25 °C, and a 16 h light/8 h dark photoperiod.

4.2. Construction of Phylogenetic Tree

GmTRAB1 homologous proteins were retrieved from the non-redundant protein sequence database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=JEV01RW2016, accessed on 31 October 2024). The amino acid sequence of the Arabidopsis bZIP proteins was downloaded from TAIR (https://www.arabidopsis.org/results?mainType=general&searchTex, accessed on 31 October 2024). The ClustalX program in MEGA7.0 software was used to perform multiple sequence alignment. The bootstrap neighbor-joining phylogenetic tree was generated by MEGA7.0 software with 500 bootstrap replicates. Ten different subgroups of AtbZIPs were used as references to classify GmTRAB1 and its homologous proteins. The accession numbers of GmTRAB1 homologous proteins and Arabidopsis bZIPs were listed in Table S2.

4.3. Quantative Real Time-PCR

The total RNA from plants was isolated with Trizol reagent, and then the genome DNA was eliminated by RNase-free DNaseI (TransGen, Beijing, China). The cDNA synthesis was performed by the TransScript One-Step RT-PCR SuperMix Kit (TransGen, Beijing, China). The qRT-PCR reactions were then conducted with a TransStart Top Green qPCR SuperMix kit using an ABI 7500 machine. The specific primers used in qRT-PCR were presented in Table S1. Atactin (At3g18780) and GmTubulin (Glyma.08G014200) were referred to as quantitative controls for Arabidopsis and soybean, respectively. The 2^−ΔΔCT method was applied for quantitative analysis. The reaction of qRT-PCR was conducted by the procedure of 95 °C (180 s), then 42 cycles of 95 °C (10 s), 57 °C (15 s), and 72 °C (45 s).

4.4. Subcellular Localization Analyses

To analyze the subcellular localization, we produced GmTRAB1-GFP constructs. GmTRAB1-GFP and OsEHD4-mCherry plasmids were introduced into the same protoplasts from Arabidopsis, and then the protoplasts were cultured in the dark (23 °C) for 12 h. OsEHD4-mCherry was applied as a nuclear marker [28]. The florescence signals of GFP and mCherry were detected with a confocal laser-scanning microscope.

4.5. Construction of Transgenic Arabidopsis and Soybean Plants

To construct GmTRAB1-OE Arabidopsis plants, the pCAMBIA1302-GmTRAB1 plasmids were generated and then introduced into Arabidopsis via the Agrobacterium-mediated floral-dip method [31].

The transgenic soybean plants were generated using the Agrobacterium rhizogene-mediated soybean hairy roots transformation system, as described previously [31,42]. To generate the GmTRAB1-RNAi vector, a 120-bp-specific DNA fragment of GmTRAB1 was cloned, and then these fragments were collected and connected to both sense and antisense orientations to flank the intron 6 of the rice zinc finger type family protein gene. The GmTRAB1-RNAi fragment was then inserted into the pCAMBIA3301 transformation vector driven by the CaMV 35S promoter. The full-length open reading frame of GmTRAB1 was introduced into pCAMBIA3301 to generate the GmTRAB1-OE vector. The GmTRAB1-RNAi and GmTRAB1-OE vectors were transformed into K599 Agrobacterium rhizogenes strains, which were then applied for infecting hypocotyls of soybean to gain transgenic soybean hairy roots.

4.6. Osmotic Stress Tolerance Assay

For Arabidopsis, homozygous T3 seedlings of the GmTRAB1-OE and WT seedlings (1-week-old) were used for drought treatment. After normal culturing in the illumination incubator for 21 d, the GmTRAB1-OE and WT seedlings were subject to drought stress without irrigation for another 17 d until remarkable leaf-wilting differences were identified.

For soybean, 2-week-old GmTRAB1-OE, EV, and GmTRAB1-RNAi soybean seedlings were grown normally in a mixed soil (1:1 vermiculite:humus) for 7 d. These seedlings were then exposed to drought stress with 15% PEG6000 irrigation for another 10 d until remarkable differences in leaf wilting were identified.

4.7. Measurements of Physiological Characteristics

Arabidopsis seedlings (4-week-old) and soybean seedlings (3-week-old) were exposed to drought treatment with irrigating 15% PEG6000 solution for 10 d. The Arabidopsis leaves and soybean hairy roots were collected for measuring physiological parameters. The MDA content, proline content, hydrogen peroxide H₂O₂ content, peroxidase POD activity, and CAT activity were detected by their corresponding detection kits.

4.8. Analysis of Seed Germination, Stomatal Closure, and Water Loss Rate

For measurement of germination rate, homozygous T3 seeds of GmTRAB1-OE and WT were sterilized and sown on 1/2-strength Murashige and Skoog media containing 0, 0.5, and 1 µM ABA. After vernalization in the dark for 3 days, the germination rates in terms of the seed radicle emergence were counted at 0, 12, 24, 36, 48, 60, 72, 84, and 96 h, respectively.

For the water loss rate analysis, the detached rosette leaves of 4-week-old GmTRAB1-OE and WT Arabidopsis were measured at 0, 30, 60, 120, 180, 21, and 240 using a 1/10,000 analytical balance, respectively. The water loss rate was computed as reported previously.

For the stomatal closure analysis, the detached rosette leaves of GmTRAB1-OE and WT Arabidopsis (4-week-old) were treated with stomata opening solution (7.5 mM iminodiacetic acid, pH = 6.15, 10 mM KCl, and 10 mM MES-Tris) and exposed to bright light. Until the stomata were fully opened, the leaves were exchanged into solutions supplemented with 0, 1, and 5 µM ABA and incubated for another 2.5 h.

4.9. Statistical Analyses

Each experiment was independently conducted three times. Data are presented as the mean ± SE of the three independent replicates. Statistical analysis was performed with the SPSS 27.0 software. The one-way analysis of variance (ANOVA) method was applied to verify significant differences, marked as *, p < 0.05.

5. Conclusions

In this study, GmTRAB1 played a positive regulatory role in improving the osmotic stress tolerance of soybean. GmTRAB1 participated in ABA-regulated stomatal closure and stress-related gene expression to regulate the drought stress response. Furthermore, GmTRAB1 was involved in activating the antioxidant defense system to promote the scavenging of ROS under drought stress. Additionally, GmTRAB1 has a role in activating drought-related genes in drought stress response. In summary, these results will provide the theoretical basis for elucidating the molecular mechanism of bZIP transcription factors-mediated drought response in soybean.