Abstract
The Cation/H+ exchanger (CAX) is an important class of transmembrane transporter protein that plays a crucial role in regulating plant Ca2+ balance, resisting abiotic stresses, and transporting heavy metal ions. The peanut CAX gene family was identified at the genome-wide level, including analyses of their physicochemical properties, phylogenetic relationships, exon-intron composition, chromosomal localization, gene duplication events, cis-acting elements, transcriptional regulatory network prediction, and expression patterns. The results showed that 10 AhCAX genes were identified in the whole genome, distributed on eight chromosomes, and five pairs of gene segment duplication events occurred. These genes encode proteins ranging from 371 to 501 amino acids, with isoelectric points of 5.1 to 8.7. Phylogenetic analysis indicated that AhCAXs were divided into two subfamilies, and members of the same subfamily exhibit similar gene structures, conserved motifs, and numbers of transmembrane domains. It was found that hormone-responsive and stress-responsive elements exist in the promoter regions of AhCAXs. The expression patterns of AhCAX genes showed that AhCAX1, AhCAX4, AhCAX7, AhCAX8, and AhCAX9 were significantly up-regulated under drought or salt stress. Notably, overexpression of the AhCAX8 gene conferred salt tolerance in transgenic Arabidopsis plants by enhancing antioxidant enzyme activity. The results provide a basis for further exploring the function of the peanut CAX genes, and provide candidate gene resources for the cultivation of stress-resistant peanut varieties.
1 Introduction
Ca2+ is an extremely important nutrient element for plant growth and development. It is an essential substance that forms the structure of cell wall and cell membrane, maintaining the stability of the cell membrane and membrane-binding proteins, and reducing or delaying membrane damage (White and Broadley, 2003; ; ). CAX (Cation/H+ exchanger antiporter), a branch of the Ca2+/cation antiporter (CaCA) superfamily, is an important class of transmembrane transporter protein that plays an extremely important role in regulating the change of plant Ca2+ content and cation transport (). CAX proteins can be divided into three categories, including type I (plants and some fungal and bacterial CAXs), type II (yeast CAXs), and type III (Escherichia coli CAXs). In type I, CAX is further divided into two different subgroups, type IA and IB (; ; ), implying a functional difference between the two subgroups (). So far, a large number of CAX genes have been identified from several plant species, including Arabidopsis (), rice (Oryza sativa) (Zou et al., 2021), apple (Malus domestica) (), poplar (Populus) (), and Saccharum (). Using bioinformatics and transgenic methods to identify members of the CAX gene family in peanut, studying their chromosome distribution, evolutionary relationships, gene structure, expression under different abiotic stress, and promoter element analysis, is of great significance for studying the function of CAX genes in peanut.
CAX is multigene family member localized on the cytoplasmic membrane or the vacuolar membrane (). Previous studies found that AtCAX1-AtCAX4 were localized on the vacuole membrane of Arabidopsis using immunohybridization and tobacco immunostaining (). Furthermore, it has been documented that Arabidopsis CAX genes were involved in responses to a variety of abiotic stresses, such as salt, osmotic stress, and heavy metals (). The Arabidopsis protein kinase SOS2 can regulate plant salt tolerance by interacting with CAX1 (). In addition, CAX1 has also been shown to function as a negative regulator in Arabidopsis anoxia stress, as cax1–1 and cax1–2 mutant lines exhibited enhanced tolerance to anoxia stress (Yang et al., 2022). CAX2 and CAX4 have been reported to not only enhance the ability of plants to store heavy metals in the vacuolar zone but also improve the capacity of plants to accumulate Ca2+, Cd2+, and Mn2+ so as to improve the plant tolerance to heavy metals stresses (; ). CAX3 positively regulates Cd tolerance by decreasing reactive oxygen species (ROS) through Ca2+ elevation in Arabidopsis (). Furthermore, overexpression of CAX4 in Arabidopsis can enhance the salt tolerance of transgenic plants (). Therefore, the abiotic stresses tolerance of plants can be widely affected by altering the expression of CAX genes.
Peanut (Arachis hypogaea) is a globally cultivated oilseed and economically significant crop, renowned for its substantial nutritional value and associated social benefits (Zhang et al., 2018). In the peanut cultivation process, abiotic stresses such as extreme temperatures, high salinity, drought, and heavy metals, often harm the metabolism and growth of peanut, leading to quality reduction and yield loss (; ). For this reason, the widespread use of resistant cultivars able to cope well with a wide range of environmental stresses under field conditions. With the availability of peanut genome sequencing data, a large number of peanut gene families have been identified (; Yu et al., 2023; ). However, the role of the peanut CAX family genes in regulating abiotic stress response has not been reported. This research analyzed the molecular characteristics of peanut CAX family genes and explored the function of AhCAX8 in regulating response to salt stress. 10 putative CAX members were identified from the peanut genome. The identified CAX members underwent a comprehensive analysis encompassing their phylogenetic relationships, gene structures, promoter regions, chromosomal locations, duplication events, expression patterns, and potential transcriptional regulatory networks, with a particular focus on the functional validation of AhCAX8 in conferring salt tolerance. Overexpression of the AhCAX8 gene in Arabidopsis conferred plants salt tolerance by enhancing antioxidant enzyme activity. These results provide theoretical basis and genetic resources for studying the function of CAXs in peanut abiotic stresses response and breeding resistant varieties.
2 Materials and methods
2.1 Identification and phylogenetic analysis of the CaCA gene family in peanut
The protein sequences of CaCA family members, including six AtCAX, five AtCCX, one AtMHX, and one AtNCL, were obtained from the Arabidopsis genome (https://www.arabidopsis.org/). The peanut whole-genome protein sequence was downloaded from the PeanutBase (https://dev.peanutbase.org/). The full-length protein sequences of Arabidopsis CaCA members were utilized as queries in a BLASTP program search against the peanut genome database, employing an E-value threshold of 1 × 10-5. Pfam () and SMART () were used for the identification of preliminary proteins containing the Na_Ca_ex domain, and proteins lacking this sequence were removed. The amino acid physicochemical properties of peanut CaCA family members such as molecular weight (Mw) and isoelectric point (pI) were obtained using ProtParam (Wilkins et al., 1999). The subcellular localization of the peanut CaCA family members was predicted through the Plant-mPLoc (). MAFFT (version 7.490) () was used to compare the peanut AhCAX, protein sequence with the CAX protein sequence of Arabidopsis. The phylogenetic tree was generated by the adjacency algorithm neighbor-joining (NJ) tree in MEGA 11 software, with a bootstrap repeat value of 1–000 times ().
2.2 Analysis of the gene structure and protein motif
GSDS 2.0 () software was used to analyze the AhCAX gene structure by comparing the full-length gene sequence and CDS sequence. MEME (version 5.5.3) () was used to analyze and predict the conserved motifs of AhCAX family members in peanut. The protein sequences of AhCAX members were submitted to MEME with the following parameter settings: the number of motifs is set to nine and the length is set to 6-50. The Na_Ca_ex conserved domain of AhCAXs family members was analyzed by SMART (). NovoPro online tool was used to predict the transmembrane region of AhCAXs family members.
2.3 Chromosomal localization, duplication event, and collinearity analysis
The position information of AhCAX genes was obtained from peanut genome. The distribution of AhCAX genes on peanut chromosomes was mapped by MG2C v2.1 (). The duplication events and collinearity of the AhCAX genes were analyzed using MCScanX (Wang et al., 2012) and TBtools (version 1.120) ().
2.4 Promoter analysis and transcriptional regulatory network prediction
To elucidate the possible molecular mechanisms regulating the AhCAX genes, these promoters were analyzed for cis-acting elements. 2kp genomic DNA sequence upstream of the start codon (ATG) of the AhCAX gene was obtained from peanut genome database. The cis-acting elements in the promoter region were identified using the PlantCARE ().
In order to predict the upstream transcription factors of AhCAX genes, the 2kb promoter sequence of AhCAX genes were submitted to PlantRegMap database. The transcription factors that regulate AhCAX genes were then predicted using Binding Site Prediction tool with p value of ≤ 10−5 ().
2.5 Growth and stress treatments of peanut plants
To analyze the expression of AhCAX genes, peanut seeds were germinated on MS medium at 25 °C for two weeks. The resulting seedlings were treated with 20% PEG 6000 (drought) or 200 mM NaCl (salt). Leaves were harvested at 0, 3, and 6 h, immediately frozen in liquid nitrogen, and stored at -80 °C. Total RNA was extracted for subsequent qRT−PCR analysis. Each treatment comprised three biological replicates.
2.6 Expression profiles and qRT-PCR of AhCAX genes
Gene expression data of peanut different plant tissues, including leaf 1, leaf 2, leaf 3, perianth, veg shoot, repr shoot, stamen, root, nodule, and pistil, was obtained from Peanut Base (accession: PRJNA291488). The expression data of peanut AhCAX genes under drought and salt stress were downloaded from NCBI (Zhang et al., 2020; Zhao et al., 2018). Using TBtools () to draw the heat map of AhCAX gene expression pattern in different tissues and under drought and salt stress.
For qRT-PCR, first-strand cDNA was synthesized using the TransGen Biotech cDNA Synthesis Kit. Quantitative real-time PCR (qRT-PCR) was subsequently carried out on an ABI 7900HT system (Applied Biosystems) with SYBR Green Master Mix (TaKaRa, Japan). Each 20 μL reaction contained a pair of gene-specific primers. Transcript levels were normalized to the peanut Actin11 gene (), and relative expression was calculated using the 2-ΔΔCT method (). All primer sequences are listed in Supplementary Table S1.
2.7 Overexpression analysis
The full-length CDS sequence of AhCAX8 gene was inserted into the expression vector pCHF3 to obtain the fusion construct pCHF3-35S::AhCAX8. The construct was then transformed into Arabidopsis by floral dip method (Zhang et al., 2006). The PCR and kanamycin were used to identified the homozygous lines. WT and two AhCAX8-OE lines (OE-7 and OE-9) that grew normally for seven days were transferred to 1/2 MS medium containing 75 and 100 mM NaCl, respectively. The medium was placed in a light incubator (23 °C, continuous light). Primary root length was measured after one week of growth.
2.8 Determination of Malondialdehyde content and enzyme activity
The levels of malondialdehyde (MDA) and the enzymatic activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined in wild−type and AhCAX8-OE plants using corresponding commercial kits (Solarbio, Beijing, China). The experiments were conducted with three biological replicates.
2.9 Statistical analysis
The GraphPad Prism 8 was used to analyze significant differences (P < 0.05). The values were determined from three biological replicates. Different lowercase letters (a, b, and c) above the bar represent statistically significant differences between columns.
3 Results
3.1 Identification of AhCAX genes
A total of 10 AhCAX members were identified from peanut genomic database using BLAST method. The 10 AhCAX genes were named from AhCAX1 to AhCAX10 based on where they are located on the chromosomes. The physiochemical features of AhCAXs such as the number of amino acids (aa), molecular weight (Mw), and isoelectric point (pI) were determined. Results showed that the length of proteins ranged from 371 (AhCAX3 and AhCAX8) to 501 (AhCAX7) aa. The Mw of these AhCAXs ranged from 40260.21 Da (AhCAX3) to 55144.08 Da (AhCAX7), while their pI varied from 5.1 (AhCAX2) to 8.7 (AhCAX3) (Supplementary Table S2).
3.2 Phylogenetic relationship and gene structure of AhCAXs
To investigate the phylogenetic relationship of the CAX gene family members, a NJ tree was constructed among CAX protein sequences from Arabidopsis and peanut. Phylogenetic analysis showed that the 10 AhCAXs were classified into two subfamilies (IA and IB) together with their Arabidopsis homologs (Figure 1). Six AhCAXs were grouped into the subfamily IA, while four AhCAXs belonged to the subfamily IB (Figure 2a), suggesting that some AhCAX genes might have undergone whole genome duplication during peanut’s evolutionary process.
Figure 1
Figure 2
The examination of the gene structure showed that most AhCAXs in same subfamily had similar gene structures (Figure 2b). A total of nine motifs were identified in AhCAX proteins. It was found that the protein motif composition of 10 AhCAX members was generally consistent (Figure 2c). The results suggest that the AhCAX members of the same subfamily might have similar functions.
3.3 Chromosomal distribution, duplication events, and syntenic analysis
The chromosome distribution map of the AhCAX genes was generated. The results indicated that 10 AhCAX genes were distributed on eight peanut chromosomes (Figure 3a). Ah-Chr1 and Ah-Chr11 possessed the maximum number of two AhCAX genes, while one AhCAX gene was found in Ah-Chr2, Ah-Chr4, Ah-Chr7, Ah-Chr12, Ah-Chr14, and Ah-Chr17. Five segmental duplication gene pairs were found in eight AhCAX genes (Figure 3b; Supplementary Table S3). The results suggest that specific AhCAX genes may have arisen through gene duplication, with segmental duplication events possibly playing a crucial role in the evolutionary development of AhCAX genes. Furthermore, analysis of the five gene pairs revealed a Ka/Ks ratio of less than 1, implying that these AhCAX genes have likely been subject to purifying selection during their evolutionary history (Supplementary Table S3).
Figure 3
In order to enhance comprehension of the phylogenetic relationships within the peanut CAX gene family, a syntenic analysis was conducted comparing the CAX genes of peanut with those of four other plant species: Arabidopsis, soybean, tomato, and rice (Figure 4). The results revealed that six, four, two, and zero AhCAX genes were synchronized with CAX genes in soybean, Arabidopsis, tomato, and rice, respectively. There were 19, 11, 2, and 0 collinear pairs between peanut and the other four plant species (soybean, tomato, Arabidopsis, and rice), indicating a close genetic relationship between peanut CAX genes and soybean CAX genes (Supplementary Table S4).
Figure 4
3.4 Cis-regulatory elements analysis and regulatory network prediction
To explore the potential functions of peanut CAX genes, cis-regulatory elements (CREs) from 2 kp of these gene promoters were analyzed using the PlantCARE tool. 22 types of CREs related to abiotic/biotic stress response, phytohormone response, and development were identified (Figure 5). For example, ABA response elements (ABREs) were found in the promoters of eight AhCAX genes, while TCA-elements were found in the promoters of six AhCAX genes. Abiotic stress response elements including STRE, TC-rich repeats, W-box, and WUN-motif were found in the promoters of seven, four, five, and six AhCAX genes, respectively. Results showed that AhCAX genes might respond to abiotic stresses in peanut.
Figure 5
To investigate the regulatory characteristics of AhCAX genes, the PlantRegMap was used to predict potential transcription factors (TFs). The results showed that transcription of the AhCAX genes is mainly regulated by 16 types of TFs (Figure 6a). Almost all AhCAX genes might be regulated by AP2, BBR-BPC, C2H2, Dof, and MIKC_MADS TFs. Furthermore, AhCAX7 was regulated by the most TFs (22), whereas AhCAX4 was regulated by the least (three). In addition, the expression patterns of these TFs encoding genes under drought and salt stress were analyzed using RNA-seq data (Figure 6b).
Figure 6
3.5 Expression profiles of AhCAXs in different tissues and response to salt stress
To assess the expression patterns of AhCAX genes, the transcriptome data of eight peanut tissues (leaf, veg shoot, repr shoot, root, nodule, perianth, stamen, and pistil) were normalized. Results showed that several AhCAX genes, including AhCAX1, AhCAX2, AhCAX3, AhCAX6, AhCAX7, and AhCAX8, were highly expressed in root tissue (Figure 7a). Considering the root tissue was an important organ in plants in response to abiotic stresses, it is plausible to suggest that these AhCAX genes could play a role in the response to abiotic stresses in peanut.
Figure 7
To further explore the response of AhCAX genes to abiotic stresses, the RNA-seq data in root tissues under drought and salt stress was analyzed. Analysis of the expression patterns indicated that three AhCAX genes, including AhCAX1, AhCAX7, and AhCAX9, were up-regulated under drought and salt stress. The expression of AhCAX5 and AhCAX10 was up-regulated under drought stress, while down-regulated under salt stress. However, AhCAX3, AhCAX4, and AhCAX8 exhibited up-regulated expression patterns under salt stress and down-regulated expression patterns under drought stress (Figure 7b). The diverse expression patterns observed in AhCAX genes indicate their distinct roles in drought or salt stress-response pathways.
3.6 Overexpression of AhCAX8 enhanced salt tolerance in Arabidopsis
To further explore the potential function of AhCAX8 in salt stress response, transgenic lines overexpressing AhCAX8 were generated in Arabidopsis plants (AhCAX8-OE). Two lines, AhCAX8-OE7 and AhCAX8-OE9, were chosen for further analysis due to the level of AhCAX8 expression in the AhCAX8-OE lines (Figure 8b). Compared with WT, the OE7 and OE9 lines exhibited a phenotype of longer roots under salt treatments (Figures 8a, c), suggesting that overexpression of AhCAX8 could enhance salt tolerance in transgenic Arabidopsis.
Figure 8
When subjected to salt stress, the OE7 and OE9 lines showed stronger antioxidant enzyme activity and significantly lower MDA content compared to those of the WT (Figure 9). The results indicated that AhCAX8-OE lines might have enhanced salt stress tolerance by enhancing antioxidant enzyme activity.
Figure 9
4 Discussion
CAX is a Ca2+/H+ antiporter and is a transmembrane protein of a class of divalent ion transporters (; ). CAX is widely found in bacteria, fungi, plants, and lower vertebrates (). Currently, the CAX gene families have been identified in multiple species. For example, there are six CAX family members in Arabidopsis (), six CAX family members in rice (Zou et al., 2021), 11 CAX family members in apple (), and 14 CAX family members in wheat (Triticum aestivum) (). However, a systematic study of peanut CAX gene family has not been reported. In this study, we identified 10 CAX gene members in the whole peanut genome, which is more than number found in the diploid plants Arabidopsis and rice. This might be because the peanut is allotetraploid, resulting in a greater number of homologous genes. In addition, these AhCAX genes encode proteins ranging from 371 to 501 amino acids, with isoelectric points of 5.1 to 8.7. Apple CAX gene families also exhibited similar physiological characteristics (). AhCAX3, AhCAX4, AhCAX5, AhCAX8, AhCAX9, and AhCAX10 belong to subfamily IA, while AhCAX1, AhCAX2, AhCAX6, and AhCAX7 belong to subfamily IB (Figure 1), suggesting a possible functional divergence of peanut CAX genes between subfamily IA and subfamily IB.
Previous studies showed that protein structure is closely related to its function. In this study, CAX members within the same subfamily all exhibited similar structures, and the gene structure and conserved motifs of these members were generally consistent with the phylogenetic analysis, and the number of transmembrane domains was also similar (Figure 2). Similar results were found in other species, such as Arabidopsis, rice (Zou et al., 2021), and apple (), suggesting that the biological function of the CAX family is relatively conserved during evolution. Gene duplication events are an important mechanism for gene family expansion (). Gene duplication includes segmental duplication and tandem duplication, both of which are significant drivers of biological evolution. Among these gene duplication events, segmental duplication is one of the major contributors to the expansion of many gene families (). In this study, five pairs of duplication events occurred in peanut CAX gene family, all of which were segmental duplication events (Figure 3b; Supplementary Table S3). Similarly, apple CAX gene families also exhibited segmental duplication events (). The results indicated that segmental duplication events may be the main driving force for the expansion of CAX gene family members during evolution. In addition, this study found that all homologous genes are in the same evolutionary clade (Figures 1, 3b), indicating the high retention rate of homology in peanut. Our phylogenetic analysis clearly divided AhCAXs into two subfamilies, IA and IB (Figure 1). Interestingly, members of subfamily IA (like AhCAX1 and AhCAX7) showed broader expression across various tissues and responded to both drought and salt stress, while subfamily IB members (like AhCAX4, AhCAX8, and AhCAX9) were more specifically induced by salt stress (Figure 7b). This suggests a potential functional divergence, where IA members may play a more general role in development and stress homeostasis, whereas IB members might be specialized for ionic stress responses, particularly salinity. This hypothesis is supported by the distinct cis-element compositions in their promoters (Figure 5) and warrants further investigation through targeted functional studies of individual members from each subfamily.
All promoters in the peanut CAX gene family members have cis-acting elements in response to abiotic stress, such as LTR responding to low temperature, ARE associated with low and oxygen stress, MBS associated with drought stress, and TC-rich in response to defense and stress (Figure 5), indicating that AhCAXs might play an important role in response to abiotic stresses. Transcription factors can regulate gene expression at the transcriptional level by binding to cis-acting elements in downstream gene promoters. Some studies have found that cis-acting elements such as MYB, W-box, MBS, and LTR play important roles in regulating plant CAX genes in response to environmental stresses (Yang et al., 2023). In this study, prediction analysis of transcriptional regulation indicated that the peanut CAX gene is regulated by several transcription factors, such as Dof, bZIP, ERF, and MIKC_MADS (Figure 6a). Notably, the expression of genes coding for these transcription factors is also induced by drought or salt stress (Figure 6b). These results indicated that AhCAX genes might be involved in peanut response to abiotic stresses by regulation of transcription factors. Moreover, the CAX gene family showed different expression patterns in a variety of tissues (Figure 7a). The AhCAX4 and AhCAX9 genes were specifically expressed in leaves, pistil, and perianth, while the AhCAX3 and AhCAX8 genes were expressed in roots, nodule, and pistil. In addition, the AhCAX1, AhCAX2, AhCAX6, and AhCAX7 genes were expressed in almost all of the tissues examined. Furthermore, the promoters of these CAX genes contain multiple cis-acting elements related with growth and development (Figure 5). The results showed that AhCAX genes play important roles in different tissues and developmental stages of peanut.
The CAX proteins of several plant species have been shown to have important roles in response to abiotic stresses (). Arabidopsis cax3-1, cax3-2, and cax1/cax3 mutants showed significant sensitivity to salt stress. Furthermore, atcax1 and atcax4 mutants showed increased sensitivity to salt stress with increasing NaCl concentration (Zhao et al., 2008). Tolerance to salt stress in lines overexpressing AtCAX1 or AtCAX4 significantly enhanced (). Under salt stress, the expression of AhCAX3, AhCAX4, AhCAX8, and AhCAX9 was significant up-regulation (Figure 7b). In addition, these four AhCAX genes were located in the same evolutionary clade together with AtCAX1, AtCAX3, and AtCAX4 (Figure 1). The Arabidopsis cax1 mutants (atcax1–3 and atcax1-4) showed increased tolerance at low temperature than the wild type, suggesting that AtCAX1 plays a negative regulatory role at low temperature (). This result is consistent with the fact that AtCAX1 overexpression of tobacco showed stronger cold sensitivity (). Promoter analysis showed that AhCAX1, AhCAX4, and AhCAX7 had low temperature stress response elements (Figure 5), indicating that these genes might play important role in regulation during cold stress. It was found that the expression of GmCAX1 was increased under drought stress (). AhCAX1, AhCAX5, AhCAX7, and AhCAX10 were significantly up-regulated under drought stress (Figure 7b), indicating that these genes may be involved in the regulation of peanut drought resistance.
In this study, the phylogenetic analysis showed that AhCAX8 and AtCAX1/3/4 were diversified into the same clade and have high homology (Figure 1). In addition, salt stress can significantly induce the expression of AhCAX8 (Figure 7b). Therefore, AhCAX8 was selected for the further functional analysis based on these results. Transgenic analysis demonstrated that the transgenic Arabidopsis lines overexpressing AhCAX8 gene displayed increased root length when exposed to salt stress, as compared to the wild-type plants (Figure 8). When plants are subjected to abiotic stress, either individually or in combination, they generate an excess of ROS, resulting in oxidative stress and disrupted redox homeostasis. This process ultimately culminates in the formation of malondialdehyde (MDA), the end product of lipid peroxidation in plant cell membranes (). Transgenic Arabidopsis lines overexpressing AhCAX8 gene under salt stress, MDA content was decreased, while activities of SOD, POD, and CAT were increased (Figure 9), suggesting that overexpression lines increased the ROS-scavenging capacity.
From a practical perspective, the enhanced salt tolerance conferred by AhCAX8 overexpression in Arabidopsis highlights its potential as a promising candidate gene for molecular breeding of stress-resilient peanut cultivars. Future work could involve overexpressing AhCAX8 in peanut itself using a root-specific or stress-inducible promoter to minimize potential pleiotropic effects. Additionally, exploring natural allelic variations of AhCAX8 in different peanut germplasm accessions could provide valuable markers for marker-assisted selection in breeding programs aimed at improving salt tolerance.
5 Conclusions
In conclusion, 10 AhCAX genes were systematically identified from the peanut genome and were analyzed comprehensively. Several AhCAX genes, including AhCAX1, AhCAX4, AhCAX7, AhCAX8, and AhCAX9, exhibited significant changes in expression levels when exposed to drought or salt stress in peanut. Notably, Arabidopsis lines overexpressing AhCAX8 displayed longer root length and stronger antioxidant enzyme activity compared to WT, indicating AhCAX8 plays an important role in regulating plant abiotic stress tolerance. Together, these findings can help us better understand peanut responses to abiotic stress and aid in the effective strategies aimed for improving abiotic stress tolerance in peanut crop. In future study, we will explore the regulatory networks that control the expression of AhCAX genes.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
Author contributions
FM: Writing – original draft, Methodology, Visualization, Conceptualization, Validation, Investigation. HW: Writing – review & editing, Investigation, Writing – original draft, Project administration. GY: Software, Writing – original draft, Investigation, Methodology, Conceptualization. CZ: Conceptualization, Project administration, Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. YX: Writing – review & editing, Formal analysis, Data curation, Resources. DC: Writing – original draft. GZ: Software, Formal analysis, Writing – review & editing, Writing – original draft, Methodology, Data curation, Project administration, Investigation.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This research was funded by the National Natural Science Foundation of China Youth Science Fund Project (32301958), the Shandong Natural Science Foundation Youth Program (ZR2021QC161), the Taishan Scholars Program (tsqnz20240844), the National Natural Science Foundation of China (32570963), the Natural Science Foundation of Qingdao, (24-4-4-zrjj-132-jch).
Conflict of interest
Author GY was employed by China Tobacco Shandong Industry Co., Ltd.
The remaining authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2026.1900784/full#supplementary-material
Supplementary Figure 1The expression patterns of AhCAX genes under drought and salt conditions. (A) The expression pattern of selected AhCAX genes in response to drought stress treatments, which was calculated as folds relative to the untreated control. (B) The expression pattern of selected AhHsf genes in response to salt stress treatments, which was calculated as folds relative to the control. Different lowercase letters (a–c) above the bar represent statistically significant differences between columns (P < 0.05).
Supplementary Table 1The primers used in this study.
Supplementary Table 2The detail information of identified cultivated peanut CAX family members.
Supplementary Table 3The detail information of segmental duplication gene pairs.
Supplementary Table 4The syntenic pairs between peanut and other four plant species.
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Summary
Keywords
CAX, drought, expression, peanut, salt
Citation
Ma F, Wang H, Yuan G, Zhang C, Xu Y, Ci D and Zhang G (2026) Identification of the cation/H+ exchanger genes in peanut and functional analysis of AhCAX8 in response to salt stress. Front. Plant Sci. 17:1900784. doi: 10.3389/fpls.2026.1900784
Received
05 June 2026
Revised
23 June 2026
Accepted
29 June 2026
Published
16 July 2026
Volume
17 - 2026
Edited by
Cun Guo, Tarim University, China
Reviewed by
Xiaolong Shi, Shenyang Agricultural University, China
Liu Yingru, Hebei Agricultural University, China
Updates
Copyright
© 2026 Ma, Wang, Yuan, Zhang, Xu, Ci and Zhang.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Guanchu Zhang, guanchuzhang@126.com
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