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Article

GsCYP93D1, a Cytochrome P450 Gene from Wild Soybean, Mediates the Regulation of Plant Alkaline Tolerance and ABA Sensitivity

1
Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500, China
2
Department of Chemistry and Molecular Biology, School of Life Science and Technology, Harbin Normal University, Harbin 150025, China
3
Key Laboratory of Saline-Alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Sciences, Northeast Forestry University, Harbin 150040, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(17), 2623; https://doi.org/10.3390/plants14172623
Submission received: 16 July 2025 / Revised: 15 August 2025 / Accepted: 20 August 2025 / Published: 23 August 2025

Abstract

Cytochrome P450 enzymes (CYPs) are crucial catalysts responsible for the oxidative modification of diverse substrates, including plant hormones, antioxidants, and compounds involved in abiotic stress responses. While CYP functions in drought and salt stress adaptation have been extensively studied, their contribution to alkaline stress tolerance, particularly concerning specific cytochrome P450 genes in wild soybean (Glycine soja), remains less explored. In this study, a cytochrome P450 gene, GsCYP93D1, was identified and isolated, and its regulatory role under alkaline stress was elucidated. Transgenic GsCYP93D1 increased Arabidopsis and soybean hairy root resistance to alkaline stress, but the Arabidopsis atcyp93d1 mutant showed a reduced capacity for alkaline tolerance. Subsequent investigation showed the enhanced antioxidant defense capabilities of GsCYP93D1 transgenic plants, as evidenced by reduced superoxide radical (O2) production under exposure to alkaline stress. Furthermore, compared to the atcyp93d1 mutant, transgenic lines of GsCYP93D1 showed sensitivity to ABA. Moreover, transcript levels of genes associated with alkaline stress response and ABA signaling pathways were elevated in both GsCYP93D1 transgenic and mutant lines. Collectively, our findings demonstrate that GsCYP93D1 positively modulates plant tolerance to alkaline stress and enhances ABA sensitivity.

1. Introduction

Plants are frequently subjected to various biotic and abiotic environmental stresses. Alkaline stress pose serious threat to plant growth and agricultural productivity [1]. Alkaline stress imposes greater damage on plants than salt stress [2,3]. In addition to ionic toxicity and osmotic stress, high pH levels severely disrupt cellular pH homeostasis, damage cell membrane structure, weaken root activity and photosynthetic efficiency under alkaline stress [1,4,5,6].
Under alkaline stress, reactive oxygen species (ROS) levels increase within plants, which can cause oxidative damage, disrupt membrane systems, and even lead to plant death in severe cases [1,7]. To counteract the toxicity of excessive ROS, plants have evolved a complex antioxidant system. This system includes antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), as well as non-enzymatic antioxidants like ascorbic acid (ASA) and glutathione [8,9]. Recent years have seen significant progress in understanding genetic regulators involved in ion homeostasis and ROS scavenging under saline–alkaline conditions [10,11]. Plants can also enhance stress tolerance by increasing the synthesis of endogenous hormones, such as abscisic acid and jasmonic acid (JA) [12]. ABA plays a critical role in plant responses to abiotic stress. Under alkaline conditions, high levels of endogenous or exogenous ABA can activate the antioxidant defense system, reducing ROS accumulation and alleviating oxidative damage [13]. For example, silencing the gene OsABA8ox1 elevates endogenous ABA levels in rice, thereby enhancing its alkaline tolerance [14].
Cytochrome P450s (CYPs) are a class of oxidoreductases utilizing heme as a cofactor. CYPs mediate NADPH-dependent oxidation reactions and play a fundamental role in both primary and secondary metabolic pathways across diverse plant species [15]. CYPs exert pivotal functions within metabolic networks by catalyzing the oxidative modification of structurally diverse substrates, including fatty acid derivatives, plant hormones, specialized defense compounds, the biopolymer lignin and protective phytoalexins [16,17]. CYPs have also been found to play a role in hormone signaling, ROS homeostasis, and controlling how plants react to different types of stress [18,19]. For example, overexpression of the CYP709B3 gene enhanced salt tolerance in transgenic Arabidopsis as indicated by cyp709b3 low resistance levels with a higher damaged seedling percentage [20]. The expression of genes from the CYP709 family was significantly upregulated in black locust (Robinia pseudoacacia) under salt stress [21]. CYP71 gene expression was activated in ginseng following exposure to nickel and cadmium [22]. During metal stress adaptation, maize plants use CYP88A-mediated gibberellin production to synchronize connections between phytohormone signaling pathways [23]. In conclusion, how CYP-mediated hormone synthesis integrates with stress signaling (such as ABA under alkaline condition) is not fully understood yet. Therefore, there is a need to investigate how CYP-regulated hormones fine-tune antioxidant defense under alkaline stress.
Wild soybean, the wild progenitor of cultivated soybean (Glycine max), exhibits strong adaptability to saline–alkali stress [24]. In our previous studies, a highly adaptable alkaline-tolerant wild soybean line was identified [25]. By using transcriptome data, we identified a candidate cytochrome P450 homolog GsCYP93D1 in response to alkaline stress. In this study, the GsCYP93D1 gene was isolated in wild soybeans, and we detected the expression profiles under alkaline stress. We further confirmed the positive roles of GsCYP93D1 in response to alkaline stress in Arabidopsis and soybean hairy roots by promoting reactive oxygen species scavenging. Moreover, GsCYP93D1 displayed a sensitive role in response to exogenous ABA treatment. In total, our results suggested that GsCYP93D1 played significant regulatory roles in plant responses to alkaline stress and ABA signal transduction.

2. Results

2.1. Spatial and Temporal Expression Patterns of GsCYP93D1 in Wild Soybean

To investigate the spatial expression dynamics of the GsCYP93D1 gene in wild soybeans, their transcript levels were analyzed across different tissues (young roots, young stems, young leaves, mature roots, mature leaves, mature stems, pods, and flowers) using quantitative real-time PCR (qRT-PCR). The results revealed that the GsCYP93D1 gene showed the highest expression in mature roots, followed by young roots. Expression levels were also comparatively higher in pods, exhibiting moderate abundance, whereas other tissues displayed only minimal expression (Figure 1A). These results suggest that GsCYP93D1 may play a primary role in root-associated functions and a secondary role in pod development in wild soybeans.
To further characterize the response of GsCYP93D1 to alkaline stress, its temporal expression dynamics were analyzed in wild soybean roots and leaves at different time points. The results showed that alkaline stress induced higher transcriptional levels of GsCYP93D1 in leaves compared to roots, particularly during the early stress phase (Figure 1B,C). In leaves, expression peaked at 6 h with a 4-fold increase, followed by a 2-fold elevation at 3 h. In roots, maximal induction (3-fold) occurred later at 24 h, with a secondary peak of 1.5-fold at 6 h. Collectively, the expression patterns demonstrated that GsCYP93D1 expression peaks earlier in leaves than in roots under alkaline stress, indicating distinct temporal regulation between the two tissues.

2.2. GsCYP93D1 Enhanced Alkaline Tolerance in Arabidopsis

To investigate the role of the GsCYP93D1 gene in Arabidopsis, the wild-type (WT), the GsCYP93D1 overexpression lines, OE4 and OE5, and the atcyp93d1 mutant were analyzed. The results showed that all lines exhibited similar growth phenotypes under control conditions (Figure 2A). However, under treatment with 0.6 or 0.8 mmol/L NaHCO3, the transgenic lines GsCYP93D1 OE4 and OE5 exhibited significantly greater stress resistance compared to that in the WT, as evidenced by longer root lengths and increased fresh weights. In contrast, the atcyp93d1 line displayed increased sensitivity (Figure 2B,C). Under alkali stress, chlorophyll content of the transgenic lines OE4 and OE5 was higher than that of WT and atcyp93d1, while malondialdehyde (MDA) content was significantly lower than that of WT and atcyp93d1 (Figure 2D,E). MDA content was related to the degree of cell membrane damage, indicating that plants adapt to the external environment by adjusting MDA content, reducing the adverse effects of alkali stress on plants. Overall, these findings support that GsCYP93D1 is a positive regulator of alkaline stress tolerance, functioning through enhancement of root growth, oxidative stress mitigation, and maintenance of photosynthetic capacity.

2.3. Modulation of Redox Homeostasis by GsCYP93D1 Confers Stress Resilience

Under external abiotic stress, plants experience a significant accumulation of ROS, including superoxide anion radicals (O2), which induce structural and functional damage to cellular components. To monitor O2 dynamics, we used a nitroblue tetrazolium (NBT) staining histochemical assay that relies on O2-mediated reduction of NBT to insoluble blue formazan precipitates (Figure 3A). Under untreated control conditions, all genotypes displayed a similar baseline staining intensity. However, under 0.6 mmol/L NaHCO3, WT plants displayed visible light blue pigmentation indicating O2 accumulation. The overexpression line of GsCYP93D1 demonstrated significantly reduced staining intensity and limited lesion expansion relative to WT, whereas atcyp93d1 loss-of-function mutants displayed intensified dark blue staining. Under 0.8 mmol/L NaHCO3, all leaf samples displayed darkened to deep blue staining, but transgenic plants still exhibited a lighter staining intensity with unstained regions remaining visible, whereas mutant plants displayed the darkest staining, with nearly all tissues turning blue. These results collectively demonstrate that GsCYP93D1 overexpression enhances the superoxide scavenging capacity in Arabidopsis under alkaline stress, while the hypersensitive phenotype of atcyp93d1 mutants confirms the enzyme’s endogenous role in ROS homeostasis.
To further validate the NBT staining results, the content of superoxide anion in alkali-stressed Arabidopsis was measured (Figure 3B). The results indicated that the content of O2 in all lines was almost the same as that in control plants. Under 0.6 mmol/L NaHCO3 treatment, the content of O2 in each line was significantly increased. Compared with the WT, the O2 accumulation in GsCYP93D1 transgenic lines was significantly lower than that in WT, while the content of O2 in atcyp93d1 was even higher than that in the WT. O2 was significantly increased under 0.8 mmol/L NaHCO3 treatment, while similar to the situation at 0.6 mmol/L NaHCO3 treatment, the content of O2 in transgenic Arabidopsis was lower, and the content of O2 in the mutant was higher.
To further investigate the role of GsCYP93D1 in oxidative stress regulation under alkaline conditions, the enzymatic activities of CAT, SOD, and POD were quantified. As shown in Figure 3C–E, no significant differences in antioxidant enzyme activity were observed among the transgenic lines, WT, or the atcyp93d1 mutant under non-stress conditions. However, following exposure to alkaline stress, the transgenic lines exhibited a marked and statistically significant increase in CAT, SOD, and POD activities. In comparison, the WT plants showed a moderate induction of enzyme activity, while the atcyp93d1 mutant displayed the lowest levels of induction. These findings suggest that GsCYP93D1 enhances the antioxidant defense system in plants, thereby mitigating cellular damage and reducing the detrimental effects of alkaline stress. These results indicate that GsCYP93D1 contributes to the maintenance of ROS homeostasis under alkaline stress, likely by modulating antioxidant pathways that limit O2 accumulation and tissue damage. The contrasting phenotypes between overexpression and mutant lines further support the notion that GsCYP93D1 functions as a positive regulator of oxidative stress tolerance in plants facing alkaline conditions.

2.4. GsCYP93D1 Regulated the Expression Levels of Stress Responsive Genes

To further elucidate the role of GsCYP93D1 in alkaline stress response, six stress-responsive marker genes, COR15A, COR47, RD29A, KIN1, H+-ATPase, and NADP-ME, were selected for expression analysis. Transcript levels were quantified using qRT-PCR in Arabidopsis plants treated with 50 mmol/L NaHCO3 for 0, 3, and 6 h (Figure 4A–F). In GsCYP93D1 transgenic lines, all six marker genes exhibited elevated expression levels following alkaline stress. Specifically, COR15A, RD29A, and KIN1 reached peak expression at 3 h post-treatment, while COR47, H+-ATPase, and NADP-ME displayed a progressive increase, with maximal expression observed at 6 h.
In contrast, the atcyp93d1 mutant showed overall lower transcript levels of the marker genes compared to the WT. Additionally, COR15A, COR47, and KIN1 in the mutant exhibited a transient induction at 3 h, followed by a decline at 6 h, suggesting a compromised and less sustained transcriptional response to alkaline stress. These results collectively suggest that GsCYP93D1 positively regulates the expression of key stress-related genes, contributing to an enhanced and sustained defense response under alkaline conditions.

2.5. GsCYP93D1 Enhanced Alkaline Tolerance in Hairy Roots of Soybean

To further assess the role of GsCYP93D1 in conferring alkaline stress tolerance in soybean, we obtained transgenic soybean hairy roots of GsCYP93D1. The transgenic and control hairy roots (K599 Agrobacterium rhizogenes) were subjected to either 0 mmol/L (control) or 60 mmol/L NaHCO3 treatment. Under non-stress conditions, no significant differences in root growth were observed between the transgenic lines and the K599 control. However, following alkaline stress treatment, all hairy roots exhibited growth inhibition to varying degrees. Notably, GsCYP93D1 transgenic hairy roots displayed significantly longer root lengths and greater biomass compared to the control, indicating a reduced sensitivity to alkali stress (Figure 5B,C). These findings are consistent with the stress-resilient phenotype observed in GsCYP93D1 transgenic Arabidopsis lines, collectively supporting the conclusion that GsCYP93D1 enhances tolerance to alkaline stress by promoting root growth and reducing growth inhibition under adverse conditions.
To further validate the alkali stress tolerance of GsCYP93D1 transgenic soybean hairy roots, we measured MDA content and activities of antioxidant enzymes (Figure 5D). Under non-stress conditions, no significant differences were observed between transgenic hairy roots and K599. Under alkali stress treatment, MDA accumulation in GsCYP93D1 transgenic hairy roots was significantly lower than in K599. The activities of the three antioxidant enzymes (CAT, SOD, and POD) in transgenic hairy roots increased significantly with stress intensity, whereas K599 hairy roots exhibited much smaller increases in enzyme activity (Figure 5E–G). These findings align with previous physiological index data from GsCYP93D1 transgenic Arabidopsis, confirming that GsCYP93D1 enhances antioxidant enzyme activity, alleviates oxidative stress impacts, reduces cellular damage, and thereby improves plant tolerance to alkali stress.

2.6. GsCYP93D1 Enhanced ABA Sensitivity in Arabidopsis

To investigate whether GsCYP93D1 is involved in abscisic-acid-related signaling pathways, we detected the roles of GsCYP93D1 exposed to exogenous ABA (Figure 6A). Under control conditions, all lines exhibited similar growth phenotypes. However, at 0.3 μmol/L ABA, GsCYP93D1 transgenic plants displayed more severe growth attenuation and stronger inhibition compared to WT, whereas atcyp93d1 mutants exhibited enhanced ABA resistance. When the ABA concentration increased to 0.5 μmol/L, transgenic plants showed greater sensitivity and intolerance to ABA than did WT, with consistent trends in phenotype, root length, and fresh weight (Figure 6B,C). In contrast, atcyp93d1 mutants displayed stronger resistance to ABA under these conditions. These results indicate that GsCYP93D1 positively regulates plant sensitivity to ABA, potentially functioning as a downstream component or modulator of ABA signaling. The hypersensitivity of transgenic lines to ABA, coupled with the ABA resistance of mutants, supports a role for GsCYP93D1 in amplifying ABA-mediated growth inhibition, possibly contributing to fine-tuning plant stress responses under adverse conditions such as alkalinity.

2.7. GsCYP93D1 Altered Expression Patterns of ABA Signal-Related Genes

To further analyze the role of GsCYP93D1 in the ABA signal transduction pathway, the ABA signal transduction pathway genes ABI1, ABI2, ABI4, ABI5, ABF2, and RAS1 were selected, and the expression levels of these genes were detected by qRT-PCR (Figure 7A–F). The results showed that the relative expression levels of ABI1, ABI2, ABI5, and RAS1 in transgenic plants were much lower than those in WT, except for ABI1 at 3 h. The levels of ABI4 and ABF2 in transgenic Arabidopsis were higher than those in WT, and the expression levels of ABI4 and ABF2 were extremely significant. The relative expression levels of ABI1, ABI2, ABI5, and RAS1 in atcyp93d1 were significantly higher than those in WT, and the differences of ABI1 at 3 h were not significant. In total, we speculated that GsCYP93D1 might suppress ABA sensitivity by regulating the expression of the ABA-signaling-related genes.

3. Discussion

Soil alkalinization is one of the major environmental factors affecting plant growth, development, and reducing global crop yield. Cytochrome P450 is one of the largest enzyme protein families. In plants, cytochrome P450 families are involved in the biosynthesis of plant hormones, the formation of secondary metabolites, and the response to external environmental stresses [26,27]. Studies have demonstrated that the cytochrome P450 gene can confer salt tolerance on plants [28]. However, the role of the cytochrome P450 genes in wild soybean under alkaline stress remains unclear. In this study, the crucial role of the GsCYP93D1 gene was detected in plant alkaline stress response and ABA signal transduction. GsCYP93D1 was found to be upregulated under alkaline stress; especially, the GsCYP93D1 gene showed predominant expression both in mature roots and seedling roots (Figure 1), aligning with prior studies showing the significant induction and sustained high expression of GsCYP82C4 under alkaline stress [29].
In the present study, GsCYP93D1 Arabidopsis lines demonstrated significantly enhanced root growth and biomass accumulation compared to WT plants under alkaline stress, particularly during the seedling stage. In contrast, atcyp93d1 mutant lines exhibited a hypersensitive phenotype, characterized by markedly shorter root lengths and reduced fresh biomass, indicating a compromised ability to cope with alkaline conditions (Figure 2A–C). Moreover, transgenic plants maintained higher chlorophyll content and exhibited lower malondialdehyde (MDA) levels than both WT and mutant plants (Figure 2D,E), suggesting that GsCYP93D1 contributes to the maintenance of photosynthetic efficiency and protection against lipid peroxidation. Similarly, overexpression of LrCYP78A5 was found to induce intensified leaf pigmentation and higher chlorophyll concentrations in Lycium, suggesting a potential role of this gene in promoting photosynthetic efficiency [27].
Alkaline-stress-induced root damage in rice is associated with excessive reactive oxygen species generation [30]. The above findings support the role of GsCYP93D1 as a positive regulator of alkaline stress tolerance, likely through enhancing root system architecture, stabilizing photosynthetic machinery, and reducing oxidative-stress-induced cellular damage. It is becoming more acknowledged that cytochrome P450 genes have a role in abiotic stress responses, specifically via influencing secondary metabolism, hormone crosstalk, and antioxidant defenses [26]. Thus, GsCYP93D1 may act as a key component in coordinating stress-responsive pathways, especially those related to ROS homeostasis and photosynthetic stability, providing new insights into the molecular mechanisms of plant adaptation to alkaline soil.
In upland cotton (Gossypium hirsutum), the cytochrome P450 gene enhances the plant’s antioxidant defense mechanisms by modulating the activity or synthesis of antioxidant enzymes [31]. Our findings are in line with this notion, demonstrating that overexpression of GsCYP93D1 elevates the antioxidant defense in wild soybean by enhancing the activities of the POD, SOD, and CAT antioxidant enzymes (Figure 3C–E). Further research has revealed that the GsCYP93D1 gene plays a role in plant cell membrane damage under alkaline stress. The degree of injury in the leaves of transgenic and mutant Arabidopsis seedlings was observed using the NBT staining method. Compared to the WT, the mutant had higher O2 levels, while transgenic Arabidopsis had relatively lower O2 levels. This is attributed to the capabilities of GsCYP93D1 to scavenge the excessive reactive oxygen species produced under alkaline stress, thereby alleviating cellular damage (Figure 3A,B). The CBRLK gene mediates ROS homeostasis in wild soybean under alkaline stress by dynamically regulating the expression of oxidative-stress-responsive genes [32]. In addition, studies also have confirmed that the wheat gene TaCYP81D5 can enhance the activity of antioxidant enzymes by accelerating the clearance of ROS, thereby increasing the salt tolerance of wheat [19].
Previous studies have proved that H+-ATPase and NADP-ME confer salt and alkaline tolerance [33,34]. A number of studies have also showed that COR15A, COR47, RD29A, and KIN1 are involved in various stresses, such as salt or drought stress [35,36]. To elucidate the molecular functional basis of GsCYP93D1 in the alkaline stress response, we analyzed the expression of these stress-responsive marker genes in transgenic Arabidopsis plants (Figure 4). qRT-PCR revealed that GsCYP93D1 overexpression significantly upregulated the expression of stress-related genes, including COR15A, COR47, RD29A, KIN1, H+-ATPase, and NADP-ME under alkaline stress. Conversely, the atcyp93d1 mutant exhibited downregulation of these genes, further supporting the role of GsCYP93D1 in enhancing alkaline stress tolerance. Consistently, studies have demonstrated that overexpression of the wild soybean gene SKP21 in Arabidopsis leads to substantial upregulation of these marker genes, contributing to enhanced alkali tolerance [37].
Agrobacterium-rhizogene-mediated genetic transformation is an effective way to obtain transgenic plants. Hairy roots of soybean transformed with GmEF8 show tolerance to drought stress [38]; hairy roots transformed with the GmPKS4 gene exhibit strong resistance to salt and alkaline stress [39]. In this study, the function of transgenic GsCYP93D1 soybean hairy roots was verified under alkaline stress. Compared with the non-transgenic K599 hairy roots, over-expressing the GsCYP93D1 gene under alkaline stress could alleviate the growth inhibition of soybeans by increasing root length, fresh biomass, and antioxidant enzyme activity (Figure 5).
ABA is a crucial phytohormone that plays a pivotal role in regulating plant growth, development, and adaptive responses to environmental stresses [12,40]. Emerging evidence indicates that cytochrome P450 (CYP) family genes participate in the ABA signaling pathway, thereby enhancing plant tolerance to abiotic stresses, including alkali and drought [13,41]. Our findings indicate that GsCYP93D1 displays enhanced sensitivity to ABA, as evidenced by pronounced growth inhibition, diminished root elongation, and reduced fresh biomass relative to the atcyp93d1 mutant (Figure 6). The ABA signaling pathway in plants may be affected by alkaline stress [42]. Previous studies have demonstrated that GsSKP21 negatively modulates the ABA signaling pathway through upregulation of ABI1 and ABI2, consequently decreasing plant sensitivity to ABA while improving alkali stress tolerance [37]. Conversely, AtMYB44 enhances ABA sensitivity and abiotic stress resistance by suppressing ABI1/ABI2 expression, thereby alleviating their inhibitory effect on ABA signaling [43]. Our findings indicate that GsCYP93D1 displays enhanced sensitivity to ABA, as evidenced by pronounced growth inhibition, diminished root elongation, and reduced fresh biomass relative to the atcyp93d1 mutant (Figure 7), resulting in reduced ABA stress tolerance but enhanced alkaline stress resistance in plants.
Taken together, the positive roles of GsCYP93D1 in response to alkaline stress were confirmed by promoting reactive oxygen species scavenging, and GsCYP93D1 displayed a sensitive role in response to exogenous ABA treatment. Further investigations should specify the roles of the GsCYP93D1 protein under alkaline stress, as well as the role of GsCYP93D1 in the crosstalk of ABA and alkaline-mediated signaling systems. Secondly, metabolomic profiling of overexpression and mutant lines under stress conditions could help identify downstream metabolic products regulated by GsCYP93D1, thereby clarifying its biochemical function. Further, to assess its translational potential, GsCYP93D1 overexpression lines should be tested under field conditions with naturally occurring alkaline soils. This will validate the gene’s function beyond controlled environments and inform its application in agricultural stress management strategies.

4. Materials and Methods

4.1. Materials and Growing Conditions

Wild soybean (G07256) seeds were sterilized in a 6% NaClO solution for 10 min, followed by rinsing 4–5 times with sterile water. Seeds were then placed on moist filter paper and germinated in the dark at 26 °C for 3 days. When the radicles of the seedlings reached approximately 1–2 cm in length, seedlings were transplanted into hydroponic containers filled with a 1/4-strength Hoagland nutrient solution. Seedlings were secured using aerated cotton, ensuring roots were submerged in the solution. The containers were placed in an artificial climate greenhouse set to the following conditions: 28 °C/23 °C day/night temperature, a 16 h light/8 h dark photoperiod, and relative humidity maintained at 65–75%. The young roots, young stems, young leaves, mature roots, mature stems, mature leaves, pods, and flowers were collected for tissue-specific localization analyses of GsCYP93D1 gene expression. Additionally, three-week-old seedlings were subjected to stress treatment with 50 mM NaHCO3 [44]. Leaf and root samples were collected at 0, 1, 3, 6, 12, and 24 h after the initiation of stress for dynamic expression analysis of GsCYP93D1. Specific primers were designed based on the complete CDS sequence of the GsCYP93D1 gene obtained from the Phytozome database. The full-length cDNA sequence was amplified and obtained using RT-PCR.

4.2. Analysis of Tissue Localization and Expression Characteristics Under Alkaline Stress

Total RNA was isolated from various tissues of wild soybean, as well as from leaf and root samples collected at different time points after treatment with 50 mM NaHCO3 stress, using a Plant Total RNA Isolation Kit (Foregene). The total RNA was reverse-transcribed into cDNA. The expression level of the GsCYP93D1 gene was detected using qRT-PCR, with GsGAPDH serving as the reference gene. The specific primer sequences used in this study are listed in Supplementary Table S1. Three biological replicates are prepared for each sample, with each biological replicate subjected to three technical replicates during qRT-PCR analysis. The 2−ΔΔCt method was used to analyze the results of the gene transcripts. Statistical analysis of the data was performed using SPSS 20.0 software.

4.3. Generation of GsCYP93D1 Transgenic Plants

The full-length cDNA of GsCYP93D1 was amplified from wild soybean leaves. The cDNA fragment was cloned into the pCAMBIA1300 vector using restriction enzymes to construct the recombinant vector. The recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101 for Arabidopsis genetic transformation (primer sequences GsCYP93D1-1300-F and GsCYP93D1-1300-R are listed in Table S1). Arabidopsis transformation was performed using the floral dip method, following the protocol as described previously [45]. Two overexpression lines, OE4 and OE5, were selected for phenotypic experiments (Figure S1A). Homozygous T-DNA insertion mutant seeds of cyp93d1 (WiscDsLoxHs035_01E) were purchased from Fuzhou Biersen Technology Co., Ltd (Fuzhou, China). (Figure S1A).

4.4. Functional Analysis of Transgenic Arabidopsis Under Alkaline Stress

To evaluate alkali-stress-induced root phenotypic changes, seeds of vernalized WT Arabidopsis, homozygous GsCYP93D1 overexpressing lines (OE4, OE5), and the cyp93d1 mutant were sown in 1/2 MS medium. After germination and growth for 5 days, the seedlings were transferred into 1/2 MS solid medium containing either 0.6 mM NaHCO3 or 0.8 mM NaHCO3 for stress treatment [29]. After 7 days of vertical growth under light, primary root length and seedling fresh weight were measured and calculated. Seeds were vernalized at 4 °C for 3 days and then sown in a soil/vermiculite mixture (1:1, w/w) (approximately 10–15 seeds per pot). Plants were grown for 30 days in a greenhouse under the following conditions: 22/20 °C day/night temperature, 16 h light/8 h dark photoperiod, and irrigated with standard Hoagland nutrient solution. Uniform 30-day-old plants were selected and subjected to alkaline stress treatment with 200 mM NaHCO3 solution. Leaves (0.1 g) from WT, transgenic, and mutant Arabidopsis plants were collected, placed into pre-labeled 1.5 mL centrifuge tubes, rapidly frozen in liquid nitrogen, and stored at −80 °C for subsequent physiological and biochemical analyses. Chlorophyll content was determined as described previously [46]. CAT and POD activities were assayed as described previously [47,48]. SOD activity was measured according to the method as described previously [49]. MDA content was assayed using the method described previously [50].

4.5. NBT Staining and Superoxide Anion Determination of Transgenic Arabidopsis Under Alkali Stress

Leaves of WT, GsCYP93D1 transgenic (OE4 and OE5), and cyp93d1 mutant Arabidopsis plants were selected and immersed in either 0.6 mM or 0.8 mM NaHCO3 solution for 12 h. Leaves immersed in distilled water served as the control. Following treatment, NBT staining was performed according to the method of [51] to detect the superoxide anion (O2) content in the leaves.

4.6. Alkaline Stress Responses in Hairy Roots of Transgenic Soybean Lines

Soybean seeds (DN50) were sown in sterilized vermiculite with a Hoagland nutrient solution. The pots were covered with plastic wrap to maintain humidity and placed in a light incubator under 26 °C with a 16 h light/8 h dark photoperiod. Seeds were grown for 5 days until cotyledon-stage seedlings suitable for infection were obtained. pCAMBIA1300-GsCYP82C4 recombinant vector was transformed into the Agrobacterium rhizogenes. Seedling hypocotyls were infected using Agrobacterium rhizogenes [52]. The infected seedlings were then transplanted into moist, sterilized vermiculite, covered with plastic wrap, and cultured under identical conditions in the light incubator. Following the establishment of the plants and the stable growth of the hairy roots (Figure S1B), an alkaline stress treatment using 60 mM NaHCO3 was administered, whereas plants with no supplementation of NaHCO3 were placed in the control group. Regular phenotypic monitoring was maintained throughout the cultivation process until notable distinctions between the control group and the stress treatment group were noted. Phenotypic changes in soybean hairy roots were observed and recorded, and root length and fresh weight were measured. Hairy root samples (0.3 g) from each treatment group were collected, placed into pre-labeled 1.5 mL centrifuge tubes, rapidly frozen in liquid nitrogen, and stored at −80 °C for subsequent physiological and biochemical analysis. MDA content was determined as described previously [53]. Antioxidant enzyme activities were assayed as described previously [47,50].

4.7. Analysis of GsCYP93D1-Regulated Gene Expression in Response to Alkaline Stress

To further elucidate the role of GsCYP93D1 in plant alkaline stress response, the alkaline-stress-responsive marker genes (COR15A, COR47, RD29A, KIN1, H+-APase, and NADP-ME) were selected. The expression levels at different time points under 50 mM NaHCO3 stress were analyzed using qRT-PCR. The transgenic lines OE4 and OE5 exhibited comparable expression levels of GsCYP93D1. Based on this equivalence, OE4 was randomly chosen as the representative specimen for qRT-PCR analyses [29]. WT, GsCYP93D1-overexpression lines, and the cyp93d1 mutant seedlings were subjected to 50 mM NaHCO3 stress treatment. The samples were collected at 0, 3, and 6 h under stress. The relative expression levels of marker genes in each sample were quantified using qRT-PCR. Actin2 was used as an internal control [54].

4.8. Functional Characterization of Transgenic Arabidopsis Under ABA Treatment

Seeds of WT, GsCYP93D1 transgenic lines (OE4 and OE5), and the cyp93d1 mutant were sown on 1/2 MS solid medium. Fifteen consistent and sturdy seedlings per line were chosen after five days of growth and moved to plates with exogenous ABA at concentrations of 0.3 μM or 0.5 μM. Seedlings grown in 1/2 MS medium without ABA served as the control. The plates were positioned vertically and cultured under light for 7 days. Phenotypes were then photographed, and the root length and fresh weight of ten seedlings per treatment were measured. To investigate the role of the GsCYP93D1 gene in the ABA signaling pathway, the expression levels of six ABA-stress-related marker genes (ABI1, ABI2, ABI4, ABI5, ABF2, and RAS1) were detected using qRT-PCR. WT, GsCYP93D1 transgenic plants, and the cyp93d1 mutant were treated with 50 μM ABA. Samples were collected at 0, 3, and 6 h after ABA application to detect the expression changes of these genes.

4.9. Statistical Analysis

All experiments included at least three independent biological replicates. Statistical significance of differences was analyzed using a one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (p < 0.05). Statistical analysis and graph generation were performed using GraphPad Prism software 9.5.1.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14172623/s1, Table S1: Gene-specific primers used in this study. Figure S1: qRT-PCR analysis of GsCYP93D1 expression in transgenic Arabidopsis, mutant Arabidopsis, and soybean hairy roots by qRT-PCR. (A) qRT-PCR analysis of GsCYP93D1 expression in transgenic Arabidopsis and mutant Arabidopsis. (B) qRT-PCR analysis of GsCYP93D1 expression in soybean hairy roots. Data are represented as means ± SD. * p < 0.05, ** p < 0.01.

Author Contributions

Conceptualization, C.C., Q.P., H.D. and H.L.; methodology, C.C. and L.X.; software, J.D.; validation, L.X. and W.Z.; formal analysis, C.C.; investigation, L.X.; resources, C.C., H.D. and H.L.; data curation, J.D.; writing—original draft preparation, J.D. and N.X.; writing—review and editing, J.D., H.D. and C.C.; visualization, W.Z.; supervision, C.C. and H.L.; project administration, C.C., H.D. and H.L.; funding acquisition, C.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Heilongjiang Province, grant number YQ2023C034, and the Science and Technology Innovation Ascent Program, grant number XPPY202305. The APC was funded by the China Postdoctoral Science Foundation, grant number 2022M711431.

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding authors upon reasonable request.

Acknowledgments

The authors wish to thank Zaib-un Nisa for language editing assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CYPsCytochrome P450 enzymes
qRT-PCRQuantitative real-time PCR
ROSReactive oxygen species
ABAAbscisic acid
SODSuperoxide dismutase
PODPeroxidase
CATCatalase
MDAMalondialdehyde
NBTNitroblue tetrazolium
O2Superoxide anion
WTWild type

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Figure 1. Expression analysis of GsCYP93D1 in wild soybean. (A) Spatial expressions of GsCYP93D1 in different tissues of wild soybean. Different lettering on bars indicates significant differences. (B,C) Transcript expressions of GsCYP93D1 in wild soybean leaves and roots at different time points in response to 50 mM NaHCO3 treatment. Statistical significance of differences was analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. Data are expressed as means ± SD. Different letters (a–e) indicate significant differences with p < 0.05.
Figure 1. Expression analysis of GsCYP93D1 in wild soybean. (A) Spatial expressions of GsCYP93D1 in different tissues of wild soybean. Different lettering on bars indicates significant differences. (B,C) Transcript expressions of GsCYP93D1 in wild soybean leaves and roots at different time points in response to 50 mM NaHCO3 treatment. Statistical significance of differences was analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. Data are expressed as means ± SD. Different letters (a–e) indicate significant differences with p < 0.05.
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Figure 2. Effect of alkaline stress on growth and germination indices in WT, atcyp93d1, and GsCYP93D1 overexpression lines. (A) Phenotype of WT, atcyp93d1, and GsCYP93D1-overexpression plants grown in ½ strength MS medium with addition of 0.6 mM or 0.8 mM NaHCO3. (B) Root lengths, (C) fresh weight, (D) MDA contents, and (E) chlorophyll contents were determined in these lines under control or alkaline stress conditions. Data are expressed as means ± SD. ns indicates no significant difference, ** p < 0.01.
Figure 2. Effect of alkaline stress on growth and germination indices in WT, atcyp93d1, and GsCYP93D1 overexpression lines. (A) Phenotype of WT, atcyp93d1, and GsCYP93D1-overexpression plants grown in ½ strength MS medium with addition of 0.6 mM or 0.8 mM NaHCO3. (B) Root lengths, (C) fresh weight, (D) MDA contents, and (E) chlorophyll contents were determined in these lines under control or alkaline stress conditions. Data are expressed as means ± SD. ns indicates no significant difference, ** p < 0.01.
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Figure 3. Effect of alkaline stress on superoxide radicals and antioxidant enzymes in WT, atcyp93d1, and GsCYP93D1-overexpression lines. (A,B) Detection of superoxide radicals in Arabidopsis leaves using NBT staining under 0.6 mM or 0.8 mM NaHCO3. (CE) CAT, POD, and SOD enzyme activity analysis in Arabidopsis leaves in response to alkaline stress. Data are expressed as means ± SD. ns indicates no significant difference, ** p < 0.01.
Figure 3. Effect of alkaline stress on superoxide radicals and antioxidant enzymes in WT, atcyp93d1, and GsCYP93D1-overexpression lines. (A,B) Detection of superoxide radicals in Arabidopsis leaves using NBT staining under 0.6 mM or 0.8 mM NaHCO3. (CE) CAT, POD, and SOD enzyme activity analysis in Arabidopsis leaves in response to alkaline stress. Data are expressed as means ± SD. ns indicates no significant difference, ** p < 0.01.
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Figure 4. Expression analysis of stress responsive marker genes in WT, atcyp93d1, and GsCYP93D1-overexpression lines under alkaline stress. Two-week-old Arabidopsis seedlings were supplemented with 50 mM NaHCO3 for 0 h, 3 h, and 6 h. (AF) The expression patterns of stress responsive genes COR15A, COR47, RD29A, KINI, H+-ATPase, and NADP-ME were determined by qRT-PCR analysis. Actin2 was used as an internal control. Data are expressed as means ± SD. ns indicates no significant difference, * p < 0.05, ** p < 0.01.
Figure 4. Expression analysis of stress responsive marker genes in WT, atcyp93d1, and GsCYP93D1-overexpression lines under alkaline stress. Two-week-old Arabidopsis seedlings were supplemented with 50 mM NaHCO3 for 0 h, 3 h, and 6 h. (AF) The expression patterns of stress responsive genes COR15A, COR47, RD29A, KINI, H+-ATPase, and NADP-ME were determined by qRT-PCR analysis. Actin2 was used as an internal control. Data are expressed as means ± SD. ns indicates no significant difference, * p < 0.05, ** p < 0.01.
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Figure 5. Effect of alkaline stress on growth parameters of K599 and transgenic soybean hairy roots. (A) Phenotype of K599 and transgenic GsCYP93D1 soybean hairy roots under normal conditions or 60 mM NaHCO3 stress. (B,C) Root lengths and fresh weight were determined in these lines under control or alkaline stress conditions. (D) Determination of MDA contents. (E) CAT, (F) SOD, and (G) POD enzyme activity analysis in soybean in response to 60 mM NaHCO3. Data are expressed as means ± SD. ns indicates no significant difference, ** p < 0.01.
Figure 5. Effect of alkaline stress on growth parameters of K599 and transgenic soybean hairy roots. (A) Phenotype of K599 and transgenic GsCYP93D1 soybean hairy roots under normal conditions or 60 mM NaHCO3 stress. (B,C) Root lengths and fresh weight were determined in these lines under control or alkaline stress conditions. (D) Determination of MDA contents. (E) CAT, (F) SOD, and (G) POD enzyme activity analysis in soybean in response to 60 mM NaHCO3. Data are expressed as means ± SD. ns indicates no significant difference, ** p < 0.01.
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Figure 6. Effect of ABA stress on growth and germination indices in WT, atcyp93d1, and GsCYP93D1-overexpression lines. (A) Phenotype of WT, atcyp93d1, and GsCYP93D1-overexpression plants grown in ½ strength MS medium with addition of 0.3 μM or 0.5 μM of ABA. (B) Root lengths and (C) fresh weight were determined in these lines under control or ABA treatment conditions. Data are expressed as means ± SD. ns indicates no significant difference, ** p < 0.01.
Figure 6. Effect of ABA stress on growth and germination indices in WT, atcyp93d1, and GsCYP93D1-overexpression lines. (A) Phenotype of WT, atcyp93d1, and GsCYP93D1-overexpression plants grown in ½ strength MS medium with addition of 0.3 μM or 0.5 μM of ABA. (B) Root lengths and (C) fresh weight were determined in these lines under control or ABA treatment conditions. Data are expressed as means ± SD. ns indicates no significant difference, ** p < 0.01.
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Figure 7. Expression analysis of stress-responsive marker genes in WT, atcyp93d1, and GsCYP93D1-overexpression lines under ABA stress. Two-week-old Arabidopsis seedlings were supplemented with 50 μM ABA for 0 h, 3 h, and 6 h. (AF) The expression patterns of stress-responsive genes ABI1, ABI2, ABI4, ABI5, ABF2, and RAS1 were determined by qRT-PCR analysis. Actin2 was used as an internal control. Data are expressed as means ± SD. ns indicates no significant difference, * p < 0.05, ** p < 0.01.
Figure 7. Expression analysis of stress-responsive marker genes in WT, atcyp93d1, and GsCYP93D1-overexpression lines under ABA stress. Two-week-old Arabidopsis seedlings were supplemented with 50 μM ABA for 0 h, 3 h, and 6 h. (AF) The expression patterns of stress-responsive genes ABI1, ABI2, ABI4, ABI5, ABF2, and RAS1 were determined by qRT-PCR analysis. Actin2 was used as an internal control. Data are expressed as means ± SD. ns indicates no significant difference, * p < 0.05, ** p < 0.01.
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Chen, C.; Dai, J.; Xu, N.; Zhou, W.; Xu, L.; Pang, Q.; Duanmu, H.; Li, H. GsCYP93D1, a Cytochrome P450 Gene from Wild Soybean, Mediates the Regulation of Plant Alkaline Tolerance and ABA Sensitivity. Plants 2025, 14, 2623. https://doi.org/10.3390/plants14172623

AMA Style

Chen C, Dai J, Xu N, Zhou W, Xu L, Pang Q, Duanmu H, Li H. GsCYP93D1, a Cytochrome P450 Gene from Wild Soybean, Mediates the Regulation of Plant Alkaline Tolerance and ABA Sensitivity. Plants. 2025; 14(17):2623. https://doi.org/10.3390/plants14172623

Chicago/Turabian Style

Chen, Chao, Jianyue Dai, Nuo Xu, Wanying Zhou, Liankun Xu, Qiuying Pang, Huizi Duanmu, and Haiying Li. 2025. "GsCYP93D1, a Cytochrome P450 Gene from Wild Soybean, Mediates the Regulation of Plant Alkaline Tolerance and ABA Sensitivity" Plants 14, no. 17: 2623. https://doi.org/10.3390/plants14172623

APA Style

Chen, C., Dai, J., Xu, N., Zhou, W., Xu, L., Pang, Q., Duanmu, H., & Li, H. (2025). GsCYP93D1, a Cytochrome P450 Gene from Wild Soybean, Mediates the Regulation of Plant Alkaline Tolerance and ABA Sensitivity. Plants, 14(17), 2623. https://doi.org/10.3390/plants14172623

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