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. 2025 Dec 26;29(2):114546. doi: 10.1016/j.isci.2025.114546

GmPLC8 enhances drought resistance in soybean by regulating stomatal closure

Di Wang 1, Mingyu Yang 1, Yuhan Liu 1, Xu Hu 1, Feiyu Hao 1, Daqian Sun 1, Nan Wang 1, Yuanyuan Dong 1, Weican Liu 1, Xiaowei Li 1, Fawei Wang 1,2,∗
PMCID: PMC12856282  PMID: 41623451

Summary

Soybean (Glycine max (L.) Merr.) is an economically important crop. Drought adversely affects nutrient acquisition and early vegetative development, thereby constraining the growth potential of soybean seedlings. Phospholipase C (PLC) participates in diverse biological processes, yet its function in soybeans has not been fully explored. In this study, we generated GmPLC8-overexpressing (OE) and GmPLC8-silenced soybean seedlings to investigate the function of GmPLC8. The results showed that GmPLC8-OE lines exhibited higher tolerance than wild-type seedlings, whereas GmPLC8-silenced seedlings were more sensitive. This was associated with reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels in GmPLC8-OE lines, and increased levels in GmPLC8-silenced seedlings. Further analysis revealed that drought caused smaller stomatal apertures and higher abscisic acid (ABA) content in GmPLC8-OE lines. Moreover, the transcription levels of ABA-related genes were upregulated in GmPLC8-OE lines. Taken together, these findings suggest that GmPLC8 enhances drought tolerance in soybean by regulating stomatal aperture.

Subject areas: Natural sciences, Plant biochemistry, Plant biology, Plant physiology

Graphical abstract

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Highlights

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    GmPLC8 enhances drought tolerance in soybean seedlings

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    GmPLC8 promotes ABA accumulation and stomatal closure under drought stress

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    Overexpression of GmPLC8 reduces MDA and H2O2 accumulation under drought stress

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    GmPLC8 enhances drought tolerance by activating ABA biosynthesis and signaling genes

Natural sciences; Plant biochemistry; Plant biology; Plant physiology

Introduction

The primary limiting factors that affect crop yield include non-biological stressors such as drought, salinity, high temperature, and cold.1 Drought is becoming a major threat to global crop production. When transpiration exceeds water absorption, drought can lead to plant dehydration.2 In situations where plants are deficient in water, the moisture within their cellular tissues progressively decreases, leading to leaf desiccation and drooping. The growth and metabolism of plants are also influenced by water deficiency, causing a decrease in nutrient absorption capacity and ultimately affecting plant growth and development. Simultaneously, drought stress can lead to the accumulation of reactive oxygen species (ROS), causing oxidative damage to cell membranes, proteins, RNA, and DNA molecules. This process is known as oxidative stress and can ultimately lead to cell destruction.3 These changes manifest in various characteristic features related to crop performance, such as diminished stem growth and plant height, leaf wilting and aging, impeded root growth and development, and a decline in yield.4,5,6 In the face of drought stress, the key to survival and reproduction in unfavorable environments is to perceive the stress and initiate adaptive signal pathways.

PLC is involved in the hydrolysis of phospholipids, resulting in the production of various second messenger molecules.7 Based on their affinity for different hydrolytic substrates, PLCs in plants can be divided into two major classes: phosphatidylinositol-specific phospholipase C (PI-PLC) and non-specific phospholipase C (NPC). PI-PLC specifically hydrolyzes phosphatidylinositol, while NPC can hydrolyze other phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylglycerol (PG).8 When plants are subjected to biological stress or abiotic stress, PI-PLC hydrolyzes the phosphatidylinositol backbone, producing two key molecules: Diacylglycerol (DAG) and Inositol trisphosphate (IP3). PI-PLC plays a pivotal role in the regulation of plant growth and development, while also providing plants with the ability to resist various stresses.9 AtPLC5 promotes the accumulation of phosphatidylinositol 4,5-diphosphate and phosphatidic acid in wheat under high temperature and osmotic stress. Specifically, these stresses induce the accumulation of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid.10 Another study has demonstrated that AtPLC5, through the production of signaling lipids such as DAG, enhances plant drought resistance by promoting the development of secondary roots in Arabidopsis.11

Phosphatidylinositol-specific PLC (PI-PLC) enzymes typically consist of four domains: an N-terminal EF-hand domain, X and Y catalytic domains, and a C-terminal C2 domain. The EF-hand domain, composed of four α-helices, specifically recognizes and binds the substrate phosphatidylinositol-4,5-bisphosphate (PIP2).12 The X and Y catalytic domains, located between the N- and C-termini, are highly conserved and essential for the catalytic activity of PI-PLC.13 The C2 domain facilitates the interaction of PI-PLC with Ca2+ and phospholipids, thereby contributing to its regulatory function in cellular signaling.14 Understanding the structural organization of PI-PLC provides a foundation for elucidating its role in plant stress responses and signal transduction pathways.

Recently, Chen et al. identified members of the GmPLC family and demonstrated that GmPI-PLC7 plays a positive role in enhancing drought tolerance in plants, providing a foundation for subsequent studies.13 Zhang et al. found that CaCl2 treatment-induced PI-PLC activation influences DcGLRs expression levels to mediate cytosolic Ca2+ influx, thus highlighting the “PI-PLC-GLRs-Ca2+” pathway in calcium signaling generation and GABA biosynthesis in shredded carrots. These studies indicate that PI-PLC acts as a key regulator involved in plant stress responses and metabolic regulation.15

Currently, PI-PLC has been identified in various plants such as rice (Oryza sativa),16 wheat (Triticum aestivum),17 soybeans (Glycine max),18 corn (Zea mays),19 cotton (Gossypium spp.),20 and tomato (Solanum lycopersicum).21 Through genome-wide expression analysis of multiple species, phospholipase C family members have been identified. In addition, certain PLC isoforms in different species are involved in the response to abiotic stress. Under drought and high-temperature stress, the expression of TaPLC1 is upregulated, and its overexpression enhances the drought resistance in Arabidopsis.22 Overexpression of BnPLC2 in Brassica napus enhances its drought resistance and promotes early flowering and maturation.23 Similarly, under salt and drought stress, overexpression of OsPLC4 enhances the abiotic stress tolerance of rice.24 AtPLC3 exhibits sensitivity to ABA during seed germination and stomatal closure.25 In conclusion, PI-PLC in plants plays a crucial role in growth, development, stomatal movement, and stress responses. However, research on soybean phospholipase C is relatively limited, and further investigation into its functions is required.

Soybeans are not only the most valuable legume crop but are also one of the primary sources of plant protein and plant oil worldwide.26 Nevertheless, drought can significantly impact the growth and yield of soybeans. At present, information on the functions of GmPLC genes in response to abiotic stress is limited. Therefore, in this study, we used Virus-Induced Gene Silencing (VIGS) to silence GmPLC8 in soybean and performed Agrobacterium-mediated genetic transformation to obtain GmPLC8-overexpressing soybean. The results demonstrated that GmPLC8 enhances drought resistance in soybean by regulating stomatal closure. This approach aimed to determine the function of the GmPLC8 and provide a theoretical basis for the discovery of candidate genes.

Results

Analysis of drought tolerance in GmPLC8-silenced soybean

To elucidate the role of GmPLC8 in soybean, a virus-induced gene silencing (VIGS) recombinant vector was constructed by selecting a specific fragment from the coding sequence (CDS) region of GmPLC8. The recombinant vector was introduced into soybean leaves via Agrobacterium-mediated infiltration. Following the confirmation of GmPLC8 silencing by quantitative RT-qPCR, plants were subjected to drought stress to assess the phenotypic and physiological responses associated with GmPLC8 knockdown (Figure S1). Under drought conditions, the GmPLC8-silenced plants (pTRV-GmPLC8) exhibited different phenotypes compared to the control (pTRV-00). Specifically, the second pair of compound leaves in the GmPLC8-silenced plants wilted more severely than those in the control group (Figure 1A).

Figure 1.

Figure 1

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Phenotypic and physiological characterizations of pTRV-GmPLC8 soybean seedlings under drought stress

(A) Phenotypic analysis of GmPLC8-silenced soybeans under drought stress. The seedlings of pTRV-00 and pTRV-GmPLC8 divided into two groups (control and drought treatment) were not irrigated for 8 days (n ≥ 20), respectively. Scale bars, 3 cm.

(B–D) The fresh weight, dry weight, relative water content were measured in pTRV-00 and pTRV-GmPLC8 after drought stress for 5 days (n = 3). The data are mean ± SD of three independent experiments. Asterisks indicate values significantly different between pTRV-00 and pTRV-GmPLC8 in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05; ∗∗, p < 0.01).

(E and F) MDA and H2O2 content of GmPLC8-silenced soybean before and after treatment (n = 3). For drought treatment, 3-week-old seedlings were grown in soil without water for 8 days. The data are mean ± SD of three independent experiments. Asterisks indicate values significantly different between pTRV-00 and pTRV-GmPLC8 in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05; ∗∗, p < 0.01).

To further investigate the reasons for phenotypic changes, we measured dry weight, fresh weight, relative water content, and MDA content of soybean leaves. The results revealed that there were no significant differences in the contents of these indices between pTRV-GmPLC8 and pTRV-00 leaves. However, the fresh weight, dry weight, and relative water content in pTRV-00 were 1.43, 1.66, and 1.87 times higher, respectively, than those in pTRV-GmPLC8 under drought stress (Figures 1B–1D). Additionally, the accumulation of MDA in pTRV-GmPLC8 was 1.38 times higher than in pTRV-00 (Figure 1E). Collectively, these findings indicate that pTRV-GmPLC8 experienced more severe damage under drought stress compared to pTRV-00.

The production of reactive oxygen species (ROS) is a common outcome of drought stress. The sustained generation of ROS can result in cellular damage and ultimately lead to cell death. To investigate the accumulation of ROS under drought conditions, we employed DAB and NBT staining to visualize ROS levels in the leaves. The results showed that the damaged area in leaves of pTRV-GmPLC8 plants was significantly larger than that in pTRV-00 plants under drought stress. However, no significant differences were observed between pTRV-GmPLC8 and pTRV-00 plants under normal growth conditions (Figure 2). Subsequently, we measured the levels of H2O2 in soybean leaves and found that the accumulation of H2O2 in pTRV-GmPLC8 was 1.39 times higher than in pTRV-00 leaves (Figure 1F). Collectively, the phenotypic changes observed in pTRV-GmPLC8 and pTRV-00 plants, along with the physiological and biochemical parameters, can be attributed to the silencing of the GmPLC8 gene. Overall, silencing of GmPLC8 reduces drought tolerance in soybean seedlings, indicating that GmPLC8 plays a positive role in soybean seedlings under drought tolerance.

Figure 2.

Figure 2

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Silencing of GmPLC8 increases the sensitivity of soybean to drought

(A) DAB staining.

(B) NBT staining of soybean seedlings subjected to drought (no irrigation) treatment for 8 days. Scale bars, 1 cm.

Analysis of drought tolerance in GmPLC8-overexpressing soybeans

To further explore the role of GmPLC8 in plant drought tolerance, we successfully obtained transgenic soybean lines with the enhanced expression of the gene. Specifically, two transgenic lines were identified, both exhibiting approximately a 4-fold increase in GmPLC8 expression compared to wild-type controls (Figure S2). The T3 generation of these transgenic lines was selected for further experimentation.

The soybeans were planted at the same depth in vermiculite and watered with a 1/2 × Hoagland nutrient solution until the second pair of compound leaves emerged. Subsequently, drought treatment was initiated by discontinuing watering. Under ample moisture conditions, both the wild-type (WT) plants and the overexpression lines (OE1 and OE2) exhibited vibrant green leaves and robust, uniform growth. After 10 days of drought stress, the WT plant showed progressive leaf yellowing and wilting from the lower to the upper parts, whereas the OE1 and OE2 lines maintained comparatively healthier growth (Figure 3A).

Figure 3.

Figure 3

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GmPLC8 enhances drought tolerance in soybean seedlings

(A) Phenotypic analysis of two overexpressed lines and WT plants after dehydration for 10 days (n ≥ 20). Scale bars, 3 cm.

(B–D) The fresh weight, dry weight, and relative water content were measured in soybean plants after drought stress for 10 days (n = 3). The data are mean ± SD of three independent experiments. Asterisks indicate values significantly different between WT and GmPLC8-OE lines in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05; ∗∗, p < 0.01).

(E and F) MDA and H2O2 content of WT and OE1/2 plants before and after treatment. For drought treatment, 3-week-old OE and WT plants were grown in soil without water for 8 days. The data are means ± SD of three independent experiments. Asterisks indicate values significantly different between WT and GmPLC8-OE lines in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05; ∗∗, p < 0.01 and ∗∗∗, p < 0.001).

Maintaining water balance within the plant is of utmost importance. To investigate the regulatory capacity of overexpressing soybean plants in response to drought stress, we measured the changes in dry weight, fresh weight, and relative water content of the soybean leaves. The results showed that, under drought stress, the fresh weight, dry weight, and relative water content of OE1 and OE2 were 1.19–1.21, 1.99–2.01 and 2.02–2.03 times higher, respectively, than those of WT (Figures 3B–3D). Lipid peroxidation occurs in plant membranes when exposed to adverse conditions. MDA is commonly used as an indicator of lipid peroxidation in cell membranes and reflects the extent of damage caused to plants by stress. Under drought stress, the accumulation of MDA in WT was 1.77 and 1.72 times higher than that in OE1 and OE2, respectively. However, under normal growth conditions, these physiological indicators did not show significant differences (Figure 3E).

Under drought stress, DAB and NBT staining revealed that the damaged area in OE1 and OE2 plants was smaller than that in WT plants (Figure 4). Consistently, the accumulation of H2O2 in OE1 and OE2 was reduced by 1.29-fold and 1.32-fold, respectively, compared to WT (Figure 3F). These observations suggest that OE1 and OE2 plants exhibit reduced accumulation of ROS. Based on the analysis of phenotype and physiological data, overexpression of GmPLC8 reduces the extent of damage in plants. These results indicate that GmPLC8-overexpressing lines (OE1 and OE2) exhibited better performance than WT under drought stress, highlighting the protective role of GmPLC8 in enhancing drought tolerance in soybean seedlings.

Figure 4.

Figure 4

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Overexpression of GmPLC8 reduces the accumulation of reactive oxygen species

(A) DAB staining.

(B) NBT staining of the WT and GmPLC8-OE lines subjected to drought (no irrigation) treatment for 10 days. Scale bars, 1 cm.

GmPLC8 regulates stomata via the abscisic acid pathway to enhance drought tolerance

When plants encounter environmental stresses, the phytohormone abscisic acid (ABA) rapidly accumulates and efficiently reduces water loss by inducing stomatal closure.27 Stomatal closure is a primary mechanism employed by plants to minimize water loss during drought periods. Under normal conditions, the degree of stomatal aperture is generally similar among plant leaves. However, when subjected to 10% PEG-induced osmotic stress simulation, the stomatal aperture of WT was 1.34-fold and 1.31-fold greater than those of OE1 and OE2, respectively (Figures 5A and 5B). Additionally, the stomatal aperture of pTRV-GmPLC8 plants was enlarged 1.71-fold larger than that of pTRV-00 plants (Figures 5D and 5E).

Figure 5.

Figure 5

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GmPLC8 regulates ABA-mediated stomatal closure to improve drought tolerance in soybean

(A) Images of epidermal stomata on leaves of GmPLC8-OE lines after 10% PEG6000 treatment observed with a bright field microscope. At least 40 stomata were observed per plant. Scale bars, 5 μm.

(B) Detection of stomatal aperture from WT and GmPLC8-OE lines under control and drought treatment (n = 10). The data are presented as mean ± SD of three independent experiments. Asterisks indicate values significantly different between GmPLC8-OE lines and wild type in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05).

(C) ABA contents of WT and GmPLC8-OE lines before and after treatment (n = 3). The data are mean ± SD of three independent experiments. Asterisks indicate values significantly different between GmPLC8-OE lines and wild type in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05).

(D) Images of GmPLC8-silenced soybean seedlings of epidermal stomata after 10% PEG6000 treatment observed with a bright field microscope. At least 40 stomata were observed in each plant. Scale bars, 5 μm.

(E) Detection of stomatal aperture from pTRV-00 and pTRV-GmPLC8 plants under control and drought treatment (n = 10). The data are presented as mean ± SD of three independent experiments. Asterisks indicate values significantly different between pTRV-00 and pTRV-GmPLC8 in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05).

(F) ABA contents of pTRV-00 and pTRV-GmPLC8 plants before and after treatment (n = 3). The data are mean ± SD of three independent experiments. Asterisks indicate values significantly different between pTRV-00 and pTRV-GmPLC8 in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05).

In response to drought stress, plants activate ABA-dependent signaling pathways to trigger the accumulation of ABA.28 ABA plays a crucial role not only in responding to drought but also in regulating stomatal movement.29 Under drought stress, we found that the ABA content in OE1 and OE2 was 1.24-fold and 1.23-fold higher, respectively, than that in WT (Figure 5C). In contrast, GmPLC8-silenced lines exhibited opposite results. Specifically, the ABA content in pTRV-GmPLC8 was 1.27-fold lower than that in pTRV-00 under drought stress (Figure 5F). These results suggest that GmPLC8 promotes stomatal closure by enhancing ABA synthesis. These findings indicate that GmPLC8 enhances ABA accumulation, thereby regulating stomatal movement.

GmPLC8 activates the abscisic acid signaling pathway in soybean

To elucidate the response mechanism of GmPLC8 in regulating the ABA signaling pathway, the expression levels of key ABA-responsive genes in soybean were measured under drought conditions. These genes include ABA biosynthesis genes (GmNCED2, GmNCED3, GmNCED5) and ABA pathway-related genes (GmOST1, GmPP2C10 and GmAAO3) (Figure 6). The results revealed that the expression levels of GmNCED2, GmNCED5, GmOST1, and GmPP2C10 were upregulated in the OE plants but downregulated in pTRV-GmPLC8 lines, indicating that these genes are potentially regulated by GmPLC8 under drought stress. In contrast, the expression levels of GmNCED3 and GmAAO3 showed a decreasing trend relative to both WT and pTRV-00, indicating that although these two genes are key genes in the ABA biosynthesis pathway, they are not responsive to regulation by GmPLC8. These findings suggest that GmPLC8 regulates the expression of specific ABA biosynthesis-related genes in stomata under drought stress conditions. This regulation enhances the responsiveness of stomata to ABA, leading to stomatal closure and reduced water loss through transpiration. Consequently, GmPLC8 improves drought tolerance in soybeans by modulating the ABA pathway.

Figure 6.

Figure 6

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The expression of ABA-responsive genes in soybean under drought stress

The data shown are mean ± SD obtained from three independent experiments. Asterisks indicate values significantly different in each group. Statistical significance was determined by ANOVA followed by Tukey’s HSD test. (∗, p < 0.05, ∗∗, p < 0.01).

Discussion

Drought stress severely impacts soybean yield, making the development of drought-resistant soybean strains a critical goal in modern agriculture.30 Virus-Induced Gene Silencing (VIGS), based on RNA interference, uses modified plant viral genomes to rapidly suppress target gene expression. This technique has become a powerful tool for analyzing gene functions across various plant species.31,32 More than 30 VIGS vectors have been developed for dicotyledonous plants, with the TRV vector being particularly effective due to its simplicity, cost-efficiency, and high silencing efficiency in multiple tissues.33,34 VIGS has successfully elucidated the functions of several soybean genes, including GmHXK2,35 GmERA1A and GmERA1B36 and has been used to improve agronomic traits such as yield, grain quality, and stress tolerance.37

In our study, we successfully generated GmPLC8-silenced soybean seedlings using VIGS and overexpressed GmPLC8 in soybeans via Agrobacterium-mediated transformation for pot experiments.38 Adequate water balance is crucial for plant survival, especially under adverse conditions, as it directly affects vital physiological processes.39 Malondialdehyde (MDA), a byproduct of lipid peroxidation, serves as an indicator of cellular damage.40 Additionally, reactive oxygen species (ROS), which are generated as byproducts of plant metabolism, can cause significant harm to plants when accumulated excessively.41 Under drought stress, their levels were lower in GmPLC8-OE lines. Consequently, GmPLC8-OE lines experienced less cellular damage, maintained better physiological status, and accumulated greater fresh and dry biomass. These results are consistent with previous findings in which the overexpression of TaLEA3 leads to reduced MDA levels under stress conditions.42 These findings highlight GmPLC8 as a potential target for improving drought tolerance in soybean seedlings.

When roots detect drought conditions, they transmit specific signals to the above-ground parts to trigger stomatal closure. This process helps regulate crop water usage through transpiration.43 Stomatal movement is largely controlled by the plant hormone ABA. Under drought conditions, ABA acts as a key regulator to mitigate water loss caused by transpiration by controlling stomatal closure.44 AtPLC3 exhibits sensitivity to ABA during seed germination and stomatal closure.25 Both AtPLC3 and GmPLC8 are involved in regulating stomatal aperture, while AtPLC3 in Arabidopsis also affects root development and seed germination. These findings suggest that the functions of PLC may be conserved. However, certain differences also exist between different species. Overall, these studies highlight the potential of PLC as a target for improving stress tolerance in crops. Previous research has demonstrated that drought stress induces ABA accumulation by activating ABA-dependent signaling pathways. The concentration of endogenous ABA gradually increases within approximately 2.5–6 h after plants experience water deficiency.45

During drought conditions, stomatal closure is the primary mechanism by which plants minimize water loss.46 This process is initiated by the synthesis of ABA within guard cells.47 Abscisic acid (ABA) is a stress hormone whose typical effect on leaves is to reduce water loss from transpiration by inducing stomatal closure.48 We found that GmPLC8 enhances drought tolerance in soybean seedlings by promoting ABA accumulation and reducing stomatal aperture, which is consistent with previous findings in rice, where OsWNK9 overexpression promoted ABA accumulation and stomatal closure under salt stress.49 Key enzymes involved in ABA biosynthesis include 9-cis-epoxycarotenoid dioxygenase (NCED) and ABA aldehyde oxidase (AAO3). NCED catalyzes a critical step in ABA biosynthesis and is essential for ABA-mediated responses.50 For example, silencing SlNCED2 in tomatoes reduces ABA content.51 AAO3, another key enzyme, facilitates the final step of ABA synthesis.52 Transient expression of AAO3 in Vicia faba guard cells induces stomatal closure.53 The protein kinase OST1 (SnRK2.6) is a pivotal component of ABA signaling in guard cells. The stomatal closure response in OST1 mutants is significantly impaired under ABA treatment and various environmental stimuli.54 In Arabidopsis, OST1 is strongly activated by ABA.55 The SnRK2 and PP2C-A families are essential components of the ABA signaling pathway and play significant roles in enhancing plant drought tolerance.56 In the study, our results indicated that GmPLC8 induces the expression of ABA biosynthesis genes (GmNCED2 and GmNCED5) and ABA signaling genes (GmOST1 and GmPP2C10). These findings suggest that GmPLC8 regulates stomatal closure through the ABA pathway, thereby reducing transpiration and enhancing drought tolerance in soybean seedlings.

Conclusions

In this study, GmPLC8-OE lines and GmPLC8-silenced lines were subjected to drought stress. Combined phenotypic and physiological analyses revealed that GmPLC8 enhances drought tolerance in soybean seedlings. Furthermore, our results demonstrate that GmPLC8 improves drought resistance by modulating stomatal behavior through the abscisic acid (ABA) signaling pathway.

Limitations of the study

This study demonstrated that GmPLC8 enhances drought tolerance in soybean seedlings by regulating ABA accumulation and stomatal closure. However, additional field data, such as pod filling, seed weight, plant height and yield are still needed to further substantiate the role of GmPLC8 in soybean drought tolerance.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Fawei Wang (fw-1980@163.com).

Materials availability

Materials generated in this study are available upon request. For further details contact the lead contact.

Data and code availability

  • •

    All data reported in this article could be shared by the lead contact upon request.

  • •

    This article does not report original code.

  • •

    Accession numbers are listed in the key resources table.

Acknowledgments

We acknowledge all the members of the research group for their helpful comments and inspiration.

Our graphical abstract was created with BioRender.com.

Funding: This work was supported by the Department of Science and Technology of Jilin Province (20250601053RC).

Author contributions

D.W. and D.Q.S. conceived and designed the research. D.W., X.H., and F.Y.H. conducted experiments. D.W., Y.H.L., and M.Y.Y. contributed to the analysis and interpretation of data. X.W.L., Y.Y.D., N.W., W.C.L., and F.W.W. participated in the revision of the article.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER
Bacterial and virus strains
DH5α Chemically Competent Cell Sangon Biotech B528413
EHA105 Chemically Competent Cell Coolaber CC403
GV3101(pSoup-p19) Chemically Competent Cell Coolaber CC407
Chemicals, peptides, and recombinant proteins
EasyScripte One-Step gDNA Removal and cDNA Synthesis SuperMix TransGen AE311-03
PerfectStart® Green qPCR SuperMix (+Universal Passive Reference Dye) TransGen A602-02
DAB Horseradish Peroxidase Color Development Kit Beyotime P0203
Hydrogen Peroxide assay kit Nanjing Jiancheng A064-2-1
BCIP/NBT Alkaline Phosphatase Color Development Kit Beyotime C3206
PEG 6000 Solarbio 25322-68-3
Plant hormone abscisic acid (ABA) ELISA Kit instruction SINOBES TBIO YX-010201P
Experimental models: Organisms/strains
Glycine max cultivar Dongnong 50 College of Life Science, Jilin Agricultural University –
Oligonucleotides
Primers used are shown in Table S1 This Paper –
Recombinant DNA
Plasmid:pCAMBIA3301-GmPLC8 This Paper –
Plasmid:pTRV2-GmPLC8 This Paper –
Software and algorithms
GraphPad Prism6 Open source https://www.graphpad.com; RRID:SCR_002798
ImageJ Open source https://ImageJ.nih.gov/ij/; RRID:SCR_003070
BioRender Open source https://BioRender.com
Deposited data
GmPLC8 (Glyma.14G059200) GenBank GenBank: NM_001248381.2
GmNCED2 (Glyma.08G096200) GenBank GenBank: NM_001254322.2
GmNCED3 (Glyma.08G176300) GenBank GenBank: XM_041018233.1
GmNCED5 (Glyma.05G140900) GenBank GenBank: NM_001254687.2
GmAAO3 (Glyma.14G045100) GenBank GenBank: XM_006595754.4
GmOST1 (Glyma.12G169800) GenBank GenBank: XM_003540111.5
GmPP2C10 (Glyma.13G106800) GenBank GenBank: XM_041008005.1
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Method details

Plant materials of GmPLC8-overexpressing plants and drought treatment

For stable genetic transformation of soybean, ‘Dongnong 50’ was selected as the experimental material. The CDS region of GmPLC8 was recombined with the pCAMBIA3301 vector through homologous recombination, and GmPLC8 overexpressing (OE) soybeans were obtained via Agrobacterium-mediated genetic transformation.

There is a method to detect drought stress tolerance, with only a small adjustment.57 Soybean seedlings grown in pots containing vermiculite at 25°C, 50% relative humidity, photoperiod (16 h day/8 h night) for 3 weeks. We subjected the soybean to drought stress for 10 days. These plant materials are used for RNA extraction qRT-PCR and physiological indicators.

Construction of VIGS vectors and drought stress for silenced plants

The procedure refers to that described by Chen with minor modifications.35 The specific fragments from CDS of GmPLC8 was amplified using primers. The primers are listed in Table S1. The products were ligated to obtain pTRV-GmPLC8 vectors. For VIGS experiment, plasmids of pTRV1, pTRV2 and pTRV-GmPLC8 vectors were transformed into A. tumefaciens strain GV3101 cells by using the freeze-thaw method. The empty plasmid pTRV2 and pTRV1 was used as control (pTRV-00). The Agrobacterium strains were inoculated into 40 mL of YEP medium as above on a shaker at 180 rpm at 28°C for 20 h to an OD600 of 1.0-1.2. Merged the bacterial solution containing pTRV1 and pTRV2 plasmids in equal volume, and then mixed pTRV1 and pTRV-GmPLC8. The Agrobacterium cells were centrifuged at 4000 rpm for 10 min at room temperature, resuspended with the infiltration solutioto a final OD600 of 1.0 and placed at room temperature in darkness for 2 h. Then the second pair of clovers of soybean were injected. After 12 hous of recovery, the seedlings were subjected to drought treatment.

DAB (3,3′-diaminobenzidine) and NBT (nitroblue tetrazolium) staining

The soybean leaves of the treatment group and the control group were stained respectively. For DAB staining, the leaves were immersed in DAB reagents (Beyotime®, China) for 14 hours, then it was placed in 90% ethanol for decolorization. For NBT staining, the leaves were soaked in NBT reagent (Beyotime®, China) for 12 hours and decolorized by 90% ethanol.

Measurement of physiological and biochemical parameters

H2O2 quantification was done with the Hydrogen Peroxide assay kit (Nanjing Jiancheng, Nanjing, China). The MDA level obtained by thiobarbituric acid reaction. The fresh weight, dry weight, and relative water content of soybean leaves were detected according to the method described by Chen et al.13

Determination of ABA content and stomatal aperture

The Plant hormone abscisic acid (ABA) ELISA Kit instruction (YX-010201P) was used to detect the content of ABA in plant samples.

Some modifications have been made to the stomatal movement analysis.58 3-week-old seedlings cultured in 1/2 Hoagland solution were treated 4 h with 10% (w/v) polyethylene glycol [PEG]6000. The determination of stomatal aperture was performed as reported by Xu et al. with a slight modification.39 Stomatal aperture width and length were measured by the open access software ImageJ (v1.37, https://imagej.nih.gov/ij/). Each experiment included at least three biological replicates, with no fewer than 60 guard cells that were measured per each sample.

RNA purification and RT-qPCR analysis

Total RNA of soybean extracted with Trizol reagent(Invitrogen™, USA). The concentration, purity, and integrity of RNA were analyzed by NanoDrop 2000c spectrophotometer (Thermo Scientific™, USA). Using 1μg total RNA as a template, 4 μL 5×TransScript®Uni All-in-One SuperMix for qPCR, 1μL gDNA Remover RNase-free Water (Trans®) to synthesis cDNA. Quantitative RT-qPCR was conduct to examine the transcription of the gene. As report by Zhou et al.59 Each sample is analyzed in triplicate, and the expression levels are calculated using the 2-ΔΔCt method. Three independent experiments were performed. The qPCR primers are listed in Table S1.

Quantification and statistical analysis

The statistical analysis was performed by ANOVA followed by Tukey’s HSD test and a p value < 0.05 was considered statistically significant.

Published: December 26, 2025

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2025.114546.

Supplemental information

Document S1. Figures S1 and S2 and Table S1
mmc1.pdf (260.2KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1 and S2 and Table S1
mmc1.pdf (260.2KB, pdf)

Data Availability Statement

  • •

    All data reported in this article could be shared by the lead contact upon request.

  • •

    This article does not report original code.

  • •

    Accession numbers are listed in the key resources table.

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