Genome-wide characterization of AhBAG genes in peanut reveals their role in bacterial wilt resistance and hormone response

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Peanut ( Arachis hypogaea L.), a globally significant oilseed and cash crop, is highly valued for its economic importance. However, systematic genome-wide analysis and functional characterization of the BAG gene family in peanut remain largely unexplored. Results In this study, we identified 13 AhBAG genes in the peanut genome, which are unevenly distributed across 11 chromosomes. Phylogenetic analysis revealed that these AhBAG genes, together with BAG family members from other plant species, are classified into four distinct clades, underscoring their evolutionary conservation. Segmental duplication was identified as a major driver of the expansion of the AhBAG gene family. Notably, AhYSVF0U exhibited significant upregulation under Ralstonia solanacearum infection and abscisic acid treatment, suggesting its potential involvement in mediating peanut resistance to bacterial wilt. Conclusions This study provides comprehensive insights into the evolutionary and functional characteristics of the peanut BAG gene family and offers valuable genetic resources for molecular breeding programs aimed at improving stress tolerance in peanut. Peanut Bacterial wilt BAG gene family Expression profile Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Peanut ( Arachis hypogaea L.), a vital oilseed and cash crop, sustains global agricultural economies and food security [ 1 , 2 ]. In 2023, the global peanut cultivation area reached approximately 31 million hectares, with an average yield of 1,755 kilograms per hectare. The total peanut production amounted to 54.3 million tons, showing an upward trend [ 3 ]. Despite its nutritional and economic value, peanut productivity is severely constrained by biotic stresses, particularly bacterial wilt (BW) caused by Ralstonia solanacearum ( R. solanacearum ). This soil-borne pathogen, notorious as the “plant cancer”, colonizes vascular tissues via root wounds, secreting extracellular polysaccharides and cell wall-degrading enzymes to obstruct water transport, ultimately leading to wilting, chlorosis, and characteristic bacterial ooze in infected plants [ 4 – 6 ]. The disease prevails in tropical and subtropical regions, with infection rates reaching 80% in severe cases, causing catastrophic yield losses [ 7 , 8 ]. Although resistant cultivars like Yuanza 9102 and Yueyou 92 have reduced incidence to < 8% in endemic areas, the narrow genetic basis of resistance and limited understanding of molecular mechanisms hinder further breeding progress [ 9 ]. Peanut resistance to BW is a polygenic trait involving complex host-pathogen interactions [ 10 , 11 ]. Upon infection, R. solanacearum employs chemotaxis to invade roots, forming biofilms in xylem vessels to evade host defenses [ 12 ]. Molecular breeding efforts have identified key quantitative trait loci (QTLs), including qBWB02.1 on chromosome B02, which co-localizes with NBS-LRR resistance gene clusters [ 13 ]. Transcriptomic analyses highlight the involvement of MAPK cascades, WRKY transcription factors, and m 6 A RNA modification in defense responses [ 14 , 15 ]. Notably, overexpression of AhRLK1 and AhRRS5 , homologs of Arabidopsis immune regulators, enhances resistance in transgenic tobacco [ 16 , 17 ]. Recent breakthroughs include synthetic hexaploid peanuts derived from wild Arachis species, demonstrating superior wilt resistance [ 18 ]. However, the precise regulatory networks governing these resistance pathways remain elusive, underscoring the need to explore novel gene families involved in stress adaptation. The Bcl-2-associated athanogene (BAG) family, evolutionarily conserved across eukaryotes, serves as a critical nexus for stress signaling and cell survival [ 19 – 21 ]. Characterized by a C-terminal BAG domain that interacts with HSP70/HSC70 chaperones, these proteins function as co-chaperones to modulate protein folding and degradation [ 22 , 23 ]. Plant BAG proteins exhibit unique structural diversification: Group I members ( AtBAG1-4 ) harbor ubiquitin-like domains for stress adaptation, while Group II ( AtBAG5-7 ) contains IQ motifs for calcium-mediated signaling [ 24 , 25 ]. Functional studies demonstrate their roles in thermotolerance ( AtBAG7 -WRKY29 interaction [ 26 ]), salinity resistance (OsBAG4-DNA methylation complex [ 27 ]), and pathogen defense ( AtBAG6 -mediated autophagy [ 28 ]). Hormonal crosstalk further links BAGs to abscisic acid (ABA) and ethylene pathways, as evidenced by promoter cis -elements and stress-inducible expression patterns [ 29 , 30 ]. Despite their characterized roles in model plants, the BAG family remains unexplored in peanuts-a gap that limits the exploitation of this regulatory hub for wilt resistance. Given their dual functions in chaperone-mediated protein homeostasis and programmed cell death (PCD), peanut BAG genes likely orchestrate critical defense responses against R. solanacearum . This study aims to fill this knowledge gap by performing a systematic genome-wide identification and functional characterization of the BAG gene family in peanut. Through integrative bioinformatics and experimental approaches, we elucidate the evolutionary relationships, structural diversification, and stress-responsive expression dynamics of AhBAG genes. Our findings provide valuable insights into the molecular mechanisms underlying BAG-mediated stress adaptation in peanut and lay the foundation for developing molecular markers and genetic engineering strategies to improve stress resilience in this vital crop. Materials and methods Plant materials and experimental treatments Two peanut cultivars-highly resistant (H108) and susceptible (H107) to BW-developed by Prof. Yin’s team at Henan Agricultural University were selected. Mature seeds were surface-sterilized with 0.1% HgCl₂ for 2 min, rinsed three times with sterile water, and germinated on moist filter paper at 25°C in darkness until radicle emergence (~ 36 h). Germinated seeds were transferred to sterilized vermiculite and cultivated in a climate-controlled chamber (37°C, 70% relative humidity, darkness) until the three-leaf stage. R. solanacearum inoculation: Root irrigation was performed with a bacterial suspension (1×10⁸ CFU/mL) following Zhao et al[ 31 ]. Hormone induction: Foliar sprays of 100 µM ABA, 2 mM SA, or 50 µM MeJA were applied[ 16 ]. Three biological replicates per treatment were harvested at 0 d, one day post-inoculation (dpi), and seven dpi post-treatment, flash-frozen in liquid nitrogen, and stored at -80°C. Identification and characterization of AhBAG genes BLASTP and HMMER ( https://www.ebi.ac.uk/Tools/hmmer/search/phmmer . PF02179 domain, E-value < 1×10⁻⁵) were employed against the Tifrunner v2 genome (PeanutBase, https://www.peanutbase.org/ ) using Arabidopsis BAG proteins (TAIR) as queries. Candidate genes were validated via NCBI Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/ ) for domain integrity. Physicochemical properties (molecular weight, pI) were analyzed using ExPASy ProtParam ( https://web.expasy.org/protparam/ ), and subcellular localization was predicted via Wolf PSORT ( https://www.genscript.com/wolf-psort.html ). Phylogenetic and structural analysis BAG protein sequences from Arabidopsis (TAIR), tomato (Phytozome), and rice (NCBI) were aligned using MUSCLE in MEGA11 [ 32 ]. A neighbor-joining phylogenetic tree (1,000 bootstrap replicates) was visualized with iTOL ( https://itol.embl.de/ ). Gene structures and chromosomal locations were mapped via TBtools. MEME was used to identify conserved motifs in the promoter regions of target genes. The analysis was performed with the following parameters: number of motifs = 10, motif width range = 6-100 bp, and E-value 0.7 (high confidence) were retained. Collinearity and cis -regulatory element analysis Interspecies collinearity between peanut, Arabidopsis , and soybean BAG genes was analyzed using TBtools. Promoter sequences (~ 2,000 bp upstream) of AhBAGs were retrieved from PeanutBase, and cis -elements were annotated via PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Tissue-specific expression profiling Public RNA-seq data from nine Tifrunner tissues (leaf, root, nodule, perianth, stamen, pistil, peg tip, fruit pat, and seed pat) were obtained from PeanutBase. Expression heatmaps were generated using TBtools. quantitative real-time PCR validation Root tissues from H108 and H107 seedlings were collected at 0, 1, and 7 days post-inoculation (dpi) under mock inoculation (ddH 2 O-treated control) and R. solanacearum -infected conditions. For phytohormone treatments, three-week-old H108 and H107 seedlings were foliar-sprayed with SA, MeJA, ABA, or ddH 2 O (control). Leaf samples were collected at 0, 1, and 7 dpi post-treatment. All samples were collected in triplicate biological replicates, flash-frozen in liquid nitrogen, and stored at -80°C until analysis. First-strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit (Takara, Dalian, China) following the manufacturer’s protocol. qRT-PCR was subsequently performed using the synthesized cDNA templates, and amplified with gene-specific primers (Table S1 ) using SYBR Green I (TB Green Premix Ex Taq II, TaKaRa) on a CFX96 Touch™ system (Bio-Rad). Cycling conditions: 95°C for 30 s; 40 cycles of 95°C for 5 s, 58°C for 30 s. Melt curves confirmed specificity. The relative expression levels were calculated using the 2 −ΔΔCt method (n = 3 biological replicates), Actin7 was used as the reference gene[ 33 ]. Error bars represent the standard error of the mean (SEM). Statistical differences were analyzed by one-way ANOVA using GraphPad Prism 8.0 (* P < 0.05, ** P < 0.01). Results Genome-wide identification and characterization of BAG genes in peanut Through systematic bioinformatic analysis of the published Tifrunner genome, we identified 13 putative BAG family genes in cultivated peanut. The coding sequence (CDS) lengths of these genes ranged from 558 bp ( Ah4JG1SZ ) to 3,711 bp ( AhYSVF0U ), encoding proteins of 185 to 1,236 amino acids (Table S2). The predicted molecular weights of BAG proteins exhibited considerable variation, spanning from 21.51 kDa ( AhJ75SJQ ) to 139.18 kDa ( AhYSVF0U ), with theoretical isoelectric points (pI) ranging from 4.89 to 9.50. Notably, eight BAG proteins were classified as acidic (pI 7), suggesting functional diversity in their molecular interactions. Protein stability analysis revealed that four members ( AhTEF0AP , AhADAM8V , Ah37JSRQ , and AhCF9SCE ) with instability indices below 40 were predicted as stable proteins, whereas the remaining nine members (instability index > 40) were categorized as unstable proteins. The aliphatic index, an indicator of thermostability, ranged from 59.68 to 81.14 across the family. All 13 BAG proteins displayed negative grand average of hydropathicity values (-0.42 to -1.05), confirming their hydrophilic nature. Subcellular localization predictions using multiple algorithms (WoLF PSORT, Plant-mPLoc, and CELLO) indicated distinct compartmentalization patterns. The majority of AhBAG proteins were predicted to localize in chloroplasts, nuclei and peroxisomes (Table S2). This differential subcellular distribution implies specialized functional roles for BAG family members in various cellular compartments. Phylogenetic evolution and chromosomal distribution of AhBAG genes To elucidate evolutionary relationships and functional diversification among BAG family members, a phylogenetic tree was reconstructed using full-length protein sequences from peanut (13 genes), Arabidopsis thaliana (7 genes), Solanum lycopersicum (10 genes), and Oryza sativa (6 genes). Neighbor Joining analysis with 1,000 bootstrap replicates resolved the BAG proteins into four distinct clades (Fig. 1 A; Table S3). Clade I contained two dicot-specific members (AtBAG7 and SlBAG10). Clade II included 14 family members (2 AtBAGs, 2 OsBAGs, 4 SlBAGs, and 6 AhBAGs). Clade III exhibited conserved orthology across species, including two peanut members (Ah35MDCZ and Ah9JS9HJ), whereas Clade IV showed the highest diversity, harboring 14 members with significant expansion in peanut (5 AhBAGs) and Arabidopsis (3 AtBAGs). This phylogenetic topology suggests both conserved evolutionary patterns and lineage-specific diversification events. Chromosomal mapping revealed that 13 AhBAG genes were unevenly distributed across 11 of 20 peanut chromosomes (Fig. 1 B). Chromosomes 4 and 14 each harbored two paralogous gene pairs ( Ah4JG1SZ / AhJ75SJQ and AhD7MIWJ / AhG65IU1 , respectively), likely resulting from segmental duplication events. The dispersed genomic distribution pattern implies multiple duplication mechanisms, contributed to BAG family expansion in peanut. Gene structure analysis uncovered significant exon-intron architecture variation across subfamilies (Fig. 1 C). Clade II members displayed the simplest structures, with 62% (4/6) containing a single exon ( AhD7MIWJ , AhG65IU1 , Ah4JG1SZ , and AhJ75SJQ ) and one member ( AhYSVF0U ) exhibiting three exons. In contrast, Clade IV genes showed conserved four-exon structures (5/5 members), while Clade III members displayed intermediate complexity (4 exons in Ah35MDCZ / Ah9JS9HJ ). The inverse correlation between phylogenetic divergence and structural complexity suggests that exon loss/gain events may have driven functional specialization during BAG family evolution. Domain architecture and motif diversification of AhBAG proteins The functional diversity of BAG proteins is intrinsically linked to their conserved domain composition [ 34 ]. Our analysis using InterProScan and SMART databases revealed that all 13 AhBAG proteins possess the hallmark BAG domain, which mediates interactions with Hsp70 ATPase to regulate chaperone activity (Fig. 2 A). Notably, two paralogous genes (AhYSVF0U and AhII6FYR) exhibited an N-terminal IQ domain, a calcium-sensing module critical for linking chaperone activity with Ca²⁺ signaling[ 25 ]. Six members (Ah35MDCZ, Ah9JS9HJ, AhTEF0AP, AhADAM8V, Ah37JSRQ, and AhCF9SCE) contained a C-terminal ubiquitin-like (UBQ) domain, known to facilitate protein degradation and stress response [ 35 ]. Strikingly, AhED3VG4 displayed a truncated UBQ domain due to a frameshift mutation in its coding sequence, suggesting potential neofunctionalization or pseudogenization during evolution. Plant BAG genes typically encode a BAG domain of 70–80 amino acid residues [ 36 , 37 ]. Analysis of conserved motifs further elucidated genetic variations among peanut BAG proteins. Ten motifs were identified across the 13 AhBAGs, predominantly composed of hydrophobic (valine V, isoleucine I, leucine L) and charged residues (glutamic acid E, aspartic acid D, arginine R, lysine K) (Fig. 2 B, C). All AhBAGs shared motif 2 and motif 3, which are likely essential for BAG protein functionality, as evidenced by their consistent presence in phylogenetically clustered members. Group IV proteins contained eight motifs (motif 1–8), with UBQ-containing members encoding complete BDs exhibiting these motifs, highlighting structural conservation. Phylogenetic analysis revealed that AhYSVF0U in Group III shares high amino acid sequence similarity with AtBAG6. Structural predictions showed that the IQ-calmodulin binding motif comprises two small α-helices connected by a hairpin loop, which interfaces with the first helix of the BAG domain-a tripartite antiparallel α-helical bundle. Intriguingly, AhYSVF0U harbors two α-helices in its IQ-calmodulin motif, contrasting with the single helix observed in AtBAG6 (Fig. 2 D). Genome synteny and cis -regulatory element analysis The evolutionary conservation of BAG genes across peanut species was investigated through comparative synteny analysis. Thirteen AhBAG genes in cultivated peanut ( A. hypogaea ) exhibited orthologous counterparts in diploid wild species ( A. duranensis and A. ipaensis ) and tetraploid wild species ( A. monticola ), suggesting functional conservation during domestication (Fig. 3 A). Notably, seven AhBAG genes distributed across seven chromosomes showed syntenic relationships with AtBAG homologs in Arabidopsis . For instance, AhYSVF0U shared collinearity with AtBAG6 , a key regulator of fungal resistance, implying conserved roles in stress adaptation. This evolutionary conservation underscores the potential functional retention of AhBAG genes in mediating stress responses. Cis -regulatory element profiling of AhBAG promoters revealed significant enrichment of hormone-responsive motifs (Fig. 3 B). Ten genes harbored ABRE (abscisic acid-responsive element), with AhYSVF0U and AhADAM8V containing five ABRE copies each, while seven genes possessed CGTCA-motifs (methyl jasmonate-responsive). Ah37JSRQ uniquely carried three MeJA-responsive elements, aligning with its rapid induction under MeJA treatment. Additionally, four AhBAG promoters contained W-box elements, which are critical for defense-related transcriptional reprogramming upon pathogen-associated molecular pattern (PAMP) recognition. These findings suggest that AhBAG genes are transcriptionally regulated through ABA/JA signaling cascades and may participate in pathogen defense. Tissue-specific expression and stress-responsive dynamics AhBAG genes exhibited distinct spatiotemporal expression patterns across tissues (Fig. 3 C; Table S4). The expression levels of different genes vary significantly across tissues. AhYSVF0U shows relatively high expression in multiple tissues, reaching 177.89 in the pistil, with elevated values also observed in leaf, root, and nodule tissues. In contrast, AhD7MIWJ exhibit extremely low expression in most tissues, with no detectable expression in root, nodule, or perianth tissues, and only minimal expression in leaf and peg tip. Additionally, some genes display strong tissue-specific expression patterns. AhII6FYR shows markedly high expression in the pistil, significantly exceeding levels in other tissues like the perianth. AhTEF0AP is expressed at 44.21 in the perianth, while maintaining relatively uniform but lower levels across other tissues. These findings suggest that these genes may play critical roles in the growth, development, or functional specialization of specific tissues. Under biotic and abiotic stresses, qRT-PCR revealed genotype- and time-dependent expression dynamics. In resistant cultivar H108, AhYSVF0U were upregulated 5-fold ( p < 0.01) in roots at 7 dpi of R. solanacearum (Fig. 4 A), consistent with their predicted roles in restricting pathogen spread via programmed cell death (PCD). ABA treatment triggered an 8-fold induction of AhYSVF0U in H108 leaves at 1 dpi, whereas MeJA rapidly activated AhTEF0AP and Ah37JSRQ (4-fold increase, Fig. 4 B, D). Intriguingly, salicylic acid (SA) suppressed AhYSVF0U expression in H108 (Fig. 4 C), highlighting antagonistic crosstalk between SA and ABA/JA pathways in modulating BAG-mediated stress responses. Protein interaction networks and functional associations STRING-based protein interaction prediction unveiled a multifaceted regulatory network centered on AhYSVF0U (Fig. 5 ). Ah0J9HE3, a HSP20-family chaperone implicated in thermotolerance, suggesting AhYSVF0U may stabilize stress-denatured proteins. AhTGXT8E, a xylanase inhibitor that fortifies cell walls by neutralizing pathogen-derived xylanases. AhK7D5N2, a calmodulin protein interacting with IQ motifs to transduce Ca 2+ signals during immune responses. This network positions AhYSVF0U as a signaling hub integrating chaperone activity, cell wall reinforcement, and calcium signaling to orchestrate multi-layered stress adaptation. Notably, the absence of direct interactions with canonical immune receptors implies that AhBAGs operate downstream of pathogen recognition. Discussion The BAG family comprises evolutionarily conserved eukaryotic proteins that orchestrate stress signaling and cell survival pathways [ 38 , 39 ]. This study presents the inaugural genome-wide identification and spatiotemporal expression profiling of BAG genes in peanut, a globally significant oilseed crop vulnerable to devastating biotic constraints such as BW caused by R. solanacearum . Our multi-dimensional analysis elucidates the evolutionary trajectories and stress-responsive regulatory networks of AhBAG genes, providing molecular blueprints for developing wilt-resistant peanut cultivars through precision breeding. Phylogenetic reconstruction of BAG orthologs from taxonomically diverse species (peanut, Arabidopsis [ 34 ], tomato[ 21 ], rice [ 40 ] revealed both ancestral conservation and species-specific divergence patterns. The four-clade classification of AhBAG proteins reflects functional diversification shaped by evolutionary selection pressures. The non-random chromosomal distribution and paralogous gene clusters suggest segmental duplication-driven family expansion, a well-documented evolutionary mechanism for environmental adaptation in plant genomes[ 41 ]. Bhat et al. detected significant chromosomal collinearity between the A and B subgenomes, particularly in the third (A03/B03) and fifth (A05/B05) chromosomes, which exhibited highly conserved gene content and arrangement order [ 42 ]. Similarly, through comparative genomic analysis, this study revealed that the 11 AhBAG genes displayed strict syntenic relationships in their genomic distribution, suggesting functional conservation of these genes during peanut domestication and evolution. Cross-species synteny conservation with wild peanut progenitors ( A. duranensis , A. ipaensis ) and Arabidopsis homologs supports functional retention of ancestral stress-adaptive mechanisms. Of particular note, AhYSVF0U demonstrates evolutionary synteny with AtBAG6, a canonical fungal resistance regulator, implying phylogenetic preservation of pathogen defense functions in peanut. Structural characterization revealed clade-specific domain architectures among AhBAG proteins, with conserved BAG and ubiquitin-like domains interspersed with lineage-specific motifs [ 5 , 43 ]. Phylogeny-correlated motif organization provides structural evidence for functional specialization during speciation. Subcellular localization predictions revealed differential targeting of AhBAG proteins to chloroplasts, nuclei, and peroxisomes, suggesting compartmentalized functional specialization. Chloroplast-localized members may regulate photosynthetic machinery protection, while nuclear isoforms could mediate stress-responsive transcriptional regulation, consistent with their predicted molecular functions. Transcriptional profiling under biotic/abiotic stresses uncovered genotype- and time-dependent expression dynamics. The pathogen-inducible expression of AhYSVF0U in resistant cultivar H108 (5-fold upregulation at 7 dpi) strongly implicates its functional involvement in PCD-mediated pathogen containment, paralleling established BAG-PCD regulatory mechanisms [ 25 ]. The rapid MeJA responsiveness of AhTEF0AP and Ah37JSRQ (4-fold induction at 1 dpi) corroborates their predicted roles in oxylipin signaling cascades. The discovery that ABA upregulates AhYSVF0U and that SA inhibits AhYSVF0U aligns with known signaling pathways in peanut defense mechanisms. Under R. solanacearum infection, the ABA receptor PYL family gene AhPYL6 was significantly upregulated, thereby enhancing plant resistance to the pathogen through promotion of defense-related gene expression [ 44 ]. When plants were treated with SA, the AhNPR3A mutation exhibited no effect on the induction of pathogenesis-related (PR) genes [ 45 ]. Notably, SA-mediated suppression of AhYSVF0U reveals antagonistic phytohormone crosstalk, highlighting the intricate hormonal regulation of BAG-mediated stress responses. The predicted interactome of AhYSVF0U with HSP20 chaperones, xylanase inhibitors, and calcium sensors suggests its central role in integrating proteostasis maintenance, cell wall fortification, and Ca 2+ signaling into a coordinated stress mitigation network. This systematic investigation establishes the molecular foundation for deciphering BAG-mediated stress adaptation in peanut. Future investigations should prioritize functional characterization through transgenic overexpression approaches. The development of transgenic peanut overexpressing stress-responsive AhYSVF0U could offer valuable resources for breeding wilt-resistant cultivars and improving peanut productivity under adverse conditions. Conclusions In this study, we systematically identified and analyzed the characteristics of BAG family genes in peanutand their roles in responses to biotic and abiotic stresses through whole-genome analysis. Using bioinformatics approaches, we identified a total of 13 AhBAG gene family members, which are widely distributed across the 11 chromosomes of peanut. Further analysis of expression patterns revealed distinct expression differences of AhBAG genes among various peanut tissues, with some genes showing higher expression levels in vegetative organs, while others are significantly expressed in reproductive organs. Moreover, these genes exhibit multi-level regulatory functions under conditions of R. solanacearum infection and various abiotic stresses. Notably, the AhYSVF0U gene shows significant regulatory potential in disease resistance responses and can be considered a priority candidate gene for peanut disease-resistant breeding. These findings provide a foundation for further investigating the biological functions and molecular mechanisms of BAG genes in peanut stress responses and offer important genetic resources for peanut disease-resistant breeding. Declarations Supplementary Materials The online version contains supplementary material available at Acknowledgments We thank Zhenyu Wang, Plant Protection Institute of Henan Academy of Agricultural Sciences for the BW strain. Author Contributions The study was designed and directed by D.Y.; K.Z., Y.L., J.W. and X.M. coordinated research; F. G., Y.T. and Z.L. analyzed the sequencing data; Z.C. and L.Z. provided reagents; figures were prepared by D.Q. and K.Z. with support from X.Z., R.R.; D.Y. and K.Z. interpreted the results, and wrote the manuscript. All authors read and approved the final manuscript. Funding This work was supported by grants from the Youth Project of Natural Science Foundation of Henan (242300420477), the Key Program of National Natural Science Foundation of China (NSFC)-Henan United Fund (No.U22A20475), Key Scientific and Technological Project of Henan Province (No.221111110500; HARS-22-05-G1), National Key Research and Development Program (2023YFD1202800), the Henan Agricultural University High Level Talent (30501418), the Key Scientific Research Project in Colleges and Universities of Henan Province of China (25A210004). Data availability All data generated and analyzed for this study are included in the manuscript and its supplementary information fles. Conflicts of Interest The authors declare no conflicts of interest. References Huai D, Xue X, Wu J, Pandey MK, Liu N, Huang L, et al. Enhancing peanut nutritional quality by editing AhKCS genes lacking natural variation. Plant Biotechnol J. 2024;22:3015. Wang Q, Zhao X, Sun Q, Mou Y, Wang J, Yan C, et al. 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Arabidopsis B-cell lymphoma2 (Bcl-2)-associated athanogene 7 (BAG7)-mediated heat tolerance requires translocation, sumoylation and binding to WRKY29. New Phytol. 2017;214:695–705. Shahid S. A DNA methylation reader with an affinity for salt stress. Plant Cell. 2020;32:3380–1. Arif M, Li Z, Luo Q, Li L, Shen Y, Men S. The Bag2 and Bag6 genes are involved in multiple abiotic stress tolerances in Arabidopsis thaliana . Int J Mol Sci. 2021;22:5856. Zhou H, Li J, Liu X, Wei X, He Z, Hu L, et al. The divergent roles of the rice bcl-2 associated athanogene (Bag) genes in plant development and environmental responses. Plants. 2021;10:2169. Gu L, Hou B, Chen X, Wang Y, Chang P, He X et al. The Bcl-2-associated athanogene gene family in tobacco ( Nicotiana tabacum ) and the function of NtBAG5 in leaf senescence. Front Plant Sci. 2023; 14. Zhao K, Ren R, Ma X, Zhao K, Qu C, Cao D, et al. Genome-wide investigation of defensin genes in peanut ( Arachis hypogaea L.) reveals AhDef2.2 conferring resistance to bacterial wilt. Crop J. 2022;10:809–19. Tamura K, Stecher G, Kumar S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38:3022–7. Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. 2013; 3: 71–85. Doukhanina EV, Chen S, Van Der Zalm E, Godzik A, Reed J, Dickman MB. Identification and functional characterization of the BAG protein family in Arabidopsis thaliana . J Biol Chem. 2006;281:18793–801. Ai G, Si J, Cheng Y, Meng R, Wu Z, Xu R, et al. The oomycete-specific BAG subfamily maintains protein homeostasis and promotes pathogenicity in an atypical HSP70-independent manner. Cell Rep. 2023;42:113391. Bansal R, Kumawat S, Dhiman P, Sudhakaran S, Rana N, Jaswal R, et al. Evolution of Bcl-2 Anthogenes (BAG) as the regulators of cell death in wild and cultivated oryza species. J Plant Growth Regul. 2023;42:348–64. Dash A, Ghag SB. Genome-wide in silico characterization and stress induced expression analysis of BcL-2 associated athanogene (BAG) family in Musa spp. Sci Rep. 2022;12:625. Liang S, Wang F, Bao C, Han J, Guo Y, Liu F, et al. BAG2 ameliorates endoplasmic reticulum stress-induced cell apoptosis in Mycobacterium tuberculosis-infected macrophages through selective autophagy. Autophagy. 2020;16:1453–67. Alekhya C, Tejaswi A, Harika G, Bomma N, Gangashetty PI, Tyagi W et al. Identification and evaluation of BAG (B-cell lymphoma-2 associated athanogene) family gene expression in pigeonpea ( Cajanus cajan ) under terminal heat stress. Front Genet. 2024; 15. Rashid Mehmood Rana. Identification and characterization of the Bcl-2-associated athanogene (BAG) protein family in rice. Afr J Biotechnol. 2011; 11. Ma J, Wang R, Zhao H, Li L, Zeng F, Wang Y, et al. Genome-wide characterization of the VQ genes in Triticeae and their functionalization driven by polyploidization and gene duplication events in wheat. Int J Biol Macromol. 2023;243:125264. Bhat RS, Shirasawa K, Gangurde SS, Rashmi MG, Sahana K, Pandey MK. Genome-wide landscapes of genes and repeatome reveal the genomic differences between the two subspecies of peanut ( Arachis hypogaea ). Crop Des. 2023;2:100029. Farid B, Saddique MAB, Tahir MHN, Ikram RM, Ali Z, Akbar W. Expression divergence of BAG gene family in maize under heat stress. BMC Plant Biol. 2025;25:16. Cao Z, Li Z, Meng L, Cao D, Zhao K, Hu S, et al. Genome-wide characterization of pyrabactin resistance 1-like (PYL) family genes revealed AhPYL6 confer the resistance to Ralstonia solanacearum in peanut. Plant Physiol Biochem. 2024;217:109295. Han S, Zhou X, Shi L, Zhang H, Geng Y, Fang Y, et al. AhNPR3 regulates the expression of WRKY and PR genes, and mediates the immune response of the peanut ( Arachis hypogaea L). Plant J. 2022;110:735–47. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS14.xlsx Cite Share Download PDF Status: Published Journal Publication published 23 Apr, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Accepted 14 Apr, 2025 Reviews received at journal 11 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Submission checks completed at journal 29 Mar, 2025 First submitted to journal 29 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6118821","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":438175755,"identity":"b8ceeaa1-118a-4634-90bf-4b6d45698938","order_by":0,"name":"Kai Zhao","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zhao","suffix":""},{"id":438175756,"identity":"68ae472e-fb75-4f6a-b2f9-e3f6107fbe9f","order_by":1,"name":"Yanzhe Li","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanzhe","middleName":"","lastName":"Li","suffix":""},{"id":438175757,"identity":"222ee1e0-41ee-4c27-9fb0-abbb1ddcc897","order_by":2,"name":"Jinzhi Wang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jinzhi","middleName":"","lastName":"Wang","suffix":""},{"id":438175758,"identity":"23ef9221-24d0-4390-b847-25f4bb3a4b54","order_by":3,"name":"Yue Tu","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Tu","suffix":""},{"id":438175759,"identity":"742c3570-7960-44bd-92a2-a4aa39d81c92","order_by":4,"name":"Zenghui Cao","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zenghui","middleName":"","lastName":"Cao","suffix":""},{"id":438175760,"identity":"bf626c99-d924-43ab-8c8e-84e07a872791","order_by":5,"name":"Xingli Ma","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xingli","middleName":"","lastName":"Ma","suffix":""},{"id":438175761,"identity":"1f8c9de4-f7a1-46c4-b962-0780fca818d4","order_by":6,"name":"Fangping Gong","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fangping","middleName":"","lastName":"Gong","suffix":""},{"id":438175762,"identity":"f277d428-3c7e-4c93-8525-dde6325d201a","order_by":7,"name":"Zhongfeng Li","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhongfeng","middleName":"","lastName":"Li","suffix":""},{"id":438175763,"identity":"2cda4404-bc20-486f-aa6f-92464658363e","order_by":8,"name":"Lin Zhang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhang","suffix":""},{"id":438175764,"identity":"488fd584-866d-4499-a2b1-06721d1a2abd","order_by":9,"name":"Ding Qiu","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ding","middleName":"","lastName":"Qiu","suffix":""},{"id":438175765,"identity":"6d3ab18d-d360-43b9-81df-57be972e64ef","order_by":10,"name":"Xingguo Zhang","email":"","orcid":"","institution":"Henan Agricultural 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Yin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYDACZgYGCYaKA1AeG9FazpCkBQgkGNtI0WJwnPfgrZvz7siZs58xYPhQdpiBf3YDAS2H+ZKtc7c9M7bsyTFgnHHuMIPEnQOEtPCYSeduO5y44UCOATNv22EGA4kEYrTMOVy/4fwbA+a/xGtpOJxgcANoCyMxWiQP8xhb5xw7bLjhxrOCgz3n0nkkbhDQwnf+jOHtnJrD8gbnkzc++FFmLcc/g4AWhQNIHBCbB796IJBvIKhkFIyCUTAKRjwAAPkeRaq6HaIPAAAAAElFTkSuQmCC","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Dongmei","middleName":"","lastName":"Yin","suffix":""}],"badges":[],"createdAt":"2025-02-27 08:08:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6118821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6118821/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-06552-4","type":"published","date":"2025-04-23T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79913139,"identity":"01946c1f-4761-467d-bb62-8e0c85762572","added_by":"auto","created_at":"2025-04-04 11:59:59","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1223087,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships and genomic features of the peanut BAG gene family. (\u003cstrong\u003eA\u003c/strong\u003e) Phylogenetic reconstruction of BAG proteins. The evolutionary tree was generated using the Neighbor-Joining method (MEGA11) with BAG protein sequences from peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e, Ah), rice (\u003cem\u003eOryza sativa\u003c/em\u003e, Os), tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e, Sl), and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (At). Bootstrap support values (1000 replicates) are displayed at branch nodes. (\u003cstrong\u003eB\u003c/strong\u003e) Chromosomal distribution of AhBAG genes. Ten AhBAG genes were mapped to 11 chromosomes using the peanut genome database. Chromosomal positions are denoted by black scale bars. (\u003cstrong\u003eC\u003c/strong\u003e) Exon-intron architecture of AhBAG genes. Gene structures were visualized using GSDS 2.0, with coding sequences (CDS, blue rectangles), untranslated regions (UTR, red rectangles), and introns (black lines). Gene lengths are proportionally scaled (scale bar = 1000 bp).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6118821/v1/416e8b8ecf04009e3105339a.jpeg"},{"id":79915155,"identity":"5a85ae0c-7711-4641-83b3-de747442035d","added_by":"auto","created_at":"2025-04-04 12:23:59","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2641091,"visible":true,"origin":"","legend":"\u003cp\u003eConserved domain architecture and structural analysis of peanut BAG proteins.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Domain organization of AhBAG proteins. All AhBAG proteins were confirmed to harbor a conserved BAG domain (deep blue rectangle) through the NCBI Conserved Domain Database (CDD). Notably, six members (Ah35MDCZ, Ah9JS9HJ, AhTEF0AP, AhADAM8V, Ah37JSRQ, and AhCF9SCE) exhibited an additional ubiquitin-like domain (red rectangle). Domain positions are annotated based on amino acid coordinates, with a scale bar representing 200 amino acids. (\u003cstrong\u003eB-C\u003c/strong\u003e) Identification of conserved motifs in AhBAG proteins. MEME analysis (E-value \u0026lt; 1e-5) revealed 10 conserved motifs (Motifs 1-10), each depicted in distinct colors. The scale bar corresponds to 200 amino acids. (\u003cstrong\u003eD\u003c/strong\u003e) Comparative three-dimensional structural modeling of BAG proteins. Homology-based structures of \u003cem\u003eArabidopsis\u003c/em\u003e AtBAG6 and peanut AhYSFV0U were generated using SWISS-MODEL, demonstrating high structural similarity. Molecular visualization was performed using PyMOL 2.5.2.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6118821/v1/a91a3f505af12d8e216c8b5e.jpeg"},{"id":79913136,"identity":"e6ebb280-2784-4188-b927-bf50d6f54a98","added_by":"auto","created_at":"2025-04-04 11:59:59","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1801891,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic evolution and functional regulatory features of BAG genes in peanut.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Collinearity network of BAG genes in the allopolyploid peanut genome. Homology relationships among BAG genes were analyzed across cultivated peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e, \u003cem\u003eA. hypogaea\u003c/em\u003e), its diploid progenitors (\u003cem\u003eA. duranensis \u003c/em\u003eand \u003cem\u003eA. ipaensis\u003c/em\u003e), wild tetraploid \u003cem\u003eA. monticola\u003c/em\u003e (\u003cem\u003eA. mon\u003c/em\u003e), and soybean (\u003cem\u003eGlycine max\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e) using MCScanX. Colored curves connect syntenic gene pairs, highlighting evolutionary conservation across species. (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eCis\u003c/em\u003e-regulatory element distribution in AhBAG promoters. Promoter regions (2,000 bp upstream) of 13 AhBAG genes were screened using the PlantCARE database, with \u003cem\u003ecis\u003c/em\u003e-element abundance visualized via TBtools. Key regulatory motifs (stress-responsive and hormone-related elements) are annotated. (\u003cstrong\u003eC\u003c/strong\u003e) Tissue-specific expression profiles of \u003cem\u003eAhBAG\u003c/em\u003e genes. Transcriptome-derived heatmap displays expression levels across nine tissues/developmental stages: leaf, root, nodule, perianth, stamen, pistil, peg tip, fruit pat, and seed pat. Expression values (log2-transformed FPKM) are color-coded, with gradients reflecting relative transcriptional activity.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6118821/v1/5b1acb307e9603cb8ab130a8.jpeg"},{"id":79913647,"identity":"bcd09c5b-903d-4f77-92c7-c4fd92e0fc3a","added_by":"auto","created_at":"2025-04-04 12:07:59","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":922457,"visible":true,"origin":"","legend":"\u003cp\u003eExpression regulation of peanut BAG genes under biotic stress and hormone treatments. (\u003cstrong\u003eA-D\u003c/strong\u003e) Dynamic expression profiles of \u003cem\u003eAhBAG\u003c/em\u003e genes based on qRT-PCR. The relative expression levels of four AhBAG genes (\u003cem\u003eAhYSVF0U\u003c/em\u003e, \u003cem\u003eAhTEF0AP\u003c/em\u003e, \u003cem\u003eAh37JSRQ\u003c/em\u003e, and \u003cem\u003eAhCF9SCE\u003c/em\u003e) were assessed at three key time points (0 d, 1 dpi, and 7 dpi) following infection with \u003cem\u003eRalstonia solanacearum\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e), treatment with abscisic acid (ABA) (\u003cstrong\u003eB\u003c/strong\u003e), salicylic acid (SA) (\u003cstrong\u003eC\u003c/strong\u003e), and methyl jasmonate (MeJA) (\u003cstrong\u003eD\u003c/strong\u003e). Relative expression levels were calculated using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method (n=3 biological replicates). Error bars represent the standard error of the mean (SEM), and statistical differences were analyzed using one-way ANOVA in GraphPad Prism 8.0 (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). NS indicates no significant difference.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6118821/v1/2f621bf5fcaeac529d13b4bc.jpeg"},{"id":79913144,"identity":"f8fa2e8a-773d-4df6-80f6-cee6a8f46483","added_by":"auto","created_at":"2025-04-04 11:59:59","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1002763,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction network and functional prediction analysis of peanut BAG proteins.\u003cstrong\u003e \u003c/strong\u003eNodes represent target proteins, while edges indicate predicted interaction relationships. The color of the nodes corresponds to the classification of different subfamilies of BAG proteins.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6118821/v1/5f6bba1210c262c61f964298.jpeg"},{"id":81569571,"identity":"3c82c0e2-8196-4c22-8d17-1e1cfe0cca92","added_by":"auto","created_at":"2025-04-28 16:07:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8461997,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118821/v1/b4e1034c-2904-4dae-9fa7-24d3c470baf4.pdf"},{"id":79913645,"identity":"0e59f8cf-b114-4e4f-8064-735bca5527cb","added_by":"auto","created_at":"2025-04-04 12:07:59","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":43918,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS14.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6118821/v1/16a3bdd67f7c9e7a5ca77b41.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide characterization of AhBAG genes in peanut reveals their role in bacterial wilt resistance and hormone response","fulltext":[{"header":"Background","content":"\u003cp\u003ePeanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.), a vital oilseed and cash crop, sustains global agricultural economies and food security [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In 2023, the global peanut cultivation area reached approximately 31\u0026nbsp;million hectares, with an average yield of 1,755 kilograms per hectare. The total peanut production amounted to 54.3\u0026nbsp;million tons, showing an upward trend [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite its nutritional and economic value, peanut productivity is severely constrained by biotic stresses, particularly bacterial wilt (BW) caused by \u003cem\u003eRalstonia solanacearum\u003c/em\u003e (\u003cem\u003eR. solanacearum\u003c/em\u003e). This soil-borne pathogen, notorious as the \u0026ldquo;plant cancer\u0026rdquo;, colonizes vascular tissues via root wounds, secreting extracellular polysaccharides and cell wall-degrading enzymes to obstruct water transport, ultimately leading to wilting, chlorosis, and characteristic bacterial ooze in infected plants [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The disease prevails in tropical and subtropical regions, with infection rates reaching 80% in severe cases, causing catastrophic yield losses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although resistant cultivars like Yuanza 9102 and Yueyou 92 have reduced incidence to \u0026lt;\u0026thinsp;8% in endemic areas, the narrow genetic basis of resistance and limited understanding of molecular mechanisms hinder further breeding progress [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePeanut resistance to BW is a polygenic trait involving complex host-pathogen interactions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Upon infection, \u003cem\u003eR. solanacearum\u003c/em\u003e employs chemotaxis to invade roots, forming biofilms in xylem vessels to evade host defenses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Molecular breeding efforts have identified key quantitative trait loci (QTLs), including qBWB02.1 on chromosome B02, which co-localizes with NBS-LRR resistance gene clusters [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Transcriptomic analyses highlight the involvement of MAPK cascades, WRKY transcription factors, and m\u003csup\u003e6\u003c/sup\u003eA RNA modification in defense responses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Notably, overexpression of \u003cem\u003eAhRLK1\u003c/em\u003e and \u003cem\u003eAhRRS5\u003c/em\u003e, homologs of \u003cem\u003eArabidopsis\u003c/em\u003e immune regulators, enhances resistance in transgenic tobacco [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recent breakthroughs include synthetic hexaploid peanuts derived from wild \u003cem\u003eArachis\u003c/em\u003e species, demonstrating superior wilt resistance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the precise regulatory networks governing these resistance pathways remain elusive, underscoring the need to explore novel gene families involved in stress adaptation.\u003c/p\u003e \u003cp\u003eThe Bcl-2-associated athanogene (BAG) family, evolutionarily conserved across eukaryotes, serves as a critical nexus for stress signaling and cell survival [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Characterized by a C-terminal BAG domain that interacts with HSP70/HSC70 chaperones, these proteins function as co-chaperones to modulate protein folding and degradation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Plant BAG proteins exhibit unique structural diversification: Group I members (\u003cem\u003eAtBAG1-4\u003c/em\u003e) harbor ubiquitin-like domains for stress adaptation, while Group II (\u003cem\u003eAtBAG5-7\u003c/em\u003e) contains IQ motifs for calcium-mediated signaling [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Functional studies demonstrate their roles in thermotolerance (\u003cem\u003eAtBAG7\u003c/em\u003e-WRKY29 interaction [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]), salinity resistance (OsBAG4-DNA methylation complex [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]), and pathogen defense (\u003cem\u003eAtBAG6\u003c/em\u003e-mediated autophagy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]). Hormonal crosstalk further links BAGs to abscisic acid (ABA) and ethylene pathways, as evidenced by promoter \u003cem\u003ecis\u003c/em\u003e-elements and stress-inducible expression patterns [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Despite their characterized roles in model plants, the BAG family remains unexplored in peanuts-a gap that limits the exploitation of this regulatory hub for wilt resistance. Given their dual functions in chaperone-mediated protein homeostasis and programmed cell death (PCD), peanut BAG genes likely orchestrate critical defense responses against \u003cem\u003eR. solanacearum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThis study aims to fill this knowledge gap by performing a systematic genome-wide identification and functional characterization of the BAG gene family in peanut. Through integrative bioinformatics and experimental approaches, we elucidate the evolutionary relationships, structural diversification, and stress-responsive expression dynamics of \u003cem\u003eAhBAG\u003c/em\u003e genes. Our findings provide valuable insights into the molecular mechanisms underlying BAG-mediated stress adaptation in peanut and lay the foundation for developing molecular markers and genetic engineering strategies to improve stress resilience in this vital crop.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and experimental treatments\u003c/h2\u003e \u003cp\u003eTwo peanut cultivars-highly resistant (H108) and susceptible (H107) to BW-developed by Prof. Yin\u0026rsquo;s team at Henan Agricultural University were selected. Mature seeds were surface-sterilized with 0.1% HgCl₂ for 2 min, rinsed three times with sterile water, and germinated on moist filter paper at 25\u0026deg;C in darkness until radicle emergence (~\u0026thinsp;36 h). Germinated seeds were transferred to sterilized vermiculite and cultivated in a climate-controlled chamber (37\u0026deg;C, 70% relative humidity, darkness) until the three-leaf stage. \u003cem\u003eR. solanacearum\u003c/em\u003e inoculation: Root irrigation was performed with a bacterial suspension (1\u0026times;10⁸ CFU/mL) following Zhao et al[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Hormone induction: Foliar sprays of 100 \u0026micro;M ABA, 2 mM SA, or 50 \u0026micro;M MeJA were applied[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Three biological replicates per treatment were harvested at 0 d, one day post-inoculation (dpi), and seven dpi post-treatment, flash-frozen in liquid nitrogen, and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentification and characterization of AhBAG genes\u003c/h3\u003e\n\u003cp\u003eBLASTP and HMMER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/hmmer/search/phmmer\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/hmmer/search/phmmer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PF02179 domain, E-value\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026times;10⁻⁵) were employed against the Tifrunner v2 genome (PeanutBase, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.peanutbase.org/\u003c/span\u003e\u003cspan address=\"https://www.peanutbase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using \u003cem\u003eArabidopsis\u003c/em\u003e BAG proteins (TAIR) as queries. Candidate genes were validated via NCBI Conserved Domain Database (CDD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for domain integrity. Physicochemical properties (molecular weight, pI) were analyzed using ExPASy ProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and subcellular localization was predicted via Wolf PSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genscript.com/wolf-psort.html\u003c/span\u003e\u003cspan address=\"https://www.genscript.com/wolf-psort.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhylogenetic and structural analysis\u003c/h3\u003e\n\u003cp\u003eBAG protein sequences from Arabidopsis (TAIR), tomato (Phytozome), and rice (NCBI) were aligned using MUSCLE in MEGA11 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A neighbor-joining phylogenetic tree (1,000 bootstrap replicates) was visualized with iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene structures and chromosomal locations were mapped via TBtools. MEME was used to identify conserved motifs in the promoter regions of target genes. The analysis was performed with the following parameters: number of motifs\u0026thinsp;=\u0026thinsp;10, motif width range\u0026thinsp;=\u0026thinsp;6-100 bp, and E-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Protein-protein interaction networks were generated using the STRING database (version 11.5, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Interactions with a confidence score\u0026thinsp;\u0026gt;\u0026thinsp;0.7 (high confidence) were retained.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollinearity and\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-regulatory element analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eInterspecies collinearity between peanut, \u003cem\u003eArabidopsis\u003c/em\u003e, and soybean BAG genes was analyzed using TBtools. Promoter sequences (~\u0026thinsp;2,000 bp upstream) of AhBAGs were retrieved from PeanutBase, and \u003cem\u003ecis\u003c/em\u003e-elements were annotated via PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eTissue-specific expression profiling\u003c/h3\u003e\n\u003cp\u003ePublic RNA-seq data from nine Tifrunner tissues (leaf, root, nodule, perianth, stamen, pistil, peg tip, fruit pat, and seed pat) were obtained from PeanutBase. Expression heatmaps were generated using TBtools.\u003c/p\u003e\n\u003ch3\u003equantitative real-time PCR validation\u003c/h3\u003e\n\u003cp\u003eRoot tissues from H108 and H107 seedlings were collected at 0, 1, and 7 days post-inoculation (dpi) under mock inoculation (ddH\u003csub\u003e2\u003c/sub\u003eO-treated control) and \u003cem\u003eR. solanacearum\u003c/em\u003e-infected conditions. For phytohormone treatments, three-week-old H108 and H107 seedlings were foliar-sprayed with SA, MeJA, ABA, or ddH\u003csub\u003e2\u003c/sub\u003eO (control). Leaf samples were collected at 0, 1, and 7 dpi post-treatment. All samples were collected in triplicate biological replicates, flash-frozen in liquid nitrogen, and stored at -80\u0026deg;C until analysis. First-strand cDNA was synthesized using the PrimeScript\u0026trade; RT Reagent Kit (Takara, Dalian, China) following the manufacturer\u0026rsquo;s protocol. qRT-PCR was subsequently performed using the synthesized cDNA templates, and amplified with gene-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) using SYBR Green I (TB Green Premix Ex Taq II, TaKaRa) on a CFX96 Touch\u0026trade; system (Bio-Rad). Cycling conditions: 95\u0026deg;C for 30 s; 40 cycles of 95\u0026deg;C for 5 s, 58\u0026deg;C for 30 s. Melt curves confirmed specificity. The relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method (n\u0026thinsp;=\u0026thinsp;3 biological replicates), \u003cem\u003eActin7\u003c/em\u003e was used as the reference gene[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Error bars represent the standard error of the mean (SEM). Statistical differences were analyzed by one-way ANOVA using GraphPad Prism 8.0 (* \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGenome-wide identification and characterization of BAG genes in peanut\u003c/h2\u003e \u003cp\u003eThrough systematic bioinformatic analysis of the published Tifrunner genome, we identified 13 putative BAG family genes in cultivated peanut. The coding sequence (CDS) lengths of these genes ranged from 558 bp (\u003cem\u003eAh4JG1SZ\u003c/em\u003e) to 3,711 bp (\u003cem\u003eAhYSVF0U\u003c/em\u003e), encoding proteins of 185 to 1,236 amino acids (Table S2). The predicted molecular weights of BAG proteins exhibited considerable variation, spanning from 21.51 kDa (\u003cem\u003eAhJ75SJQ\u003c/em\u003e) to 139.18 kDa (\u003cem\u003eAhYSVF0U\u003c/em\u003e), with theoretical isoelectric points (pI) ranging from 4.89 to 9.50. Notably, eight BAG proteins were classified as acidic (pI\u0026thinsp;\u0026lt;\u0026thinsp;7) while five exhibited basic properties (pI\u0026thinsp;\u0026gt;\u0026thinsp;7), suggesting functional diversity in their molecular interactions. Protein stability analysis revealed that four members (\u003cem\u003eAhTEF0AP\u003c/em\u003e, \u003cem\u003eAhADAM8V\u003c/em\u003e, \u003cem\u003eAh37JSRQ\u003c/em\u003e, and \u003cem\u003eAhCF9SCE\u003c/em\u003e) with instability indices below 40 were predicted as stable proteins, whereas the remaining nine members (instability index\u0026thinsp;\u0026gt;\u0026thinsp;40) were categorized as unstable proteins. The aliphatic index, an indicator of thermostability, ranged from 59.68 to 81.14 across the family. All 13 BAG proteins displayed negative grand average of hydropathicity values (-0.42 to -1.05), confirming their hydrophilic nature. Subcellular localization predictions using multiple algorithms (WoLF PSORT, Plant-mPLoc, and CELLO) indicated distinct compartmentalization patterns. The majority of AhBAG proteins were predicted to localize in chloroplasts, nuclei and peroxisomes (Table S2). This differential subcellular distribution implies specialized functional roles for BAG family members in various cellular compartments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenetic evolution and chromosomal distribution of AhBAG genes\u003c/h3\u003e\n\u003cp\u003eTo elucidate evolutionary relationships and functional diversification among BAG family members, a phylogenetic tree was reconstructed using full-length protein sequences from peanut (13 genes), \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (7 genes), \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (10 genes), and \u003cem\u003eOryza sativa\u003c/em\u003e (6 genes). Neighbor Joining analysis with 1,000 bootstrap replicates resolved the BAG proteins into four distinct clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; Table S3). Clade I contained two dicot-specific members (AtBAG7 and SlBAG10). Clade II included 14 family members (2 AtBAGs, 2 OsBAGs, 4 SlBAGs, and 6 AhBAGs). Clade III exhibited conserved orthology across species, including two peanut members (Ah35MDCZ and Ah9JS9HJ), whereas Clade IV showed the highest diversity, harboring 14 members with significant expansion in peanut (5 AhBAGs) and \u003cem\u003eArabidopsis\u003c/em\u003e (3 AtBAGs). This phylogenetic topology suggests both conserved evolutionary patterns and lineage-specific diversification events. Chromosomal mapping revealed that 13 AhBAG genes were unevenly distributed across 11 of 20 peanut chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Chromosomes 4 and 14 each harbored two paralogous gene pairs (\u003cem\u003eAh4JG1SZ\u003c/em\u003e / \u003cem\u003eAhJ75SJQ\u003c/em\u003e and \u003cem\u003eAhD7MIWJ\u003c/em\u003e / \u003cem\u003eAhG65IU1\u003c/em\u003e, respectively), likely resulting from segmental duplication events. The dispersed genomic distribution pattern implies multiple duplication mechanisms, contributed to BAG family expansion in peanut.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene structure analysis uncovered significant exon-intron architecture variation across subfamilies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Clade II members displayed the simplest structures, with 62% (4/6) containing a single exon (\u003cem\u003eAhD7MIWJ\u003c/em\u003e, \u003cem\u003eAhG65IU1\u003c/em\u003e, \u003cem\u003eAh4JG1SZ\u003c/em\u003e, and \u003cem\u003eAhJ75SJQ\u003c/em\u003e) and one member (\u003cem\u003eAhYSVF0U\u003c/em\u003e) exhibiting three exons. In contrast, Clade IV genes showed conserved four-exon structures (5/5 members), while Clade III members displayed intermediate complexity (4 exons in \u003cem\u003eAh35MDCZ\u003c/em\u003e / \u003cem\u003eAh9JS9HJ\u003c/em\u003e). The inverse correlation between phylogenetic divergence and structural complexity suggests that exon loss/gain events may have driven functional specialization during BAG family evolution.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDomain architecture and motif diversification of AhBAG proteins\u003c/h2\u003e \u003cp\u003eThe functional diversity of BAG proteins is intrinsically linked to their conserved domain composition [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our analysis using InterProScan and SMART databases revealed that all 13 AhBAG proteins possess the hallmark BAG domain, which mediates interactions with Hsp70 ATPase to regulate chaperone activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Notably, two paralogous genes (AhYSVF0U and AhII6FYR) exhibited an N-terminal IQ domain, a calcium-sensing module critical for linking chaperone activity with Ca\u0026sup2;⁺ signaling[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Six members (Ah35MDCZ, Ah9JS9HJ, AhTEF0AP, AhADAM8V, Ah37JSRQ, and AhCF9SCE) contained a C-terminal ubiquitin-like (UBQ) domain, known to facilitate protein degradation and stress response [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Strikingly, AhED3VG4 displayed a truncated UBQ domain due to a frameshift mutation in its coding sequence, suggesting potential neofunctionalization or pseudogenization during evolution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlant BAG genes typically encode a BAG domain of 70\u0026ndash;80 amino acid residues [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Analysis of conserved motifs further elucidated genetic variations among peanut BAG proteins. Ten motifs were identified across the 13 AhBAGs, predominantly composed of hydrophobic (valine V, isoleucine I, leucine L) and charged residues (glutamic acid E, aspartic acid D, arginine R, lysine K) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). All AhBAGs shared motif 2 and motif 3, which are likely essential for BAG protein functionality, as evidenced by their consistent presence in phylogenetically clustered members. Group IV proteins contained eight motifs (motif 1\u0026ndash;8), with UBQ-containing members encoding complete BDs exhibiting these motifs, highlighting structural conservation. Phylogenetic analysis revealed that AhYSVF0U in Group III shares high amino acid sequence similarity with AtBAG6. Structural predictions showed that the IQ-calmodulin binding motif comprises two small α-helices connected by a hairpin loop, which interfaces with the first helix of the BAG domain-a tripartite antiparallel α-helical bundle. Intriguingly, AhYSVF0U harbors two α-helices in its IQ-calmodulin motif, contrasting with the single helix observed in AtBAG6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome synteny and\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-regulatory element analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe evolutionary conservation of BAG genes across peanut species was investigated through comparative synteny analysis. Thirteen AhBAG genes in cultivated peanut (\u003cem\u003eA. hypogaea\u003c/em\u003e) exhibited orthologous counterparts in diploid wild species (\u003cem\u003eA. duranensis\u003c/em\u003e and \u003cem\u003eA. ipaensis\u003c/em\u003e) and tetraploid wild species (\u003cem\u003eA. monticola\u003c/em\u003e), suggesting functional conservation during domestication (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Notably, seven \u003cem\u003eAhBAG\u003c/em\u003e genes distributed across seven chromosomes showed syntenic relationships with \u003cem\u003eAtBAG\u003c/em\u003e homologs in \u003cem\u003eArabidopsis\u003c/em\u003e. For instance, \u003cem\u003eAhYSVF0U\u003c/em\u003e shared collinearity with \u003cem\u003eAtBAG6\u003c/em\u003e, a key regulator of fungal resistance, implying conserved roles in stress adaptation. This evolutionary conservation underscores the potential functional retention of AhBAG genes in mediating stress responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCis\u003c/em\u003e-regulatory element profiling of AhBAG promoters revealed significant enrichment of hormone-responsive motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Ten genes harbored ABRE (abscisic acid-responsive element), with \u003cem\u003eAhYSVF0U\u003c/em\u003e and \u003cem\u003eAhADAM8V\u003c/em\u003e containing five ABRE copies each, while seven genes possessed CGTCA-motifs (methyl jasmonate-responsive). \u003cem\u003eAh37JSRQ\u003c/em\u003e uniquely carried three MeJA-responsive elements, aligning with its rapid induction under MeJA treatment. Additionally, four AhBAG promoters contained W-box elements, which are critical for defense-related transcriptional reprogramming upon pathogen-associated molecular pattern (PAMP) recognition. These findings suggest that AhBAG genes are transcriptionally regulated through ABA/JA signaling cascades and may participate in pathogen defense.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTissue-specific expression and stress-responsive dynamics\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAhBAG\u003c/em\u003e genes exhibited distinct spatiotemporal expression patterns across tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Table S4). The expression levels of different genes vary significantly across tissues. \u003cem\u003eAhYSVF0U\u003c/em\u003e shows relatively high expression in multiple tissues, reaching 177.89 in the pistil, with elevated values also observed in leaf, root, and nodule tissues. In contrast, \u003cem\u003eAhD7MIWJ\u003c/em\u003e exhibit extremely low expression in most tissues, with no detectable expression in root, nodule, or perianth tissues, and only minimal expression in leaf and peg tip. Additionally, some genes display strong tissue-specific expression patterns. \u003cem\u003eAhII6FYR\u003c/em\u003e shows markedly high expression in the pistil, significantly exceeding levels in other tissues like the perianth. \u003cem\u003eAhTEF0AP\u003c/em\u003e is expressed at 44.21 in the perianth, while maintaining relatively uniform but lower levels across other tissues. These findings suggest that these genes may play critical roles in the growth, development, or functional specialization of specific tissues.\u003c/p\u003e \u003cp\u003eUnder biotic and abiotic stresses, qRT-PCR revealed genotype- and time-dependent expression dynamics. In resistant cultivar H108, \u003cem\u003eAhYSVF0U\u003c/em\u003e were upregulated 5-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in roots at 7 dpi of \u003cem\u003eR. solanacearum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), consistent with their predicted roles in restricting pathogen spread via programmed cell death (PCD). ABA treatment triggered an 8-fold induction of \u003cem\u003eAhYSVF0U\u003c/em\u003e in H108 leaves at 1 dpi, whereas MeJA rapidly activated \u003cem\u003eAhTEF0AP\u003c/em\u003e and \u003cem\u003eAh37JSRQ\u003c/em\u003e (4-fold increase, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D). Intriguingly, salicylic acid (SA) suppressed \u003cem\u003eAhYSVF0U\u003c/em\u003e expression in H108 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), highlighting antagonistic crosstalk between SA and ABA/JA pathways in modulating BAG-mediated stress responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProtein interaction networks and functional associations\u003c/h2\u003e \u003cp\u003eSTRING-based protein interaction prediction unveiled a multifaceted regulatory network centered on AhYSVF0U (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Ah0J9HE3, a HSP20-family chaperone implicated in thermotolerance, suggesting AhYSVF0U may stabilize stress-denatured proteins. AhTGXT8E, a xylanase inhibitor that fortifies cell walls by neutralizing pathogen-derived xylanases. AhK7D5N2, a calmodulin protein interacting with IQ motifs to transduce Ca\u003csup\u003e2+\u003c/sup\u003e signals during immune responses. This network positions AhYSVF0U as a signaling hub integrating chaperone activity, cell wall reinforcement, and calcium signaling to orchestrate multi-layered stress adaptation. Notably, the absence of direct interactions with canonical immune receptors implies that AhBAGs operate downstream of pathogen recognition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe BAG family comprises evolutionarily conserved eukaryotic proteins that orchestrate stress signaling and cell survival pathways [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This study presents the inaugural genome-wide identification and spatiotemporal expression profiling of BAG genes in peanut, a globally significant oilseed crop vulnerable to devastating biotic constraints such as BW caused by \u003cem\u003eR. solanacearum\u003c/em\u003e. Our multi-dimensional analysis elucidates the evolutionary trajectories and stress-responsive regulatory networks of \u003cem\u003eAhBAG\u003c/em\u003e genes, providing molecular blueprints for developing wilt-resistant peanut cultivars through precision breeding.\u003c/p\u003e \u003cp\u003ePhylogenetic reconstruction of BAG orthologs from taxonomically diverse species (peanut, \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], tomato[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], rice [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] revealed both ancestral conservation and species-specific divergence patterns. The four-clade classification of AhBAG proteins reflects functional diversification shaped by evolutionary selection pressures. The non-random chromosomal distribution and paralogous gene clusters suggest segmental duplication-driven family expansion, a well-documented evolutionary mechanism for environmental adaptation in plant genomes[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Bhat et al. detected significant chromosomal collinearity between the A and B subgenomes, particularly in the third (A03/B03) and fifth (A05/B05) chromosomes, which exhibited highly conserved gene content and arrangement order [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Similarly, through comparative genomic analysis, this study revealed that the 11 AhBAG genes displayed strict syntenic relationships in their genomic distribution, suggesting functional conservation of these genes during peanut domestication and evolution. Cross-species synteny conservation with wild peanut progenitors (\u003cem\u003eA. duranensis\u003c/em\u003e, \u003cem\u003eA. ipaensis\u003c/em\u003e) and \u003cem\u003eArabidopsis\u003c/em\u003e homologs supports functional retention of ancestral stress-adaptive mechanisms. Of particular note, AhYSVF0U demonstrates evolutionary synteny with AtBAG6, a canonical fungal resistance regulator, implying phylogenetic preservation of pathogen defense functions in peanut.\u003c/p\u003e \u003cp\u003eStructural characterization revealed clade-specific domain architectures among AhBAG proteins, with conserved BAG and ubiquitin-like domains interspersed with lineage-specific motifs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Phylogeny-correlated motif organization provides structural evidence for functional specialization during speciation. Subcellular localization predictions revealed differential targeting of AhBAG proteins to chloroplasts, nuclei, and peroxisomes, suggesting compartmentalized functional specialization. Chloroplast-localized members may regulate photosynthetic machinery protection, while nuclear isoforms could mediate stress-responsive transcriptional regulation, consistent with their predicted molecular functions.\u003c/p\u003e \u003cp\u003eTranscriptional profiling under biotic/abiotic stresses uncovered genotype- and time-dependent expression dynamics. The pathogen-inducible expression of \u003cem\u003eAhYSVF0U\u003c/em\u003e in resistant cultivar H108 (5-fold upregulation at 7 dpi) strongly implicates its functional involvement in PCD-mediated pathogen containment, paralleling established BAG-PCD regulatory mechanisms [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The rapid MeJA responsiveness of \u003cem\u003eAhTEF0AP\u003c/em\u003e and \u003cem\u003eAh37JSRQ\u003c/em\u003e (4-fold induction at 1 dpi) corroborates their predicted roles in oxylipin signaling cascades. The discovery that ABA upregulates \u003cem\u003eAhYSVF0U\u003c/em\u003e and that SA inhibits \u003cem\u003eAhYSVF0U\u003c/em\u003e aligns with known signaling pathways in peanut defense mechanisms. Under \u003cem\u003eR. solanacearum\u003c/em\u003e infection, the ABA receptor PYL family gene \u003cem\u003eAhPYL6\u003c/em\u003e was significantly upregulated, thereby enhancing plant resistance to the pathogen through promotion of defense-related gene expression [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. When plants were treated with SA, the \u003cem\u003eAhNPR3A\u003c/em\u003e mutation exhibited no effect on the induction of pathogenesis-related (PR) genes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Notably, SA-mediated suppression of \u003cem\u003eAhYSVF0U\u003c/em\u003e reveals antagonistic phytohormone crosstalk, highlighting the intricate hormonal regulation of BAG-mediated stress responses. The predicted interactome of \u003cem\u003eAhYSVF0U\u003c/em\u003e with HSP20 chaperones, xylanase inhibitors, and calcium sensors suggests its central role in integrating proteostasis maintenance, cell wall fortification, and Ca\u003csup\u003e2+\u003c/sup\u003e signaling into a coordinated stress mitigation network.\u003c/p\u003e \u003cp\u003eThis systematic investigation establishes the molecular foundation for deciphering BAG-mediated stress adaptation in peanut. Future investigations should prioritize functional characterization through transgenic overexpression approaches. The development of transgenic peanut overexpressing stress-responsive \u003cem\u003eAhYSVF0U\u003c/em\u003e could offer valuable resources for breeding wilt-resistant cultivars and improving peanut productivity under adverse conditions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we systematically identified and analyzed the characteristics of BAG family genes in peanutand their roles in responses to biotic and abiotic stresses through whole-genome analysis. Using bioinformatics approaches, we identified a total of 13 AhBAG gene family members, which are widely distributed across the 11 chromosomes of peanut. Further analysis of expression patterns revealed distinct expression differences of \u003cem\u003eAhBAG\u003c/em\u003e genes among various peanut tissues, with some genes showing higher expression levels in vegetative organs, while others are significantly expressed in reproductive organs. Moreover, these genes exhibit multi-level regulatory functions under conditions of \u003cem\u003eR. solanacearum\u003c/em\u003e infection and various abiotic stresses. Notably, the \u003cem\u003eAhYSVF0U\u003c/em\u003e gene shows significant regulatory potential in disease resistance responses and can be considered a priority candidate gene for peanut disease-resistant breeding. These findings provide a foundation for further investigating the biological functions and molecular mechanisms of BAG genes in peanut stress responses and offer important genetic resources for peanut disease-resistant breeding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Zhenyu Wang, Plant Protection Institute of Henan Academy of Agricultural Sciences for the BW strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was designed and directed by D.Y.; K.Z., Y.L., J.W. and X.M. coordinated research; F. G., Y.T. and Z.L. analyzed the sequencing data; Z.C. and L.Z. provided reagents; figures were prepared by D.Q. and K.Z. with support from X.Z., R.R.; D.Y. and K.Z. interpreted the results, and wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Youth Project of Natural Science Foundation of Henan (242300420477), the Key Program of National Natural Science Foundation of China (NSFC)-Henan United Fund (No.U22A20475), Key Scientific and Technological Project of Henan Province (No.221111110500; HARS-22-05-G1), National Key Research and Development Program (2023YFD1202800), the Henan Agricultural University High Level Talent (30501418), the Key Scientific Research Project in Colleges and Universities of Henan Province of China (25A210004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed for this study are included in the manuscript and its supplementary information fles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHuai D, Xue X, Wu J, Pandey MK, Liu N, Huang L, et al. 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Front Genet. 2024; 15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRashid Mehmood Rana. Identification and characterization of the Bcl-2-associated athanogene (BAG) protein family in rice. Afr J Biotechnol. 2011; 11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa J, Wang R, Zhao H, Li L, Zeng F, Wang Y, et al. Genome-wide characterization of the VQ genes in \u003cem\u003eTriticeae\u003c/em\u003e and their functionalization driven by polyploidization and gene duplication events in wheat. Int J Biol Macromol. 2023;243:125264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhat RS, Shirasawa K, Gangurde SS, Rashmi MG, Sahana K, Pandey MK. Genome-wide landscapes of genes and repeatome reveal the genomic differences between the two subspecies of peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e). Crop Des. 2023;2:100029.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarid B, Saddique MAB, Tahir MHN, Ikram RM, Ali Z, Akbar W. Expression divergence of BAG gene family in maize under heat stress. BMC Plant Biol. 2025;25:16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao Z, Li Z, Meng L, Cao D, Zhao K, Hu S, et al. Genome-wide characterization of pyrabactin resistance 1-like (PYL) family genes revealed \u003cem\u003eAhPYL6\u003c/em\u003e confer the resistance to \u003cem\u003eRalstonia solanacearum\u003c/em\u003e in peanut. Plant Physiol Biochem. 2024;217:109295.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan S, Zhou X, Shi L, Zhang H, Geng Y, Fang Y, et al. \u003cem\u003eAhNPR3\u003c/em\u003e regulates the expression of WRKY and PR genes, and mediates the immune response of the peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L). Plant J. 2022;110:735\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Peanut, Bacterial wilt, BAG gene family, Expression profile","lastPublishedDoi":"10.21203/rs.3.rs-6118821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6118821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eThe BAG gene family, encoding Bcl-2-associated anti-apoptotic proteins, plays pivotal roles in regulating plant growth, development, and stress responses. Peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.), a globally significant oilseed and cash crop, is highly valued for its economic importance. However, systematic genome-wide analysis and functional characterization of the BAG gene family in peanut remain largely unexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e In this study, we identified 13 \u003cem\u003eAhBAG\u003c/em\u003e genes in the peanut genome, which are unevenly distributed across 11 chromosomes. Phylogenetic analysis revealed that these \u003cem\u003eAhBAG\u003c/em\u003egenes, together with BAG family members from other plant species, are classified into four distinct clades, underscoring their evolutionary conservation. Segmental duplication was identified as a major driver of the expansion of the \u003cem\u003eAhBAG\u003c/em\u003e gene family. Notably, \u003cem\u003eAhYSVF0U\u003c/em\u003e exhibited significant upregulation under \u003cem\u003eRalstonia solanacearum\u003c/em\u003e infection and abscisic acid treatment, suggesting its potential involvement in mediating peanut resistance to bacterial wilt.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e This study provides comprehensive insights into the evolutionary and functional characteristics of the peanut BAG gene family and offers valuable genetic resources for molecular breeding programs aimed at improving stress tolerance in peanut.\u003c/p\u003e","manuscriptTitle":"Genome-wide characterization of AhBAG genes in peanut reveals their role in bacterial wilt resistance and hormone response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 11:59:54","doi":"10.21203/rs.3.rs-6118821/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-04-14T05:05:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-11T09:47:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22653665890821964496222949317755109864","date":"2025-04-04T00:02:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T23:59:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-29T09:29:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-03-29T08:19:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d5052d50-7250-460d-a8a3-52d1bc885ef1","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T16:00:17+00:00","versionOfRecord":{"articleIdentity":"rs-6118821","link":"https://doi.org/10.1186/s12870-025-06552-4","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-04-23 15:57:18","publishedOnDateReadable":"April 23rd, 2025"},"versionCreatedAt":"2025-04-04 11:59:54","video":"","vorDoi":"10.1186/s12870-025-06552-4","vorDoiUrl":"https://doi.org/10.1186/s12870-025-06552-4","workflowStages":[]},"version":"v1","identity":"rs-6118821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6118821","identity":"rs-6118821","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}