The glutathione-related metabolism and the AhGST23 gene mediate drought and salt stresses tolerance in peanut (Arachis hypogaea L.)

Abstract

Background

Abiotic stresses such as drought and salinity significantly constrain peanut productivity. However, their underlying molecular response mechanisms remain unclear.

Results

This study identified the AhGST23 gene, together with its involvement in glutathione metabolism and the ascorbic acid-glutathione (AsA-GSH) cycle, as a key component in alleviating drought and salt stresses in peanut. Under drought (15% PEG6000 for 6 days) and salt (200 mM NaCl for 6 days) stresses, peanut seedlings exhibited a marked reduction in biomass, net photosynthetic rate, transpiration rate, and chlorophyll fluorescence parameters. Concurrently, levels of H₂O₂, O₂⁻, and malondialdehyde (MDA), as well as the activities of antioxidant enzymes (APX, GR, SOD, POD, and CAT), were significantly elevated in both leaves and roots. RNA-seq analysis identified 3,780 and 5,019 shared differentially expressed genes (DEGs) in leaves and roots, respectively, which were enriched in pathways including plant hormone signal transduction, starch and sucrose metabolism, glutathione metabolism, and MAPK signaling. The key gene families involved in the glutathione metabolism (AhGSTs, AhGPXs) and AsA-GSH cycle (AhGRs, AhAPXs, and AhMDHARs) were highlighted as central players in the antioxidant system. Silencing AhGST23 disrupted glutathione-related metabolism in peanut. This disruption was manifested by reduced contents of ascorbic acid (AsA), dehydroascorbate (DHA), glutathione (GSH), and oxidized glutathione (GSSG), as well as decreased activities of ascorbate eroxidase (APX), glutathione S-transferases (GST), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR). These disruptions impaired ROS scavenging capacity and heightened sensitivity to drought and salt stresses.

Conclusions

In summary, the glutathione-related metabolism, with AhGST23 as a key functional gene, plays an essential role in conferring drought and salt stresses tolerance in peanut. These findings offer novel insights into antioxidant defense mechanisms and provide valuable genetic resources for enhancing peanut stress resilience.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08491-0.

Introduction

Drought and salt stresses significantly constrain crop productivity by impairing plant growth and development, thereby posing serious threats to global agricultural production [1, 2]. These stresses disrupt the uptake of essential macronutrients such as K⁺, Ca²⁺, and Mg²⁺, while salt stress specifically leads to cytotoxic Na⁺ hyperaccumulation, aggravating ionic imbalance and disruption of osmotic homeostasis. The consequent decline in root-zone water potential, coupled with diminished hydraulic conductivity, exacerbates cellular dehydration and ultimately induces osmotic stress [3–5]. Critically, both stressors trigger a burst of reactive oxygen species (ROS), resulting in oxidative damage to biomolecules (nucleic acids, proteins) and organelles (plasma membranes, chloroplasts, mitochondria). Accumulation of these harmful compounds affects various physiological traits including net photosynthetic rate, respiration, and energy metabolism, thus culminating in arrested growth, reproductive failure, and reduced yield [6–8].

To mitigate these injurious effects of drought and salt, plants employ multifaceted adaptive strategies: (1) upregulating the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and the contents of antioxidants (glutathione, ascorbate) to restore redox equilibrium; (2) synthesizing osmoprotectants, such as proline and soluble sugar; (3) fine-tuning the ion transporters (SOS1, NHX) for maintaining the K⁺/Na⁺ and Ca²⁺/Mg²⁺ homeostasis; (4) activating ABA and MAPK cascades for stress signaling; and (5) reprogramming stress-responsive transcriptome via NAC, MYB, and WRKY transcription factors [9–11].

Within this molecular framework, the role of glutathione metabolism and ascorbate-glutathione (AsA-GSH) cycle are pivotal in responding to oxidative stress by directly scavenging ROS [11–13].

Notably, plants have evolved divergent adaptive mechanisms in response to drought and salt stresses. For instance, salinity imposes ionic toxicity and osmotic stress which are countered by activating SOS pathway for Na⁺ extrusion and vacuole sequestration in salt-tolerant species [14]. Conversely, drought tolerant species prefer to conserve water through ABA-mediated stomatal closure and remodel root architecture to absorb more water from deeper soil layers. Roots also display stress-specific plasticity: drought often promotes the formation of deeper roots, whereas salinity triggers avoidance strategies [15]. Therefore, it is imperative to elucidate the shared core mechanisms and unique aspects of crop responses to drought and salt stresses.

Peanut (Arachis hypogaea L.) is a major global oil and economic crop. Its seeds are used for both oil extraction and direct consumption, while the residual plant biomass can be utilized as animal feed [16]. Additionally, peanuts restore soil fertility by establishing symbiotic relationships with rhizobia for biological nitrogen fixation, which significantly reduces their reliance on chemical fertilizers [17]. Consequently, peanut cultivation is characterized by high yield, low input costs, and environmental friendliness [18, 19]. Although peanuts are of great agricultural importance, the molecular mechanisms underlying their responses to drought and salt stresses remain unclear. In particular, the similarities and differences in these molecular mechanisms merit in-depth investigation [20].

This study focuses on investigating the similarities and differences in peanut responses to drought and salt stresses. The stress treatments were calibrated via preliminary dose-response assays to achieve similarity in concentration. We comprehensively assessed peanut responses to drought and salt stresses by characterizing various morphological and physiological parameters. Transcriptomic data were also collected to corroborate the molecular and physiological responses. Finally, the role of a key gene was validated using virus-induced gene silencing (VIGS)-mediated functional analysis. The results demonstrate that the glutathione metabolism and ‌AsA-GSH cycle‌ play a pivotal role in peanut adaptation to both drought and salt stresses, with AhGST23 emerging as a key regulatory gene.‌ These molecular insights not only elucidate the mechanistic basis of peanut stress adaptation but also identify the AhGST23 as a candidate gene for enhancing drought and salt tolerance in peanut.

Methods

Plant material cultivation and stress treatments‌

The widely cultivated peanut cultivar Yuhua 37 (developed by the Henan Academy of Agricultural Sciences) was selected for this study. Seeds produced through controlled selfing were used in this study. Seeds of uniform size were surface-sterilized with 3% H₂O₂ for 10 min followed by rinsing three times with distilled water, and germinated on moist filter paper under controlled conditions: 10 h light/14 h dark cycle, 27 ± 2℃ day/25 ± 2℃ night, photosynthetic photon flux density of 350 µmol/(m²·s), and 75% relative humidity. After nine days, a total of 54 uniformly grown seedlings were transplanted into nine hydroponic containers (25 cm×20 cm×15 cm), with six seedlings in each container. These containers were randomly divided into three treatment groups (control, drought, and salt stress), with three containers (biological replicates) per group. All plants were initially acclimated by keeping in half-strength modified Hoagland solution for three days. Stress treatments were imposed for six days. A stepwise stress regime was implemented with increments at two-day intervals. For drought stress, PEG6000 concentrations were increased from 5% to 10% and 15%; for salt stress, NaCl concentrations were increased from 100 mM to 150 mM and 200 mM, respectively. All treatment solutions were replenished daily and maintained in an aerated state to ensure oxygen supply.

Determination of phenotypic traits, photosynthetic performance, and chlorophyll fluorescence parameters‌

After six days of treatment, three peanut seedlings were randomly selected from each container. The photosynthetic performance‌ (net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, and transpiration rate) and ‌chlorophyll fluorescence parameters‌ of the first fully expanded leaf from the upper part of the seedlings were recorded using LI-6800 portable photosynthesis system (LI-COR Biosciences, USA). Root systems were scanned using an Epson Expression 12000XL scanner, and ‌total root length, root surface area, and root volume were analyzed with the WinRHIZO 2007 root analysis system (Regent Instruments, Canada). Weight of fresh roots, stems, and leaves was recorded using an electronic balance (0.00001 g precision). Seedlings were kept at 105 °C for 30 min, followed by oven-drying at 80 °C to constant weight, and re-weighed to determine dry biomass. For the remaining three seedlings in each treatment, leaves and roots were thoroughly rinsed with deionized water, separated, and stored at -80 °C for subsequent analyses, including ‌physiological index determination, ‌transcriptome sequencing‌, and ‌qRT-PCR validation.

Determination of physiological indices

The O₂⁻ and H₂O₂ contents were quantified using biochemical kits (Solarbio Science & Technology Co., Ltd, Beijing, BC1290, BC3595). The MDA contents were determined by applying the thiobarbituric acid method, the activities of SOD and POD were determined using the nitroblue tetrazolium method and the guaiacol method, respectively, and the activities of CAT, APX, GR, GST were measured using the UV spectrophotometric method, as described by Liu et al. [21].

Transcriptome sequencing and qRT-PCR validation‌

Transcriptome sequencing was performed using established molecular biology protocols. Total RNA was extracted from peanut root tissues using an RNA extraction kit (CoWin Bioscience, Item No. CW0599S). Quality and quantity of RNA were confirmed before synthesizing cDNA. The mRNA was then purified from the total RNA and sheared into short fragments for sequencing. Subsequently, first-strand cDNA was synthesized from the fragmented mRNA templates followed by second-strand cDNA synthesis to create double-stranded DNA. The cDNA underwent purification to remove reaction components followed by end repair to create blunt ends. A poly-A tail was added to facilitate sequencing, and then adapters were ligated to enable PCR amplification. Finally, the library was amplified through PCR to generate sufficient DNA template for high-throughput sequencing. The constructed libraries were sequenced using an Illumina NovaSeq™ X Plus system (LC-Bio Technology Co., Ltd., Hangzhou, China). Gene expression levels were quantified as Fragments Per Kilobase Million mapped reads (FPKM), and differentially expressed genes (DEGs) were identified with thresholds of |fold change| ≥ 2 and q-value < 0.05. Subsequently, based on these DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed [22].

Total RNA was reverse-transcribed into cDNA for qRT-PCR following the manufacturer’s instructions for the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Cat. No. Q311-03). Ten DEGs were randomly selected for qRT-PCR validation to confirm the accuracy of transcriptome data [22].

Virus-induced gene silencing of AhGST23

Peanut AhGST23-silenced plants were generated using virus-induced gene silencing (VIGS). Briefly, a pTRV2-AhGST23 silencing vector was constructed and transformed into Agrobacterium tumefaciens strain EHA105. The Agrobacterium suspension was infiltrated into peanut leaves using a syringe. Silencing efficiency was confirmed by qRT-PCR. Detailed protocols followed our previous publication [22]. The contents of AsA, dehydroascorbate ( ), glutathione (GSH), oxidized glutathione (GSSG), and the activities of dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) were determined using biochemical kits (Solarbio Science & Technology Co., Ltd, Beijing, Item No. BC1235, BC1245, BC1175, BC1185, BC0665 and BC0655), respectively.

Statistical analysis‌

Data were statistically analyzed using IBM SPSS Statistics 22. Figures were constructed with Microsoft Office PowerPoint, GraphPad Prism 5, and Excel software.

Results and analysis

Drought and salt stresses significantly inhibit peanut growth‌

Both drought and salt stresses negatively affected the morphology of peanut seedlings (Fig. 1). Salt stress caused more severe suppression of shoot growth, as evidenced by a significant reduction in seedling height and aboveground biomass (Table 1). In contrast, drought stress led to sharp reduction in root biomass (Table 1). The whole-plant dry weight decreased by 16.33% under drought stress and by 30.61% under salt stress, with the latter being significantly higher. Conversely, the reduction in root dry weight was more severe under drought (31.25%) than under salt stress (18.75%). These results indicate that while both stresses inhibit peanut growth, salt stress primarily constrains shoot development, whereas drought stress more severely disrupts root biomass.

Fig. 1.

Observation on the growth of peanut seedlings under drought and salt stresses

Table 1.

Determination of growth indices

	Plant height (cm)	Fresh weight (g)			Dry weight (g)			Root length (cm)	Surface area (cm2)	Root volume (cm3)
		Whole plant	Aboveground	Root	Whole plant	Aboveground	Root
Control	9.80 a	7.06 a	4.55 a	2.51 a	0.98 a	0.83 a	0.16 a	899.02 a	141.57 a	1.89 a
Drought	6.93 b	4.52 b	3.15 b	1.37 c	0.82 b	0.71 b	0.11 b	568.48 b	94.78 b	1.26 b
NaCl	5.93 c	4.10 b	2.37 c	1.72 b	0.68 c	0.55 c	0.13 b	407.19 c	78.96 b	1.19 b

Different lowercase letters represent significant differences between groups (P < 0.05), the same applies below

Drought and salt stresses elevate ROS levels and antioxidant activity in peanut seedlings

Under drought stress, the production rate of O₂⁻ in peanut leaves increased by 123.22%, while MDA, a key indicator of membrane damage resulting from lipid peroxidation, increased by 63.24% compared to the control (Fig. 2A and C). Interestingly, the H₂O₂ content decreased by 21.56% (Fig. 2B), indicating the enhanced enzymatic detoxification of H₂O₂, potentially through CAT or glutathione-dependent pathways. This was supported by increased activities of CAT (112.10%) and GR (121.92%) (Fig. 2F and H), suggesting a robust antioxidant response to counteract ROS accumulation. Additionally, SOD and POD activities increased by 14.58% and 27.92%, respectively, further reflecting activation of enzymatic defence systems (Fig. 2D and E).

Fig. 2.

Determination results of ROS and antioxidant enzyme related indicators in peanut leaves. A O2- content; B H2O2 content; C MDA content; D SOD activity; E POD activity; F CAT activity; G APX activity; H GR activity

Under salt stress, the oxidative and antioxidant responses followed a different pattern. The H₂O₂ and MDA contents increased sharply by 53.70% and 130.60% (Fig. 2B and C), respectively, indicating substantial ROS accumulation and membrane damage. Notably, the O₂⁻ production rate did not change significantly, possibly due to efficient elimination by upregulated SOD activity (18.75%, Fig. 2D) which converts O2- into H2O2. To mitigate H2O2 toxicity, POD activity significantly increased by 70.05% (Fig. 2E), alongside APX by 44.39% (Fig. 2G) and GR by 80.82% (Fig. 2H). However, CAT activity remained unchanged (Fig. 2F).

In roots, drought stress induced marked ROS accumulation and severe oxidative damage. Specifically, the O₂⁻ production rate, as well as the contents of H₂O₂ and MDA, increased by 309.91%, 153.26%, and 80.91%, respectively, reflecting enhanced ROS generation and lipid peroxidation (Fig. 3A-C). In response, the antioxidant system was strongly activated: SOD, CAT, APX, and GR increased by 44.34%, 237.50%, 39.67%, and 497.87%, respectively (Fig. 3D-H). The pronounced upregulation of CAT (2.4-fold) and GR (5.0-fold) underscores the importance of both H₂O₂ scavenging and glutathione metabolism in drought tolerance.

Fig. 3.

Determination results of ROS and antioxidant enzyme related indicators in peanut roots. A O₂- content; B H₂O₂ content; C MDA content; D SOD activity; E POD activity; F CAT activity; G APX activity; H GR activity.

In contrast, salt stress also induced oxidative stress in peanut roots, though to a lesser extent than drought. The O₂⁻ production rate, as well as the contents of H₂O₂ and MDA increased by 93.79%, 57.86%, and 46.65%, respectively, indicating moderate ROS accumulation (Fig. 3A-C). The SOD, POD, CAT, APX, and GR activities increased by 27.46%, 12.10%, 343.62%, 21.87%, and 68.35%, respectively (Fig. 3D-H). Notably, CAT activity increased 3.4 fold more than the other enzymes, highlighting its crucial role in H₂O₂ detoxification under salt conditions. Collectively, both drought and salt stress increased the content of O₂⁻, H₂O₂, and MDA, as well as the activities of SOD, POD, CAT, APX, and GR in peanut plants, with this trend being particularly pronounced under drought stress.

Drought and salt stresses reduced photosynthetic performance in peanut leaves‌

Both drought and salt stresses significantly decreased the net photosynthetic rate and transpiration rate in peanut seedlings, indicating a marked inhibition of photosynthetic activity (Table 2). In contrast, both drought and salt stresses elevated the intercellular CO₂ concentration (Table 2).

Table 2.

Measurement results of photosynthetic performance parameters

Control	Net photosynthesis rate (µmol CO2/(m2·s))	Transpiration rate (mmol/(m2·s))	Stomatal conductance (mol/(m2·s))	Intercellular CO2 concentration (µmol/mol)
	6.70 a	0.003 a	395.68 a	288.26 b
Drought	0.59 b	0.001 b	398.48 a	360.91 a
Salt	0.63 b	0.002 b	398.49 a	362.49 a

Different lowercase letters represent significant differences between groups (P < 0.05)

Both stresses also significantly reduced the potential activity of PSII (Fv/Fo) and the photochemical performance index (PI abs), indicating impaired photosynthetic capacity (Fig. 4). Notably, salt-stressed seedlings exhibited lower PI abs values than those under drought. The coordinated decline in Pn, Tr, and PSII activity, along with elevated Ci, further reflects an overall reduction in photosynthetic capacity and water use efficiency under stress conditions. Overall, both drought and salt stresses suppressed photosynthetic performance in peanut seedlings, with salt stress exerting a stronger negative impact on the photochemical efficiency of PSII.

Fig. 4.

Measurement results of chlorophyll fluorescence parameters

RNA-seq data output and quality assessment

RNA sequencing was conducted on a total of 18 peanut samples, comprising leaves and roots subjected to control, drought, and salt treatments. The sequencing output and quality metrics demonstrated high sequencing reliability and consistency across all samples. The number of raw reads per sample ranged from 41.97 to 50.82 million, corresponding to a total base count between 6.38 and 7.62 gigabases (Gb), indicating a sequencing depth sufficient for accurate gene expression analysis. After filtering, the percentage of high-quality reads remained consistently high, ranging from 93.08% to 97.09%. In addition, Q30 scores were uniformly high (97.30%-98.06%), further confirming the high quality of the sequencing data. The GC contents across all samples ranged from 43.50% to 45.50% as expected for plant transcriptomic data (Table 3).

Table 3.

The output statistics of RNA seq data

Sample	Raw Data		Valid Data		Valid Ratio (%)	Q20 (%)	Q30 (%)	GC content (%)
	Read	Base	Read	Base
Control_L1	45,811,618	6.87 G	44,051,680	6.61 G	96.16	99.95	97.83	44.5
Control_L2	43,753,050	6.56 G	41,819,538	6.27 G	95.58	99.97	98.05	45.5
Control_L3	42,884,020	6.43 G	41,075,086	6.16 G	95.78	99.97	98.06	44.5
Control_R1	48,148,844	7.22 G	46,485,530	6.97 G	96.55	99.95	97.71	44.0
Control_R2	44,151,058	6.62 G	42,684,540	6.40 G	96.68	99.95	97.64	44.5
Control_R3	48,931,230	7.34 G	47,508,260	7.13 G	97.09	99.97	97.84	44.5
Drought_L1	42,513,658	6.38 G	40,558,748	6.08 G	95.40	99.95	97.92	44.5
Drought_L2	42,811,120	6.42 G	40,569,784	6.09 G	94.76	99.94	97.88	44.5
Drought_L3	44,020,806	6.60 G	41,648,160	6.25 G	94.61	99.94	97.91	43.5
Drought_R1	44,392,754	6.66 G	42,602,560	6.39 G	95.97	99.96	97.67	44.0
Drought_R2	42,915,388	6.44 G	41,128,376	6.17 G	95.84	99.96	97.67	44.0
Drought_R3	43,798,054	6.57 G	41,757,596	6.26 G	95.34	99.96	97.65	44.5
Salt_L1	42,863,772	6.43 G	40,362,816	6.05 G	94.17	99.96	97.69	44.5
Salt_L2	41,971,184	6.30 G	39,067,614	5.86 G	93.08	99.96	97.76	45.0
Salt_L3	43,790,978	6.57 G	41,837,192	6.28 G	95.54	99.94	97.61	43.5
Salt_R1	49,941,354	7.49 G	48,049,324	7.21 G	96.21	99.94	97.30	44.0
Salt_R2	50,824,958	7.62 G	49,034,578	7.36 G	96.48	99.93	97.30	44.0
Salt_R3	45,566,844	6.84 G	43,803,036	6.57 G	96.13	99.95	97.51	44.5

Sample correlation analysis revealed high reproducibility among biological replicates. Most treatment groups showed correlation coefficients greater than 0.98. Slightly lower correlations were observed in the Drought_Leaf (0.91) and Salt_Leaf (0.90) groups, which may be attributed to increased biological variability in leaf tissues under stress conditions. Nevertheless, these values remain within an acceptable range for transcriptomic studies. Overall, the results confirm that biological replicates were consistent and reliable for transcriptome analysis.

Identification and functional analysis of differentially expressed genes‌

A total of 66,979 genes and 84,455 transcripts were annotated from the sequencing data (Table S1). Among these, 5,063 DEGs were identified in leaves under drought stress, including 3,305 upregulated and 1,758 downregulated genes (Fig. 5A, Table S2). In contrast, salt stress triggered a more extensive response in leaves, with 13,329 DEGs (8,247 upregulated and 5,082 downregulated). In roots, the expression pattern was reversed: drought stress induced 15,734 DEGs (5,599 upregulated, 10,135 downregulated), whereas salt stress resulted in 8,004 DEGs (3,671 upregulated, 4,333 downregulated). These results indicate that drought stress has a greater impact on root transcriptomes, whereas salt stress exerts stronger effects on leaves.

Fig. 5.

Statistical analysis (A) and Venn analysis (B) of DEGs in comparison group. Drought_L, Salt_L, Control_L, Drought_R, Salt_R and Control_R stand for Drought_Leaf, Salt_Leaf, Control_Leaf, Drought_Root, Salt_Root and Control_Root, respectively. DL, SL, CL, DR, SR, and CR stand for Drought_Leaf, Salt_Leaf, Control_Leaf, Drought_Root, Salt_Root, and Control_Root, respectively, same as below

Notably, the DEG profiles contrasted markedly between leaves and roots under the two stresses (Fig. 5A). Leaves under salt stress showed the highest number of DEGs, consistent with the stronger growth inhibition observed in aboveground tissues. Conversely, roots under drought stress exhibited the highest DEG count, aligning with the more severe physiological damage in root systems. Furthermore, upregulated genes outnumbered downregulated ones in leaves under both stresses, whereas downregulated genes predominated in roots, especially under drought.

Analysis also revealed that 2,103 DEGs in leaves and 2,905 DEGs in roots were commonly expressed under both drought and salt stresses conditions (Fig. 5B). A total of 3,780 DEGs under drought and 5,019 under salt stress were commonly regulated in both leaves and roots. The higher number of shared DEGs under salt stress implies a broader systemic transcriptional reprogramming under salt stress. Moreover, the total number of stress-responsive genes was greater in roots than in leaves, consistent with the direct exposure of roots to PEG6000 and NaCl solutions, positioning them as the primary site of stress perception and response. Importantly, 636 DEGs were consistently differentially expressed across all treatment comparisons, across both stress types and tissues, highlighting their role in countering both stresses in peanut.

GO and KEGG enrichment analysis of DEGs

To elucidate the biological function of DEGs involved in peanut responses to drought and salt stresses, GO and KEGG pathway enrichment analyses were performed across all treatment groups (Table S3). The top ten significantly enriched terms in each category, biological process (BP), cellular component (CC), and molecular function (MF), were shown in Fig. 6A. For example, in the BP category, DEGs were primarily enriched in transcription regulation, oxidation-reduction processes, defense response, and response to water deprivation. Within the CC category, DEGs were strongly associated with the nucleus, plasma membrane, cytoplasm, and chloroplasts. In the MF category, significantly enriched terms included protein binding, ATP binding, metal ion binding, and protein serine/threonine kinase activity.

Fig. 6.

A and KEGG (B) enrichment analysis statistics of DEGs. The numbers in the chart represent the number of enriched DEGs

KEGG pathway analysis revealed that DEGs were significantly enriched in several stress-related pathways (Table S4, Fig. 6B). These included plant hormone signal transduction, plant-pathogen interaction, starch and sucrose metabolism, phenylpropanoid biosynthesis, MAPK signaling pathway, and glutathione metabolism. In summary, these enrichment analyses reveal a highly coordinated molecular response in peanuts under drought and salt stresses, involving transcriptional control, signal transduction, metabolic adjustment, and antioxidant defense.

The GO (Table S3) and KEGG (Table S4) enrichment results of the 636 common DEGs responsive to both drought and salt stress indicated that these DEGs were mainly enriched in plant hormone signal transduction, protein processing in the endoplasmic reticulum, ribosome biogenesis in eukaryotes, MAPK signaling pathway, glutathione metabolism, and TCA cycle (Fig. S1), highlighting the important and shared roles of these metabolic pathways in peanuts responses to drought and salt stress, thereby laying a foundation for the subsequent screening and validation of key metabolic pathways and gene families.

Phylogenetic and expression analysis of ROS and antioxidant enzyme-related gene families

Based on physiological and transcriptomic data, we conducted phylogenetic and expression analyses of key gene families involved in antioxidant metabolism. The study specifically focused on genes associated with the glutathione metabolism and AsA-GSH cycle, which had been significantly enriched in our KEGG analysis. Results revealed that gene families such as AhGRs, AhGSTs, AhGPXs, and AhAPXs were broadly and significantly induced under both drought and salt stresses. Notably, a large proportion of the DEGs from these families were strongly upregulated, particularly members of the AhGST family (Fig. 7A). For example, the expression of the AhGST23 gene (Gene ID: G3R5KE) in leaves was upregulated by 2.33-fold and 28.12-fold under drought and salt stress, respectively, while in roots it increased by 22.98-fold and 13.17-fold, respectively (Fig. 7A).

Fig. 7.

Phylogenetic and expression multiples analysis of AsA-GSH cycle (A) and AhRbohs gene families (B). The numbers in the figure represent the multiples of expression level among the comparison groups, the red, blue, and yellow represent significant upregulation, significant downregulation, and no significant difference, respectively

In addition, most AhRbohs genes were upregulated under stress conditions, indicating their active involvement in ROS production (Fig. 7B). Collectively, these findings elucidated the important role of AhRbohs in ROS generation, while the glutathione metabolism and AsA-GSH cycle facilitates efficient scavenging of excess ROS, thereby preventing oxidative damage and maintaining cellular redox balance.

qRT-PCR validation of transcriptome sequencing results‌

To validate the transcriptome sequencing results, qRT-PCR was performed on 10 selected DEGs involved in the glutathione-related metabolism and ROS regulation, including six AhGSTs, two AhRbohs, one AhGR, and one AhGPX. The expression patterns of all ten genes, as validated by qRT-PCR, aligned with the RNA-seq data. Specifically, genes that were up- or down-regulated in RNA-seq showed corresponding directional changes in qRT-PCR (Fig. 8), which validated the transcriptomic results. Although the overall expression trends were consistent, minor discrepancies in fold-change values were observed between the two methods. These differences can be attributed to the distinct computational approaches used for gene expression quantification: RNA-seq data were analyzed using the FPKM method, which normalizes for transcript length and sequencing depth, whereas qRT-PCR data were processed using the 2^−ΔΔCT method, which relies on cycle thresholds and reference gene normalization. These methodological differences typically affected the magnitude of fold changes but did not alter the overall expression patterns. In summary, the strong concordance in gene expression trends between qRT-PCR and RNA-seq supports the accuracy and reproducibility of the transcriptome sequencing data.

Fig. 8.

qRT-PCR analysis of some DEGs

Disruption of the glutathione metabolism and AsA-GSH cycle in AhGST23-silenced peanuts reduces drought and salt tolerance

To further investigate the functional role of AhGSTs in conferring drought and salt stresses tolerance, particularly through its involvement in regulating glutathione metabolism and AsA-GSH cycle, a VIGS approach was employed. The AhGST23 gene (Gene ID: G3R5KE), a representative member of the Glutathione S-transferase family, was selected based on its significant upregulation under both stresses. A gene silencing vector (pTRV2-AhGST23) was constructed and used to generate 17 AhGST23-silenced peanut plants through leaf infiltration. The pTRV2-AhGST23 treated plants and control (infiltrated with the empty pTRV2 vector) were subjected to drought and salt stresses treatments.

Under drought and salt stresses, AhGST23-silenced plants exhibited earlier and more severe wilting, along with a significant increase in water loss (Fig. 9A, B), and a marked reduction in fresh weight by 10.98% and 9.16%, respectively (Fig. 9C). Also, under control conditions, the contents of AsA, DHA, GSH and GSSG in peanut AhGST23-silenced plants were decreased by 22.30%, 35.74%, 40.86% and 29.79%, respectively, compared to the control plants (Fig. 10A-D). Under drought and salt stresses, the contents of AsA, DHA, GSH and GSSG in both control and AhGST23-silenced plants showed a significant increasing trend, but the elevation was much lower in silenced plants.

Fig. 9.

Phenotypic observation (A and B) and biomass analyse (C) of peanut AhGST23 silenced plant under drought and salt stresses

Fig. 10.

The contents of AsA (A), DHA (B), GSH (C), GSSG (D) and the activities of APX (E), GST (F), DHAR (G), MDHAR (H) in peanut AhGST23 silenced plants under drought and salt stresses

The activities of APX, GST, DHAR, and MDHAR followed the similar trends as observed for antioxidant contents. Compared to the empty-vector control plants, the AhGST23-silenced plants showed significantly reduced activities of APX (by 42.20%), GST (50.17%), DHAR (35.48%), and MDHAR (41.67%) under non-stress conditions (Fig. 10E-H). Under drought and salt stresses, while APX and GST activities increased in both control and silenced plants, the extent of increase was markedly lower in the silenced plants. Furthermore, under stress conditions, the activities of DHAR and MDHAR in AhGST23-silenced plants did not differ significantly from their levels under non-stress conditions (Fig. 10).

In summary, our results showed that silencing AhGST23 expression not only reduced the GST activity and GSH content in peanut plants, but also significantly diminished key antioxidants and enzymes involved in the glutathione metabolism and AsA-GSH cycle. The disruption of the glutathione-related metabolism impaired the plants’ ability to effectively scavenge excess ROS, ultimately leading to a substantial decline in drought and salt stresses tolerance.

Discussion

In recent years, scientists have identified a series of key transcription factors and enzymes involved in the response of peanuts to drought and salt stress. For instance, under salt stress conditions, the transcription factor AhbHLH121 enhances antioxidant enzyme activity in peanut plants by promoting the expression of AhPOD, AhCAT, and AhSOD genes [23]. Heterologous overexpression of the transcription factors AhLBD33 and AhLBD67 has been shown to improve plant tolerance to salt stress [24]. Conversely, silencing the B-box (BBX) family gene AhBBX6 in peanuts weakens their tolerance to both salt and drought stress [25]. Additionally, the leucine-rich repeat receptor-like kinase AhLRR-RLK265, the drought-induced 19 (Di19) family gene AhDi19-3B, as well as 12-oxo-phytodienoic acid reductase AhOPR6 and trehalose-6-phosphate phosphatase (TPP), have all been demonstrated to play significant roles in peanut responses to drought and salt stress [26–29]. In the present study, we identified two key components involved in the antioxidant defense of peanuts under drought and salt stresses: glutathione-related metabolism and NADPH oxidase-mediated ROS regulation, with a particular focus on the roles of AhGST23 and AhRbohs gene families.

The glutathione-related metabolism and the AhGST23 gene in peanut stress tolerance

Glutathione metabolism and the AsA-GSH cycle play vital roles in plant defence against abiotic stresses by effectively scavenging ROS, thus maintaining redox homeostasis within cells. In this cycle, ASA is oxidized by APX during the reduction of H₂O₂ to water. The oxidized forms of ASA, monodehydroascorbate (MDHA) and dehydroascorbate (DHA), are regenerated by MDHA reductase (MDHAR) and DHA reductase (DHAR), respectively, ensuring continuous antioxidant capacity [30]. This pathway constitutes a central mechanism for ROS scavenging in stressed plants [13, 23]. In this study, gene expression profiling revealed a significant upregulation of the glutathione metabolism and AsA-GSH cycle related gene families, AhGRs, AhGSTs, AhGPXs, and AhAPXs, under both drought and salt stresses conditions (Fig. 7). These results align with previous reports on the role of glutathione metabolism and the AsA-GSH cycle in conferring drought and salt tolerance in maize and rice [31–33]. Among these, the AhGSTs family was most markedly upregulated, with AhGST23 showing the greatest increase, suggesting a central role for this gene within the family. This was further confirmed through VIGS experiments: silencing AhGST23 significantly reduced the contents of AsA, DHA, GSH, and GSSG, as well as the activities of APX, GST, DHAR, and MDHAR. These disruptions led to dysfunction of the glutathione-related metabolism, which impaired the plants’ ability to scavenge ROS. Consequently, severe oxidative stress accumulated, rendering the plants more vulnerable to drought and salt stresses. These findings demonstrate that the AhGST23 plays a pivotal role in peanuts responses to drought and salt stresses by participating in the glutathione metabolism. The successful identification and functional characterization of the AhGST23 gene provide a theoretical foundation for the future use of this gene in developing salt-tolerant peanut germplasm.

AhRbohs-mediated ROS production impacts peanut stress responses

ROS production in plants is primarily mediated by NADPH oxidases (also known as respiratory burst oxidase homologs, Rbohs), along with enzymes such as peroxidases, amine oxidases, and aldehyde oxidases [34, 35]. Rbohs catalyze the transfer of electrons from NADPH to oxygen, generating O₂⁻, which is then converted into H₂O₂ [36, 37]. While low to moderate ROS levels serve as signalling molecules to trigger defence and acclimation responses, their excessive accumulation leads to oxidative stress and cellular damage [34, 38]. Studies have reported dual roles of Rbohs in stress responses: CmoRbohD1/D2 overexpression enhanced cucumber salt tolerance via ROS signaling [39], while OsRbohA knockout improved rice salt tolerance by reducing ROS and balancing K⁺/Na⁺ [40]. In peanuts, AhRbohs have been implicated in ROS burst under arsenic stress [41], and prior research has shown that silencing AhRbohB6 and AhRbohC18 significantly enhanced salt tolerance by reducing ROS levels [22].

In this study, RNA-seq analysis revealed that expression of AhRbohs was significantly higher than that of the other genes belonging to ROS-related families under drought and salt stresses, with most DEGs being significantly upregulated (Fig. 7). However, excessive ROS production can be detrimental, suggesting that selective downregulation or genetic silencing of key AhRboh genes could be an effective breeding strategy to minimize the oxidative damage and enhance peanut resilience.

Differential responses of peanut to drought and salt stresses

This study compared the effects of PEG-induced drought and NaCl-induced salt stress of comparable intensity on peanut plants. Phenotypically, salt stress caused more severe overall growth inhibition than drought stress. Notably, salt stress primarily impaired shoot development, whereas drought stress caused a greater reduction in root elongation (Fig. 1; Table 1). RNA-seq results supported these phenotypic observations: salt-stressed leaves exhibited 8,266 more DEGs than drought-stressed leaves, while drought-stressed roots showed 2,405 more DEGs than salt-stressed roots (Fig. 5). This tissue-specific enrichment of DEGs reflects differences in tissue-specific signal perception and transcriptional reprogramming patterns.and transcriptional reprogramming patterns.

Functional enrichment analysis further revealed distinct stress- and tissue-specific metabolic adaptations. In leaves, DEGs under salt stress were significantly enriched in pathways such as glutathione metabolism, MAPK signalling, flavonoid biosynthesis, and isoflavonoid biosynthesis, which are closely associated with antioxidant defence and cellular signaling. In roots, DEGs under drought stress showed significant enrichment in flavonoid and isoflavonoid biosynthesis, indicating a prominent role of secondary metabolites in root-specific drought adaptation. These findings highlight the central role of glutathione metabolism and the AsA-GSH cycle in leaf responses to salt stress, particularly through GST-mediated detoxification, and emphasize the contribution of flavonoid pathways in roots under drought.

Interestingly, the most affected tissues under each stress condition (leaves under salt stress and roots under drought) also displayed the strongest enrichment of adaptive metabolic pathways. This pattern suggests that stress-responsive gene networks are selectively activated in a tissue-specific manner to respond to environmental challenges. Future research should focus on dissecting these key metabolic pathways and their core regulatory genes to understand their precise molecular mechanisms in drought and salt tolerance.

Conclusions

In summary, this study elucidates the molecular and physiological mechanisms underlying peanut responses to drought and salt stresses. Although both stresses significantly suppressed plant growth, salt stress more strongly inhibited shoot development, whereas drought stress caused greater impairment to root development, reflecting distinct adaptive strategies under different abiotic conditions. Drought and salt stresses induced the expression of AhRbohs genes, leading to ROS overaccumulation. Concurrently, key family genes involved in the glutathione metabolism and AsA-GSH cycle, including AhGSTs (especially AhGST23), AhGRs, AhGPXs, and AhAPXs, were markedly upregulated, thereby facilitating ROS detoxification and alleviating oxidative damage (Fig. 11). Silencing of AhGST23 led to a significant decline in GST activity and content, along with reduced levels of key antioxidants and enzymes involved in the glutathione-related metabolism, ultimately disrupting redox homeostasis and increasing plant sensitivity to both drought and salt stresses.This study not only elucidates the critical role of glutathione-related metabolism and the AhGST23 gene in peanut responses to drought and salt stress, but also establishes a crucial theoretical foundation and provides valuable genetic resources for the molecular breeding of drought- and salt-tolerant peanut varieties.

Fig. 11.

The schematic diagram of the role of glutathione-related metabolism in peanut response to drought and salt stresses. Under drought and salt stresses, peanut plants exhibit upregulated expression of AhRbohs family genes, which mediate ROS production and result in the accumulation of H₂O₂ and O₂-. Meanwhile, multiple gene families involved in maintaining the dynamic balance of the GSH/GSSG ratio and the glutathione-related metabolism, including AhPGDs, AhGRs, AhGPXs, AhGSs, AhGSTs, and AhAPXs, exhibit upregulated expression. These changes enhance the capacity of ASA-GSH cycle to scavenge H₂O₂ and O₂-, thereby alleviating the damage caused by drought and salt stresses in peanuts. The red, blue, and yellow represent significant upregulation, significant downregulation, and non-significant changes, respectively

Authors’ contributions

Zengqiang Li conducted experiments, analyzed data, and wrote the original draft. Zhiyong Zhang conceptualized the project, and reviewed the manuscript. Muhammad Hafeez Ullah Khan, Mehboob-ur Rahman, Khudayberdi Nazarov, Xiangjun Kong and Xin Zhang participated in manuscript revision. Peipei Jia and Zhenkun Cui enrolled in this research. Haifang Dai, Mengyao Shao and Lijie Li conducted data analysis.

Data availability

The datasets generated during the current study are available in the National Center for Biotechnology Information: Submission ID PRJNA1392150. All data, with the exception of the mRNA-Seq data, are available in the article and its supplementary information.

Declarations

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Competing interests

The authors declare no competing interests.