Genome-wide identification, Characterization, and Expression Analysis of the CsBAG family in Citrus sinensis (L.) Osbeck

Genome-wide identification, Characterization, and Expression Analysis of the CsBAG family in Citrus sinensis (L.) Osbeck | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification, Characterization, and Expression Analysis of the CsBAG family in Citrus sinensis (L.) Osbeck Tianli Wu, Leyi Long, Yongting Liu, Kaidong Liu, Lanyan Zheng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4348725/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) proteins constitute a multifunctional family of co-chaperone regulators. They play pivotal roles in modulating various processes, encompassing plant growth and development and response to biotic and abiotic stress. However, despite advancements in our understanding of plant BAGs, the underlying mechanisms remain unelucidated. Therefore, this study aims to systematically examine citrus BAG and their respond to hormonal treatment and Huanglongbing infection. In this study, we conducted a genome-wide in silico analysis of the CsBAG gene family in a globally significant citrus crop to explore its potential roles in fruit trees. We identified and characterized 10 CsBAGs and eight CsBAGs, revealing their distribution across five of the nine citrus chromosomes. Results: All 10 proteins exhibited a characteristic BAG domain. CsBAG2, 4, 5, and CsBAG8 possess an additional ubiquitin-like domain, while CsBAG1 and CsBAG6 feature a calmodulin-binding motif (IQ motif). Most CsBAGs are predicted to be localized in the nucleus, mitochondria, or chloroplasts. Phylogenetic analysis revealed four major clusters, further categorized into G1–G4 groups. Cis-regulatory elements within all CsBAG promoters were identified and categorized, and the associated transcription factors were predicted. The findings suggest the involvement of these genes in defense against biotic and abiotic stresses, photoperiodic control, hormonal responses, growth, and development. This notion was further supported by gene expression analysis, revealing varying degrees of responsiveness to treatment with plant hormones (GA 3 and ABA) and infections with the citrus Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus ( C Las). Segmental duplications contributed to the expansion of the CsBAG gene family in citrus. Conclusions : Our findings suggest that certain members of the CsBAG gene family may have roles in stress response and pathogen immunity. This study could help to comprehensively analyze the characteristics of the citrus BAG gene family, and the results will offer additional target genes for molecular disease resistance breeding of citrus HLB, laying a theoretical and practical foundation for the future rational utilization of BAG genes. CsBAG Citrus hormones Huanglongbing expression analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background The B-cell lymphoma 2 (Bcl-2)-associated athanogene ( BAG ) gene was initially discovered while screening a small mouse embryonic cDNA library, where recombinant human Bcl-2 protein was utilized as bait to identify Bcl-2 interactors. This gene was designated as BAG1 (Takayama et al., 1995 ). BAG1 and Bcl-2 synergistically promote cell survival, emerging as a novel anticell death gene within the programmed cell death pathway. After its discovery, similar sequences of the BAG gene have been identified across various plants and animals, such as BAG1-6 in mammals (Doong et al., 2002 ), AtBAG1-8 in Arabidopsis thaliana (Yan et al., 2003 ), OsBAG1-6 in Oryza sativa L. (Rana et al., 2012 ), MusaBAG1-13 in bananas (Dash and Ghag, 2022 ), SlBAG1-7 in tomatoes (He et al., 2021 ), TaBAG and TaBAG2 in wheat (Ge et al., 2016 ), and HSG1 in grape (Kobayashi et al., 2012 ). Sequence analysis revealed that these genes are members of the BAG family. In plants and animals, the protein encoded by the BAG gene possesses a BAG domain comprising 110 amino acid residues at the C-terminus. This frequently includes a ubiquitin-like domain (UBL) and various other motifs, such as the isoleucine glutamine motif (IQ), proline-rich repeat (PXXP), TXSEEX repeat, nuclear localization signal (NLS), and the WW domain (Doong et al., 2002 ; Kabbage and Dickman, 2008 ). Among these, IQ motifs are exclusive to plants, potentially indicating functional variations in BAG between plants and animals (Doukhanina et al., 2006 ). Most BAG family members participate in plant responses to abiotic stresses, such as salt, heat, cold, drought, ultraviolet radiation, and plant hormones. Plant BAG genes have evolved independently, leading to diverse spatiotemporal expression patterns and functions across different species. Lee et al. ( 2016 ) observed that transgenic seedlings of A. thaliana AtBAG1 exhibited increased sensitivity to salt treatment (Lee et al., 2016 ). Fang et al. ( 2013 ) demonstrated the involvement of AtBAG2 in A. thaliana in plant responses to environmental stress (Fang et al., 2013 ). The AtBAG2 gene is also induced by ABA, ACC, SA, heat, salt, and drought stress (Nawkar et al., 2017 ; Arif et al., 2021 ). Transcript levels of AtBAG3 increase in response to salt, MeJA, ABA, and SA, with cold responses inhibited in the roots (Nawkar et al., 2017 ). Overexpression of AtBAG4 in tobacco enhances tolerance to UV, cold, oxidants, and salt treatments (Doukhanina et al., 2006 ). Transcription of AtBAG6 is induced by stress-related plant hormones (such as ACC, SA, MeJA, and ABA), heat stress, mannitol, and programmed cell death (PCD) inducers (Echevarría-Zomeño et al., 2016 ; Nawkar et al., 2017 ; Fu et al., 2019 ). AtBAG6 mutations enhance short-term heat tolerance in fes1a mutants (where Fes1A mutations decrease heat stress tolerance); however, this improved heat tolerance is diminished by calmodulin inhibitor treatment (Fu et al., 2019 ). AtBAG7 expression is reduced under salt stress conditions; however, ACC treatment counteracts the salinity-induced effects (Pan et al., 2016 ). In rice, ABA, IAA, and high-temperature treatments induce the expression of OsBAG1 and OsBAG3 (Zhou et al., 2021a ). Transgenic rice overexpressing OsBAG4 exhibits enhanced tolerance to salt stress (Eckardt, 2006 ). Overexpressing TaBAG2 in wheat has been shown to increase heat tolerance in A. thaliana (Ge et al., 2016 ). The expression of HSG1 in grapes significantly rises under heat stress, and when overexpressed in A. thaliana , it leads to substantial resistance to high temperature (Kobayashi et al., 2012 ). Furthermore, heterologous overexpression of tomato SlBAG9 heightens the sensitivity of A. thaliana to drought, salinity, and ABA (Jiang et al., 2022 ). These findings underscore the significant roles played by BAG family members in plant responses to abiotic stress. Pathogens infection such as fungi, bacteria, viruses, insects, and nematodes are believed to be major triggers of plant BAG gene expression. In Arabidopsis , AtBAG2 expression was specifically suppressed in response to the necrotic fungus Botrytis cinerea , semi-living trophic fungus Phytophthora infestans , and bacterium Pseudomonas syringae pv. phaseolicola. The expression of AtBAG3 and AtBAG6 is moderately induced by the toxic Pseudomonas syringae pv. tomato DC3000 and necrotizing fungal pathogen Botrytis cinerea (Nawkar et al., 2017 ). Correspondingly, atbag6 mutants display heightened sensitivity to the necrotizing fungus B. cinerea (Doukhanina et al., 2006 ). Transgenic plants overexpressing MusaBAG1 in bananas exhibit increased resistance to wilt disease (caused by fungi or bacteria) (Ghag et al., 2014 ). BAG4 is triggered in the leaf tissue of Medicago truncatula upon infection with the bacterial pathogen Xylella fastidiosa (Dash and Ghag, 2022 ). In rice, accumulation of OsBAG4 in ebr1 mutants or plants overexpressing OsBAG4 initiates autoimmunity and broad-spectrum disease resistance (against pathogens such as Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae ) (You et al., 2016 ). Soybean GmBAG7 exhibits a dual effect on Arabidopsis - Phytophthora capsici interactions, acting as a susceptibility factor in the endoplasmic reticulum but conferring resistance to Phytophthora capsici in the nucleus (Zhou et al., 2021b ). Several studies have highlighted the significant role of BAG in plant resistance against fungal and bacterial stress. BAG not only combat pathogenic microorganisms but also contributes to defense against insect predation. In rice, the expression of OsBAG1-3 is significantly induced by feeding from the brown planthopper (BPH) insect (Zhou et al., 2021a ). Overexpressing soybean GmBAG6a in A. thaliana confers resistance to nematode infection (Yeckel, 2012 ). In addition, BAG plays a role in plant antiviral responses. Gayral et al. discovered that AtBAG7 is crucial for the localized accumulation of Plantago asiatica mosaic virus in the endoplasmic reticulum-to-nuclear signaling pathway and is linked to host resistance against Turnip mosaic virus (Gayral et al., 2020 ). In our previous study, transcriptome sequencing of citrus roots infected with Huanglongbing (HLB) revealed the regulation of key genes within the disease resistance pathway by HLB infection, and a gibberellin induced gene CcGASA4 was identified. Transcription and metabolome analyses of transgenic citrus plants overexpressing CcGASA4 revealed that the differentially expressed genes predominantly included pathogens, stress responses, hormones, and growth and development-related genes. By employing CcGASA4 as bait, the citrus yeast expression library underwent screening, resulting in the identification of the interacting BAG protein family member PRPBAG6-A. The interaction between these two proteins has been elucidated in vitro through yeast double hybridization and bimolecular fluorescence complementation (Wu et al., 2020 ). This result showed that BAG may have a significant role in the citrus response to HLB infection through its interaction with CcGASA4. However, despite progress made in understanding plant BAGs, the underlying mechanisms remain unelucidated. Therefore, this study aims to systematically analyze citrus BAG and investigate its role in the response to hormonal treatment and HLB infection. Methods Identification of putative Citrus sinensis BAG genes The C. sinensis genome and protein sequences were obtained from the Pan-genome to Breeding Database (CPBD, http://citrus.hzau.edu.cn ) (Xu et al., 2013 ). The genome and protein sequences of A. thaliana , O. sativa , Malus domestica , and Carica papaya were retrieved from the Phytozome online database ( https://phytozome-next.jgi.doe.gov ) (Goodstein et al., 2012 ). BAG proteins were identified through Hidden Markov Model (HMM) searches of sequences in the downloaded peptide sequence FASTA file using the HMMER 3.0 program ( https://www.ebi.ac.uk/Tools/hmmer/ ) with default parameters (Eddy, 1998 ). A comprehensive search was also conducted using the amino acid sequences of eight previously reported Arabidopsis BAG proteins (Doukhanina et al. 2006 ). The integrity of the BAG domain (Accession: PF02179) in all putative C. sinensis BAG sequences was confirmed using the NCBI Conserved Domain Database ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) (Marchler-Bauer et al., 2015 ). Only nonredundant putative protein sequences with conserved BAG domains were retained for subsequent analysis. All CsBAG protein coding sequences, genomic regions, and associated information, such as accession numbers and chromosomal positions, were obtained from the CPBD database. In addition, the physical location of each CsBAG gene in the genome was mapped using MapChart software. The CDS sequences of CsBAG and their respective genomic sequences were compared and analyzed to identify introns and exons using the Gene Structure Display Server 2.0 (GSDS2.0, http://gsds.gao-lab.org/ ) (Hu et al., 2015 ). Analysis of physicochemical properties of the citrus BAG proteins The physicochemical parameters of the CsBAG proteins were computed using ProtParam on the ExPASy server ( http://web.expasy.org/protparam ) (Gasteiger, 2003 ). Putative protein secondary and tertiary structures were predicted using SOPMA ( https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html ) and the SWISS MODLE engine ( https://swissmodel.expasy.org/interactive ), respectively. Subcellular protein localizations were predicted using the WOLF PSORT program ( https://wolfpsort.hgc.jp ), while transmembrane helices were predicted using the DeepTMHMM server v1.0.24 ( https://dtu.biolib.com/DeepTMHMM:1.0.24/ ). Analysis of protein phylogenetic relationships and gene structures A phylogenetic tree was constructed using 62 BAG protein sequences from different plant species, including C. sinensis (10), A. thaliana (8), O. sativa (10), M. domestica (15), Solanum lycopersicum (10), and Carica papaya (9). The phylogenetic tree was generated using the Maximum Likelihood (ML) method with MEGA 7.0 software. The ML method parameters included 1000 bootstrap replications, the “Jones-Taylor-Thornton” model, “Uniform rates,” “Complete deletion,” and “Nearest-Nerghbor-Interchange” method. Conserved motifs within the CsBAG proteins were identified using the MEME ( https://meme-suite.org/tools/meme ) website, with motif parameters set to 10. The phylogenetic tree and motif images were visualized using the Chiplot website ( https://www.chiplot.online/ ) for visualization. Promoter analysis of CsBAG genes The 2.0-kb long promoter sequence upstream of the start codon (ATG) for each CsBAG was obtained from the CPBD database (Xu et al., 2013 ). Analysis of the cis-regulatory elements within the CsBAG gene promoters was conducted using the online program Plant Cis-Acting Regulatory DNA Elements ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) (Lescot, 2002 ). Prediction of transcription factor (TFs) networks was performed online, with a threshold parameter p-value ≤ 1e-5 on the Plant Transcriptional Regulatory Map (PTRM) website ( http://plantregmap.gao-lab.org/regulation_prediction.php ), utilizing all CsBAG promoter sequences as input. Cytoscape 3.9.1 software was employed to visualize the TFs regulatory network. Expression analysis of citrus CsBAG genes Biotic stress treatment involved selecting 3-year-old C. sinensis trees, onto which branches infected with HLB were grafted onto stems. The control group comprised tree shoots grafted without HLB bacteria. The 2-month-old C. sinensis seedlings were planted in plastic garden pots for use as hormone treatment materials. For hormone treatment, trees were sprayed with 100 µΜ of GA 3 and ABA. Root, stem, and leaf samples were harvested at 3 h, 6 h, 12 h, 24 h and 48 h. Control trees were sprayed with sterile water, and root, stem, and leaf tissues were harvested at the same time points as those in the hormone treatment group. Samples were flash-frozen in liquid nitrogen and stored at -80°C until use. Three biological replicates were utilized for each treatment. Total RNA was extracted from frozen leaf samples using the TRIzol extraction method (TIANGEN, China). RNA extraction process involved the following steps: 1. 50–100 mg of leaf tissue was fully ground with liquid nitrogen in a 2.0 ml centrifuge tube; 1 ml of Trizol was added to fully homogenize the tissue, and the mixture was allowed to stand at 25°C for 5 min; 2. 0.2 mL of chloroform was added, the tube was shaken for 15 s, and allowed to stand for 2 min. 3. The mixture was centrifuged at 12000 g for 15 min at 4°C, and the supernatant was collected; 4. Subsequently, 1:1 isopropanol was added, mixed gently, and incubated at 25°C for 10 min; 5. Subsequently, the mixture was centrifuged at 12000 g for 10 min at 4°C, and the supernatant was discarded. 6. Following that, 1 mL of 75% ethanol was added to wash the pellet gently. Afterward, the mixture was centrifuged at 7500 g for 5 min at 4°C, and the supernatant was discarded (this step was repeated twice). 7. The pellet was air-dried, followed by the addition of 60 uL DEPC water to dissolve it. RNA concentrations were determined using a NanoDrop 2000C (ThermoFisher Scientific, USA), and RNA quality was assessed using the OD260/OD280 ratio and agarose gel electrophoresis. cDNA synthesis, quantitative real-time PCR (qRT-PCR), and data analysis followed the procedure outlined by Wu et al. (Wu et al., 2021 ). Supplementary Table S1 lists the Primers utilized in this study. Actin (GenBank accession number: Cs_ont_1g004160.1) served as the reference gene. Due to the prediction that the three proteins, CsBAG4.1, CsBAG4.2, and CsBAG4.3, originated from a single gene sequence (Cs_ont_5g040580) through alternative splicing, and because the coding sequences of CsBAG4.1 and CsBAG4.3 were identical, while only the noncoding sequences differed, designing primers to distinguish CsBAG4.3 from CsBAG4.1 was not feasible. Therefore, the final expression results of CsBAG4.1 and CsBAG4.3 were combined. For quality controls, three technical replicates were also employed in addition to three biological replicates. Synteny analysis and calculation of the Ka/Ks ratio for duplicated genes Potential duplicated citrus CsBAG genes were identified using the MCScanX function of the TBtools software, and results were visualized using the same software. Chromosomal location information for all CsBAG genes was obtained from the annotation gff3-file. Collinear pairs were extracted using TBtools software to identify syntenic blocks and duplications within the BAGs across the whole genomes of six species: C. sinensis , A. thaliana , Oryza sativa , M. domestica , Solanum lycopersicum , and Carica papaya . A collinearity map between these species was generated using MCScan X (TBtools software). Results Genome-wide identification and characterization of BAG genes in C. sinensis In the C. sinensis genome, HMMER searches revealed 10 CsBAG proteins containing a conserved BAG domain (Accession: PF02179) (Table 1 ). This resulted in the identification of eight CsBAG genes. These CsBAG genes were designated as CsBAG1 – CsBAG8 based on their chromosomal locations. Upon comparing CsBAG4.1, CsBAG4.2, and CsBAG4.3 proteins, it was observed that they were encoded by the same gene, Cs_ont_5g040580 ( CsBAG4 ). Ten CsBAGs were distributed across the five chromosomes of the C. sinensis genome (Fig. 1 ). Chromosomes 2, 5, and 9 contained two BAG genes, while chromosomes 6 and 7 contained one BAG gene. Gene structure diagrams of CsBAG genes, depicting exon-intron components, were generated using the Gene Structure Display Server (GSDS) (Fig. S1). CsBAG6 was analyzed to comprise a single exon, while two CsBAG genes ( CsBAG1 and CsBAG3 ) contained two exons. In contrast, four CsBAG genes ( CsBA G2, CsBAG4.2 , CsBAG5 , and CsBAG8 ) within the same group contained four exons. The deduced protein sequences of the ten CsBAG transcripts ranged in length from 218–645 amino acid residues (Table 1 ). The predicted molecular weights (MWs) ranged from 24.68–70.81 kDa, with isoelectric points (pIs) spanning 4.48–9.40. All CsBAGs exhibited hydrophilic characteristics, indicated by their negative grand average of hydropathicity (GRAVY) value. The aliphatic index values of the CsBAG proteins ranged from 60.89–94.68. A high aliphatic index suggests thermostability across a broad temperature range (Ikai, 1980 ). Most of the proteins were predicted to be unstable, with eight of them—except for CsBAG4.1 and CsBAG4.3—having instability index values exceeding 40. Table 1 Characteristics of the CsBAG family gene in Citrus sinensis . Gene Name Transcript ID Chr Start Sit End Sit Strand No. of Exons CDS (bp) MW (kDa) Protein (A.A) pl GRAVY II AI Subcellular localization CsBAG1 Cs_ont_2g000960.1 Chr2 536380 538956 reverse 2 657 25.32 218 7.09 -0.7 47.9 69.31 nucl, cyto, mito CsBAG2 Cs_ont_2g007530.1 Chr2 4538601 4540720 reverse 4 768 28.66 255 9.02 -0.446 49.53 87.49 chlo, mito, nucl, cyto CsBAG3 Cs_ont_5g003990.1 Chr5 2567670 2569909 forward 2 1938 70.81 645 4.48 -0.649 42.12 87.04 cyto, nucl, cysk, golg CsBAG4.1 Cs_ont_5g040580.1 Chr5 43283953 43285952 forward 3 657 24.68 218 8.77 -0.432 37.48 94.68 chlo, mito, cyto, nucl CsBAG4.2 Cs_ont_5g040580.2 Chr5 43283953 43285952 forward 4 864 32.62 287 9.05 -0.596 43.66 88.54 chlo, nucl, mito, plas CsBAG4.3 Cs_ont_5g040580.3 Chr5 43283953 43285952 forward 3 657 24.68 218 8.77 -0.432 37.48 94.68 chlo, mito, cyto, nucl CsBAG5 Cs_ont_6g014260.1 Chr6 9178620 9180654 reverse 4 861 31.94 286 5.39 -0.925 40.35 69.86 nucl, cyto, mito, plas CsBAG6 Cs_ont_7g021670.1 Chr7 24928099 24929870 forward 1 1146 42.74 381 4.74 -1.081 55.47 60.89 nucl, chlo, mito CsBAG7 Cs_ont_9g023720.1 Chr9 28052851 28056522 forward 3 1197 45.46 398 9.16 -0.564 47.55 78.17 chlo, nucl, cyto, plas, mito CsBAG8 Cs_ont_9g025230.1 Chr9 29328941 29331515 reverse 4 1068 39.79 355 9.4 -0.761 45.24 72.76 nucl, cyto, vacu, chlo, mito, plas, E.R. Note: CDS, coding DNA sequence; AA, amino acid; MW, molecular weight; pI, isoelectric point; kDa, kilodalton; Instability index (II); Aliphatic index (AI); pI isoelectric point, GRAVY, grand average of hydropathicity. chlo, chloroplast; mito, mitochondria; cyto, cytoplasm; extr, extracellular; vacu, vacuoles; nucl, nucleus; golg, golgiosome; plas, plastid; ER, endoplasmic reticulum. The predicted subcellular localization of citrus CsBAG family proteins primarily included the nucleus, cytoplasm, mitochondria, and chloroplast, with 10, 8, 9, and 7 members, respectively. However, some CsBAGs were potentially localized in the cytoskeleton, Golgi, plastids, vacuoles, and endoplasmic reticulum (Table 1 ). The TMHMM server confirmed the absence of a transmembrane helix (Supplementary Fig. S2). 3D structure prediction revealed that almost all CsBAG proteins exhibited flexibilility, characterized by random coils at the N-terminus and C-terminus. In the middle of the CsBAG sequences, several α-helices were observed adjacent to random coils and extended strands or beta-turn (Fig. 2 , Supplementary Table S2). Phylogenetic relationship and structural features of BAG proteins in citrus To assess the evolutionary relationships among BAG family proteins in C. sinensis and other species, a phylogenetic tree was generated using 62 full-length BAG protein sequences from six plant species ( 10 from sweet orange, eight from Arabidopsis , 10 from rice, 15 from apple, nine from papaya, and 10 from tomato) using the maximum likelihood (ML) method (Fig. 3 , Supplementary Table S3). All six species analyzed exhibited homologs within the four branches, with BAG proteins distributed unevenly across four groups: G1, G2, G3, and G4. Among citrus CsBAG proteins, CsBAG2 and CsBAG5 clustered with G1, CsBAG8 was grouped within the fewest clade G2, and CsBAG1, CsBAG3, CsBAG6, and CsBAG7 were classified within the largest clade, G3 (Fig. 3 ). The G4 group, including CsBAG4.1, CsBAG4.2, and CsBAG4.3, stood out from the other BAG genes by forming an individual clade, indicating its specificity to sweet orange. Unlike rice and Arabidopsis , there were no homolog members in this clade, suggesting that they resulted from duplication after the segregation of Arabidopsis and Citrus ancestors. To further explore the evolution of the BAG gene family, we examined the structural characteristics of the BAG genes in C. sinensis . Exon–intron organization analysis revealed a relatively conserved arrangements pattern within the same group (Fig. S1). Among these, CsBAG3 exhibited the longest gene structure, spanning 1938 bp, while CsBAG4.1/4.3 had the shortest structure, measuring 657 bp (Table 1 ). Among all the BAG genes, CsBAG2, 4.2, 5, and 8 exhibited the highest number of exons, with four each. The CsBAG4.1, 4.3, and 7 had three exons each. CsBAG6 lacked introns entirely. Additionally, most BAG genes displayed a similar exon/intron structure, with intron phases predominantly clustered within the same group (Fig. S1). To assess the sequence features of C. sinensis BAGs, conserved motifs and domains were examined. It was found that all CsBAG proteins contained a BAG domain situated at the terminus of the CsBAG proteins (Fig. S3). In addition to the BAG domain, CsBAG2, 4.2, 5, and 8 contained ubiquitin-like domains similar to that in their animal counterparts. However, CsBAG1 and CsBAG6 featured an IQ calmodulin-binding domain situated at the N-terminus. This characteristic is exclusive to plants.To explore genetic variation in C. sinensis , 10 conserved motifs among the 62 BAG proteins were examined using the MEME program (Fig. 3 ). The lengths of these conserved motifs ranged from 18–50 amino acids, displaying a highly diverse distribution (Fig. S4). CsBAG1, 3, 6, and 7 exhibited only three motifs, while most BAGs contained eight motifs ( 1, 2, 3, 4, 5, 6, 7, and 8) arranged in a similar order. The composition and arrangement of conserved motifs among BAG protein sequences within the same phylogenetic branch were similar. For example, within the G3 group, most members featured two uniquely spaced motifs: motifs 9 and 10 were interspersed with motif 2, indicating specialized functions that benefited this group. The citrus G1, G2, and G4 members exhibited greater diversity than those of G1 (Fig. 3 ). In addition, while most G1 and G2 members contained eight motifs, the spacing between motif 6 and the adjacent motif 3 was wider in G2 than in G1. Some citrus CsBAG proteins should be structurally or functionally compromised due to missing one or two motifs compared to their respective group members. For instance, CsBAG5 lacked motif7, while CsBAG4.1 and 4.3 were devoid of motif3. Overall, most CsBAGs in adjacent branches displayed similar exon-intron compositions and motif and domain distributions. CsBAG promoters and their possible activators Figure 4 illustrates the cis-regulatory elements identified in the promoters of CsBAG genes. Many elements, such as MBS (drought-inducibility), ARE (antioxidant response element), WUN-motif (wound responsiveness), LTR (low-temperature responsiveness), GC-motif (enhancer-like element involved in anoxic-specific inducibility), MYC (abiotic element), WRE3 (wound-response element 3), DRE core (cold and dehydration responsiveness), box S (elicitation, wounding, and pathogen responsiveness), W box (wounding and pathogen responsiveness), and MYB (abiotic element). MYB and MYC, crucial for abiotic responsiveness, were found in all CsBAG promoters. The antioxidant response element (ARE) was present in seven CsBAG promoters, except for CsBAG2, 5, and 6. The promoters of CsBAG genes contained various hormone-responsive elements such as ABRE (responsive to ABA), P-box and GARE-motif (responsive to GA), AuxRR-core, and TGA-element (responsive to AUX), ERE (responsive to ET), TCA-element, and TCA (responsive to SA), as well as CGTCA- and TGACG-motif (responsive to MeJA). Light response cis-elements, including the G-box, Box 4, AE-box, Sp1, I-box, Box II, ACE, LS7, LAMP-element, GATA-, GA-, ATCT-, GT1-, TCT-, GTGGC-, and TCCC-motif, were also prevalent in the CsBAG promoters. In addition, CsBAG promoters also harbored plant growth- and development-associated cis-elements, including the meristem-specific expression element CAT-box, plant endosperm-specific negative expression element AAGAA-motif, zein metabolism regulation element O2-site, plant cell cycle control-related element MSA-like, and palisade mesophyll cell differentiation element HD-Zip 1. The analysis of the potential transcriptional regulatory network of CsBAG genes revealed a large number of TFs capable of binding to the identified cis-elements (Fig. S5 and Supplementary Table S4). Predominantly, TFs included ERF, bHLH, MYB, WRKY, and MIKC_MADS, with 78, 36, 21, 21, and 18 members identified respectively. In addition, 32 Dofs, 2 ARFs, and 14 C2H2s were identified as activators of CsBAG genes. Functionally, these TFs were primarily linked to abiotic and biotic stresses, such as pathogen attack, heat shock, and drought stress. Expression pattern of CsBAGs under HLB pathogen and plant hormone (ABA and GA 3 ) stresses To investigate the tissue specificity of CsBAG expression, qRT-PCR was conducted on various tissues, including the leaves, stems, and roots of 2-month-old C. sinensis (Fig. 5 ). The results showed that CsBAG1 , CsBAG2 , CsBAG3 , CsBAG4.2 , and CsBAG8 exhibited higher expression levels in stems compared to leaves. CsBAG4.1 and CsBAG4.3 exhibited predominant expression in leaves, with comparatively lower expression levels in roots. In contrast, CsBAG7 showed the highest expression in roots, followed by moderate expression levels in stems and leaves. CsBAG5 and CsBAG6 displayed similar expression levels across roots, stems, and leaves. To explore the potential functions of CsBAGs in response to hormone treatment, we examined the expression patterns of CsBAG genes following treatments with GA 3 and ABA (Fig. 6 ; Fig. S6, S7). Upon GA 3 treatment, CsBAG1 , 2 , 3 , 4.2 , 6 , 7 , and CsBAG8 exhibited significant upregulation in citrus leaves. CsBAG1 exhibited the highest upregulation, approximately 208-fold, following 3 h of GA 3 treatment. CsBAG2 showed the second highest induction, approximately 197-folds, after 6 h of GA 3 treatment. CsBAG3 , CsBAG4.2 , and CsBAG7 were upregulated approximately 10-fold. The expression of CsBAG5 and CsBAG8 increased initially in stems, peaked in the middle, and then declined towards the end of the treatment, while the expression of CsBAG3 and CsBAG4.2 decreased at 3 h, 6 h, 12 h, and 24 h, but increased at 48 h. In the stems under GA 3 treatment, CsBAG6 and CsBAG7 showed gradual upregulation, while CsBAG1 and CsBAG2 exhibited gradual downregulation. In GA 3 -treated roots, CsBAG4.1 and CsBAG4.3 were significantly enriched, while CsBAG6 and CsBAG7 were downregulated. CsBAG7 expression sharply decreased from 0–12 h and then gradually decreased to its lowest level after 48 h of treatment. CsBAG6 expression exhibited a V-shaped pattern, while CsBAG7 expression gradually decreased. During ABA treatment, the expression of CsBAG1 , 2 , 3 , 4.2 , 5 , 7 , and CsBAG8 exhibited an inverted V-shaped pattern, while CsBAG6 showed downregulation initially and then increased again by the end of the treatment in citrus leaves. In stems, CsBAG2 and CsBAG3 were upregulated in the middle, while CsBAG4.2 and CsBAG6 were initially decreased but returned to normal levels by the end of the treatment (Fig. S6). The transcript levels of the remaining CsBAG genes in the stems exhibited no significant changes, suggesting that these genes might not play significant roles under GA 3 treatment. In the roots, the expression of CsBAG1 , 2 , 3 , 4.1 , 4.2 , 4.3 , and CsBAG5 slightly increased at 24 h after ABA treatment. In contrast, CsBAG6 , 7 and CsBAG8 were significantly downregulated by ABA, with CsBAG7 and CsBAG8 (Fig. S7). The responsiveness of CsBAG genes to HLB was examined. In Fig. 7 , the transcript levels of most CsBAG genes exhibited a substantial increase under HLB stress. Six genes, CsBAG1 , 4.1 , 4.2 , 4.3 , 5 , and 6 , were significantly induced by HLB, with CsBAG4.3 showing the highest induction, surpassing a 3-fold increase, and CsBAG5 exhibiting a 1.7-fold increase. CsBAG2 expression was significantly suppressed by HLB infection (Fig. 7 ). The expression levels of two CsBAGs ( CsBAG3 and CsBAG7 ) remained unchanged in all tested samples, suggesting a lack of involvement in HLB infection. Except for CsBAG2 and CsBAG5 , these results align with the RNA-seq data analysis of five CsBAGs ( CsBAG 1/2/4/6/8), which exhibited predominantly induced expression in HLB-infected tissues (Supplementary Table. S6). Overall, the varied expression patterns of CsBAG genes across citrus tissues imply their multifaceted roles in citrus development. Evolution of citrus CsBAG genes Analysis of all CsBAG genes revealed potential duplication events. Specifically, an interspersed segmental duplication event was identified involving CsBAG4 on chromosome 5 and CsBAG8 on chromosome 9 (Fig. 8 ). A comprehensive analysis of orthologous BAG genes across the genomes of C. sinensis , A. thaliana , Oryza sativa , M. domestica , Solanum lycopersicum , and Carica papaya revealed 43 collinearity events between C. sinensis and the other five species. We observed that 6, 5, 16, 9, and 7 CsBAG genes in C. sinensis shared synteny with those in A. thaliana , rice, apple, tomato, and papaya, respectively (Fig. 9 and Supplementary Table S6). In addition, CsBAG4.2 , 5 , and 8 exhibited collinearity with the BAG genes of the other five species, suggesting their pivotal roles in expanding the BAG family. Discussion The evolutionarily conserved BAG family significantly influences plant growth, development, and stress responses (Thanthrige et al., 2020 ). As new plant genome sequences are continually published, additional BAG family genes are being identified in plants. Currently, research on plant BAG function primarily centers on model plants such as Arabidopsis thaliana and rice, with fewer studies exploring the function of the BAG family in other species. Our previous study showed an interaction between citrus BAG6 protein and HLB-induced GASA4, which made us interested in the citrus CsBAG gene family (Wu et al., 2020 ). Therefore, it is necessary to identify and characterize BAG family proteins in citrus fruit trees through a combination of bioinformatics tools and expression studies. This study aims to comprehensively characterize the genome-wide functions of CsBAG genes and proteins in citrus. To the best of our knowledge, this is the first study to offer insights into the function of BAG in C. sinensis . We identified 8 BAG genes through sequence alignment and HMMER database searches, with detailed information for these genes (Table 1 ). Among the 10 BAG proteins discovered, BAG4.1, 4.2, and 4.3 were encoded by the same gene, Cs_ont_5g040580 . We analyzed the protein structures and evolutionary relationships of the ten BAG proteins with those in Arabidopsis, rice, apple, papaya, and tomato. CsBAG proteins were categorized into groups G1, G2, G3, and G4 (Fig. 3 ), all featuring a BAG domain. The C-terminus of the BAG protein hosts the BAG domain, facilitating interaction with the ATPase domain of constitutive and inducible heat shock protein Hsp70, while the N-terminus enables interaction with various proteins involved in protein degradation, cell migration, proliferation, and apoptosis. This domain also regulates chaperones through UBL and other motifs (Takayama et al., 1995 ; Doong et al., 2002 ). For example, AtBAG4 binds to HSP70 in the cytoplasm, inhibiting abiotic stress-induced cell death (Thanthrige et al., 2020 ). Upon pathogen infection, Arabidopsis AtBAG6 interacts with bag-associated GRAM protein (BAGP1) and asparagylprotease-cleaved BAG protein (APCB1), forming complexes that initiates autophagy and induce PCD, thereby enhancing fungal disease resistance (Li et al., 2016 ). In addition to the BAG domain, group members of G1 (CsBAG5, CsBAG2), G2 (CsBAG8), and G4 (CsBAG4.1, CsBAG4.2, CsBAG4.3) contain a UBL domain. The domain interacts with the 26S proteasome and is integral to BAG1 during stress response (Alberti et al., 2002 ). The UBL domains in the G1, G2, and G4 groups suggest their potential involvement in the degradation of some proteins as molecular bridges. The IQ motif can bind to CaM, influencing complex formation between CaM and its target protein (Kang et al., 2006 ). The two CsBAGs (CsBAG1 and CsBAG6) in G3 feature a specific CaM-binding domain, the IQ motif. Ca 2+ can modulate the binding affinity of AtBAG6 to CaM, thereby regulating the AtBAG6-mediated cell death process (Kang et al., 2006 ). Functioning as a signaling hub, AtBAG5 links the Ca 2+ signaling network with the Hsc70 chaperone system, thereby regulating plant defense and senescence (Li et al., 2016 ). Therefore, citrus CsBAG1 and CsBAG6 potentially play roles in mediating plant defense and senescence through regulating Ca 2+ signaling. The results of this study show the involvement of CsBAG in plant defense processes. The hydrophilic and unstable nature of these CsBAG proteins indicates predicted expression in the nucleus (Table 1 ), suggesting potential roles as TFs functions. Secondly, the analysis of cis-elements revealed the presence of various stress- and hormone-related elements across all CsBAG promoters. These include MBS, ARE, WUN-motif, LTR, GC-motif, MYC, WRE3, MYB, DRE core, box S, W box, ABRE, P-box, GARE-, CGTCA-, and TGACG-motif (Fig. 4 ). These results align with those of previous studies where similar cis-elements were identified in the upstream promoter regions of SlBAG genes in tomato (Jiang et al., 2022 ) and AtBAG genes in Arabidopsis (Nawkar et al., 2017 ). This suggests their potential roles in responding to various stresses, such as cold, antioxidant, drought, and wound stress. For example, AtBAG7 interacts with the transcription factor WRKY29 in the nucleus, where WRKY29 binds to the W-box of the AtBAG7 promoter, triggering transcription of AtBAG7 and other chaperone proteins to enhance stress tolerance (Li et al., 2017 ). In addition, analysis of TFs interacting with these CsBAG promoter elements revealed several TFs linked to stress responses (Fig. 4 ). Third, accumulating evidence shows that citrus CsBAG expression can be induced by treatments with stress-related hormones (ABA and GA 3 ) as well as by the biotic pathogen HLB (Figs. 6 and 7 and Supplementary Fig. S5). The ABA signaling pathway is crucial in helping plants manage abiotic stress and senescence (Gao et al., 2016 ). However, further investigation is required to understand the role of ABA in the CsBAG-mediated HLB protection pathway. These findings reveal the involvement of the CsBAG family in citrus response to abiotic stresses, such as ABA and GA 3 , with potential involvement of HLB signals in these pathways. However, the molecular mechanisms underlying CsBAG-mediated disease resistance remain unclear. Gene family expansion primarily arises from DNA duplication, which can occur through interspersed segmental or tandem duplication (Samonte and Eichler, 2002 ). Here, eight CsBAG genes were identified in C. sinensis . Approximately 8, 15, 6, 10, and 9 CsBAGs were found in rice, apple, Arabidopsis, tomato, and papaya, respectively (Table S3). The number of BAG genes in apples is approximately twice that of citrus, rice, Arabidopsis, tomato, and papaya. This significant difference could be attributed to the recent genome-wide duplication (WGD) specific to apples, which has not occurred in the other species (Velasco et al., 2010 ). Similarly, a comparable pattern emerged with the EIN3/EIL genes. In species such as Arabidopsis, tobacco, tomato, rice, peach, mei, and strawberry, which lacked a recent WGD, the number of these genes remained at 4–5. However, in pear, which shared a recent WGD with apples, the count doubled to 10 (Xu et al., 2013 ; Cao et al., 2017 ). Given the preserved structure, function, and expression patterns, we summarized the primary biological roles of CsBAG protein in plant development and disease resistance. Conclusions This study identified and analyzed 10 CsBAG proteins from the C. sinensis genome, focusing on their potential roles in citrus defense. Bioinformatics analysis revealed varying degrees of structural and functional divergence among members of the CsBAG gene family, suggesting potential diverse roles in the growth and development of citrus. Experimental evidence showed that the expression of G4 group members (CsBAG4.1/4.2/4.3) is highly responsive to bacterial infection and ABA treatment. This suggests their potential significance in Citrus response to biotic stresses. Further studies are needed to elucidate the roles of these proteins in growth and stress responses and consider them as candidate genes for engineering transgenic citrus plants with enhanced agronomic traits. Abbreviations ARE antioxidant response element BAG B-cell lymphoma 2 (Bcl-2)-associated athanogen BPH brown planthopper CLas Candidatus Liberibacter asiaticus GSDS Gene Structure Display Server GRAVY grand average of hydropathicity HMM Hidden Markov Model HLB Huanglongbing IQ isoleucine glutamine motif ML maximum likelihood MWs molecular weights NLS nuclear localization signal PTRM Plant Transcriptional Regulatory Map PXXP proline-rich repeat PCD programmed cell death TFs transcription factor UBL ubiquitin-like domain WGD wide duplication. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests The authors declare no conflict of interest. Funding This research was financially supported by the Guangdong Provincial Basic and Applied Basic Research Fund Project (2022A1515111183), Lingnan Normal University Talent Project (ZL22013), and an open competition program of the top nine critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province [2023SDZG06]. Authors’ contributions T.L.W. was responsible for most experiments, analysis, writing, and revision of the original manuscript, and for obtaining funding. L.Y. L. and Y.T.L. participated in the qRT-PCR analysis of CsBAG gene expression. L.Y. Z. extracted RNA from citrus leaves treated with HLB. K.D.L. and Y.Z. provided guidance on the experimental design and revised the manuscript. All the authors have read and agreed to the published version of the manuscript. Acknowledgments The authors thank Dr. Yanyan Ma and Dr. Feiyan Wang for their valuable help and advice in writing this manuscript. References Takayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA, et al. Cloning and Functional Analysis of BAG-l: A Novel Bcl-2-Binding Protein with Anti-Cell Death Activity. Cell. 1995;80:279–84. Doong H, Vrailas A, Kohn EC. What’s in the ‘BAG’? – a functional domain analysis of the BAG- family proteins. Cancer Lett. 2002;188:25–32. Yan J, He C, Zhang H. The BAG-family proteins in Arabidopsis thaliana. Plant Sci. 2003;165:1–7. Rana RM, Dong S, Ali Z, Khan AI, Zhang HS. Identification and characterization of the Bcl-2-associated athanogene (BAG) protein family in rice. Afr J Biotechnol. 2012;11:88–99. 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. He M, Wang Y, Jahan MS, Liu W, Raziq A, Sun J, et al. Characterization of SlBAG Genes from Solanum lycopersicum and Its Function in Response to Dark-Induced Leaf Senescence. Plants. 2021;10:947. Ge S, Kang Z, Li Y, Zhang F, Shen Y, Ge R, et al. Cloning and function analysis of BAG family genes in wheat. Funct Plant Biol. 2016;43:393. Kobayashi M, Takato H, Fujita K, Suzuki S. HSG1, a grape Bcl-2-associated athanogene, promotes floral transition by activating CONSTANS expression in transgenic Arabidopsis plant. Mol Biol Rep. 2012;39:4367–74. Kabbage M, Dickman MB. The BAG proteins: a ubiquitous family of chaperone regulators. Cell Mol Life Sci. 2008;65:1390–402. 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. Lee DW, Kim SJ, Oh YJ, Choi B, Lee J, Hwang I. Arabidopsis BAG1 Functions as a Cofactor in Hsc70-Mediated Proteasomal Degradation of Unimported Plastid Proteins. Mol Plant. 2016;9:1428–31. Fang S, Li L, Cui B, Men S, Shen Y, Yang X. Structural insight into plant programmed cell death mediated by BAG proteins in Arabidopsis thaliana . Acta Crystallogr D Biol Crystallogr. 2013;69:934–45. Nawkar GM, Maibam P, Park JH, Woo SG, Kim CY, Lee SY, et al. In silico study on Arabidopsis BAG gene expression in response to environmental stresses. Protoplasma. 2017;254:409–21. 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. Echevarría‐Zomeño S, Fernández‐Calvino L, Castro‐Sanz AB, López JA, Vázquez J, Castellano MM. Dissecting the proteome dynamics of the early heat stress response leading to plant survival or death in Arabidopsis. Plant Cell Environ. 2016;39:1264–78. Fu C, Hou Y, Ge J, Zhang L, Liu X, Huo P, et al. Increased fes1a thermotolerance is induced by BAG6 knockout. Plant Mol Biol. 2019;100:73–82. Pan Y-J, Liu L, Lin Y-C, Zu Y-G, Li L-P, Tang Z-H. Ethylene Antagonizes Salt-Induced Growth Retardation and Cell Death Process via Transcriptional Controlling of Ethylene-, BAG- and Senescence-Associated Genes in Arabidopsis. Front Plant Sci. 2016;7. 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. Eckardt NA. Programmed Cell Death in Plants: A Role for Mitochondrial-Associated Hexokinases. Plant Cell. 2006;18:2097–9. Jiang H, Ji Y, Sheng J, Wang Y, Liu X, Xiao P, et al. Genome-Wide Identification of the Bcl-2 Associated Athanogene (BAG) Gene Family in Solanum lycopersicum and the Functional Role of SlBAG9 in Response to Osmotic Stress. Antioxidants. 2022;11:598. Ghag SB, Shekhawat UKS, Ganapathi TR. Native cell-death genes as candidates for developing wilt resistance in transgenic banana plants. AoB PLANTS. 2014;6:plu037–plu037. You Q, Zhai K, Yang D, Yang W, Wu J, Liu J, et al. An E3 Ubiquitin Ligase-BAG Protein Module Controls Plant Innate Immunity and Broad-Spectrum Disease Resistance. Cell Host Microbe. 2016;20:758–69. Zhou Y, Yang K, Cheng M, Cheng Y, Li Y, Ai G, et al. Double-faced role of Bcl-2-associated athanogene 7 in plant– Phytophthora interaction. J Exp Bot. 2021;72:5751–65. Yeckel GJ. Characterization of a soybean BAG gene and its potential role in nematode resistance. Dr Diss Univ Mo-Columbia USA. 2012. Gayral M, Arias Gaguancela O, Vasquez E, Herath V, Flores FJ, Dickman MB, et al. Multiple ER‐to‐nucleus stress signaling pathways are activated during Plantago asiatica mosaic virus and Turnip mosaic virus infection in Arabidopsis thaliana . Plant J. 2020;103:1233–45. Wu T, Cheng C, Zhong Y, Lv Y, Ma Y, Zhong G. Molecular characterization of the gibberellin-stimulated transcript of GASA4 in Citrus. Plant Growth Regul. 2020;91:89–99. Xu Q, Chen L-L, Ruan X, Chen D, Zhu A, Chen C, et al. The draft genome of sweet orange (Citrus sinensis). Nat Genet. 2013;45:59–66. Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012;40:D1178–86. Eddy SR. Profile hidden Markov models. Bioinformatics. 1998;14:755–63. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, et al. CDD: NCBI’s conserved domain database. Nucleic Acids Res. 2015;43:D222–6. Hu B, Jin J, Guo A-Y, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31:1296–7. Gasteiger E. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003;31:3784–8. Lescot M. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30:325–7. Wu T, Zhong Y, Chen M, Wu B, Wang T, Jiang B, et al. Analysis of CcGASA family members in Citrus clementina (Hort. ex Tan.) by a genome-wide approach. BMC Plant Biol. 2021;21:565. Ikai A. Thermostability and Aliphatic Index of Globular Proteins. J Biochem (Tokyo). 1980. https://doi.org/10.1093/oxfordjournals.jbchem.a133168. Thanthrige N, Jain S, Bhowmik SD, Ferguson BJ, Kabbage M, Mundree S, et al. Centrality of BAGs in Plant PCD, Stress Responses, and Host Defense. Trends Plant Sci. 2020;25:1131–40. Li Y, Kabbage M, Liu W, Dickman MB. Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants. Plant Cell. 2016;28:233–47. Alberti S, Demand J, Esser C, Emmerich N, Schild H, Höhfeld J. Ubiquitylation of BAG-1 Suggests a Novel Regulatory Mechanism during the Sorting of Chaperone Substrates to the Proteasome. J Biol Chem. 2002;277:45920–7. Kang CH, Jung WY, Kang YH, Kim JY, Kim DG, Jeong JC, et al. AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants. Cell Death Differ. 2006;13:84–95. Li Y, Williams B, Dickman M. Arabidopsis B‐cell lymphoma2 (Bcl‐2)‐associated athanogene 7 ( BAG 7)‐mediated heat tolerance requires translocation, sumoylation and binding to WRKY 29. New Phytol. 2017;214:695–705. Gao S, Gao J, Zhu X, Song Y, Li Z, Ren G, et al. ABF2, ABF3, and ABF4 Promote ABA-Mediated Chlorophyll Degradation and Leaf Senescence by Transcriptional Activation of Chlorophyll Catabolic Genes and Senescence-Associated Genes in Arabidopsis. Mol Plant. 2016;9:1272–85. Samonte RV, Eichler EE. Segmental duplications and the evolution of the primate genome. Nat Rev Genet. 2002;3:65–72. Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, et al. The genome of the domesticated apple (Malus × domestica Borkh.). Nat Genet. 2010;42:833–9. Cao Y, Han Y, Meng D, Li D, Jin Q, Lin Y, et al. Genome-wide analysis suggests high level of microsynteny and purifying selection affect the evolution of EIN3/EIL family in Rosaceae. PeerJ. 2017;5:e3400. Additional Declarations No competing interests reported. Supplementary Files TableS17.xlsx Additional file 1: Table S1. Primers used for qRT-PCR. Table S2 Secondary structures of BAG proteins. Table S3. Protein, CDS, and genomic sequences of Oryza sativa, Malus domestica, Arabidopsis thaliana, Solanum lycopersicum, and Carica papaya. Table S4. Cis-regulatory elements in the promoter sequences of CsBAG genes. Table S5. Analysis of RNA-seq data for CsBAG genes. Table S6. Collinearity link of BAG family genes in Citrus sinensis , Arabidopsis thaliana , Oryza sativa , Malus domestica , Solanum lycopersicum, and Carica papaya . FigureS17.zip Additional file 2: Figure S1. Gene structure diagram depicting exons (green boxes) and introns (black line between the red boxes). Additional file 2: Figure S2. Transmembrane topology analysis of CsBAG protein. The Y-axis represents probability, and the X-axis represents the number of amino acid residues. Additional file 2: Figure S3. Alignment diagram displaying BAG protein sequence. Additional file 2: Figure S4. Sequence logos of the ten conserved BAG sequences were generated using the MEME program. Additional file 2: Figure S5. Visualization of the interaction between the CsBAG promoter with predicted transcription factors (TFs). Additional file 2: Figure S6. Expression patterns of eight CsBAG genes during GA 3 and ABA treatment in sweet orange stems. Data represent the mean ± SE of three qRT-PCR experiments and three biological replicates. Statistical significance (P<0.05) based on Duncan’s least significant difference multiple range test is represented by different lowercase letters (a–c) on the bars. Error bars indicate the standard deviation calculated from three independent experiments, with the citrus actin gene serving as an internal control. Additional file 2: Figure S7. Expression patterns of eight CsBAG genes during GA 3 and ABA treatments in sweet orange roots Data represent the mean ± SE of three qRT-PCR experiments and biological replicates. Statistical significance (P<0.05) based on Duncan’s least significant difference multiple range test is represented by different lowercase letters (a–c) on the bars. Error bars indicate standard deviation calculated from three independent experiments, with the citrus actin gene serving as an internal control. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4348725","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":300714599,"identity":"d8f77bb7-54c3-47c3-9ee1-37feb9166fda","order_by":0,"name":"Tianli Wu","email":"","orcid":"","institution":"Life Science and Technology School, Lingnan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Tianli","middleName":"","lastName":"Wu","suffix":""},{"id":300714601,"identity":"588e254a-ab04-4eb0-94b1-5ec5d11fc633","order_by":1,"name":"Leyi Long","email":"","orcid":"","institution":"Life Science and Technology School, Lingnan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Leyi","middleName":"","lastName":"Long","suffix":""},{"id":300714603,"identity":"fade2672-3f22-45ff-ac77-570ccf89fd30","order_by":2,"name":"Yongting Liu","email":"","orcid":"","institution":"Life Science and Technology School, Lingnan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yongting","middleName":"","lastName":"Liu","suffix":""},{"id":300714605,"identity":"4f57f3f1-c49f-42e6-8c3a-796820ed0616","order_by":3,"name":"Kaidong Liu","email":"","orcid":"","institution":"Life Science and Technology School, Lingnan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Kaidong","middleName":"","lastName":"Liu","suffix":""},{"id":300714607,"identity":"be5f7ed9-4650-4505-8151-cc69b292e558","order_by":4,"name":"Lanyan Zheng","email":"","orcid":"","institution":"Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lanyan","middleName":"","lastName":"Zheng","suffix":""},{"id":300714609,"identity":"13f3ea17-0b7c-4a4a-b811-904c6f8b8699","order_by":5,"name":"Yun Zhong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACCSjNz97AwPDBwMaOeC2SPQcYGGcUpCUTr8XgRgIDM8+HQ4wNhHTIz24+9vBr2+E8gxvJzx7bGBxgZmA/fHQDPi2Mc46lG8u2HS6WPPPM3DjH4A4fA09a2g18WpglcsykJdsOJ/Ydz2GTzjF4xswgwWOGVwsbTEvDAaAWC4PDjA2EtPAAtUh+BGqZcAKohYEYLRISaWnSDOfSE2f2HDOT7DFIS2Yj5Bf5GcnHJH+UWSf2szc/k/jxx8aOn/3wMbxaQICZlw3Zd4SUgwDjjz/EKBsFo2AUjIIRCwBdqEqBf7IPsQAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yun","middleName":"","lastName":"Zhong","suffix":""}],"badges":[],"createdAt":"2024-04-30 11:30:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4348725/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4348725/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56351446,"identity":"e4643493-b3ef-4907-91fb-3de6d44a9b17","added_by":"auto","created_at":"2024-05-13 04:16:11","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":150848,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal location of \u003cem\u003eCsBAG\u003c/em\u003e genes in \u003cem\u003eCitrus sinensis\u003c/em\u003e genome. Each bar represents a Chromosome (Chr), with the number indicated above. Chromosome size is depicted by relative length (Mb) sourced from CPBD data. Genes of \u003cem\u003eC. sinensis \u003c/em\u003eare depicted by black lines within chromosomes. The start position of each\u003cem\u003e CsBAG\u003c/em\u003e gene is marked on the left side.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/a0b85558c711e2e81da0a400.jpeg"},{"id":56351447,"identity":"7e81b38a-fdfc-41de-9bbc-2f5fd614100e","added_by":"auto","created_at":"2024-05-13 04:16:11","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22137987,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted three-dimensional (3D) structures of CsBAG proteins. The ribbon diagram displays different colors representing the structural model derived from the SWISS MODLE, highlighting the predominantly helical structure characteristic of the BAG protein fold.\u003c/p\u003e","description":"","filename":"Figure2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/2fe682d0ddd165550c52f6bd.jpeg"},{"id":56351445,"identity":"cdf30050-8a9a-42f7-b1df-46d4042f0c75","added_by":"auto","created_at":"2024-05-13 04:16:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10856543,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree and conserved motifs of the CsBAG family members.\u003cstrong\u003e a. \u003c/strong\u003ePhylogenetic tree analysis of 62 complete CsBAG protein sequences in \u003cem\u003eCitrus sinensis\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eMalus domestica\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum,\u003c/em\u003e and \u003cem\u003eCarica papaya \u003c/em\u003eusing the maximum-likelihood method with 1000 bootstrap values. The numbers indicate the bootstrap values as a percentage of trees obtained from 1000 replicates.\u003cstrong\u003e \u003c/strong\u003eSymbols represent different species\u003cstrong\u003e: \u003c/strong\u003ecircle (Arabidopsis), cross (rice), diamond (apple), square (citrus), five-pointed star (papaya), and triangle (tomato). \u003cstrong\u003eb.\u003c/strong\u003e Ten conserved motifs of the CsBAG proteins in \u003cem\u003eC. sinensis\u003c/em\u003e. Each box color represents a different motif number, and the length of the box indicates motif length.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/686270d021489dddb11d49a8.png"},{"id":56351454,"identity":"25b8ca46-b613-4f62-9c94-f5b3b3b04648","added_by":"auto","created_at":"2024-05-13 04:16:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":626462,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of cis-elements in the \u003cem\u003eCsBAG\u003c/em\u003e genes. Colors indicate the relative abundance of each transcript in each sample compared to the median expression value of that gene in the whole sample set: red indicates higher abundance, while white represents lower abundance.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/f62da966fdeb42efe9641097.png"},{"id":56351443,"identity":"7edc3737-4c37-4b43-ba36-e8c86ef21895","added_by":"auto","created_at":"2024-05-13 04:16:10","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2868820,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003eCsBAG\u003c/em\u003e gene expression in various organs of \u003cem\u003eC. sinensis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/daeff74194c24e384cac8ec3.jpeg"},{"id":56351597,"identity":"5beee876-b2ee-48e3-b93f-0ead602881cb","added_by":"auto","created_at":"2024-05-13 04:24:12","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3294593,"visible":true,"origin":"","legend":"\u003cp\u003eThe qRT-PCR analysis of selected \u003cem\u003eC. sinensis\u003c/em\u003e \u003cem\u003eCsBAG\u003c/em\u003e genes in response to GA\u003csub\u003e3\u003c/sub\u003e and ABA stress. Bars represent the mean±standard deviation of three replicates. Different letters indicate significant differences at different time points, determined by Duncan’s LSD multiple range test (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/5f1e7fd5c4cebfe648fd64f1.jpeg"},{"id":56351448,"identity":"d226cb16-6849-4079-956f-809249c67364","added_by":"auto","created_at":"2024-05-13 04:16:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":43382,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression level of CsBAGs in citrus leaves inoculated with Huanglongbing (HLB). Mean±standard error of three replicates is shown. Different lowercase letters (a–e) on the bars indicate statistically significant differences (P\u0026lt;0.05) based on Duncan’s LSD multiple range test.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/48cbd3e16e2dfa0ca596d96d.png"},{"id":56351452,"identity":"b2ae3f8a-978a-4b56-a9d0-d8fea2f12e5d","added_by":"auto","created_at":"2024-05-13 04:16:12","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2643213,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution and synteny analysis of citrus \u003cem\u003eCsBAG\u003c/em\u003e gene family members. Syntenic and chromosomal regions are depicted in different colors.\u003c/p\u003e","description":"","filename":"Figure8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/03b0abc6827214c9f6312ed6.jpeg"},{"id":56351450,"identity":"c7d71763-97ca-47a3-94f3-8009690e9eef","added_by":"auto","created_at":"2024-05-13 04:16:12","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":6847495,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analysis of \u003cem\u003eCsBAG\u003c/em\u003e genes among citrus, Arabidopsis, apple, and grape. The chromosomes of \u003cem\u003eC. sinensis\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eM. domestica\u003c/em\u003e,\u003cem\u003e S. lycopersicum,\u003c/em\u003e and \u003cem\u003eC. papaya \u003c/em\u003eare represented, indicating the relative positions of the\u003cem\u003e CsBAG\u003c/em\u003e genes. Gray lines in the background represent collinear blocks within different species, while blue lines highlight syntenic BAG gene pairs\u003c/p\u003e","description":"","filename":"Figure9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/cc7df17ea228c8379fdfe387.jpeg"},{"id":63892533,"identity":"490b0b82-081b-41b4-b156-197f0fa7ea7d","added_by":"auto","created_at":"2024-09-03 12:32:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":52861887,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/944ef4fa-5950-400a-a6aa-260a7f8c17e4.pdf"},{"id":56351451,"identity":"60245ea6-ca66-4814-8c08-d46cc36d02f2","added_by":"auto","created_at":"2024-05-13 04:16:12","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":245025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Table S1.\u003c/strong\u003e Primers used for qRT-PCR.\u003cstrong\u003e Table S2\u003c/strong\u003e Secondary structures of BAG proteins.\u003cstrong\u003e Table S3. \u003c/strong\u003eProtein, CDS, and genomic sequences of \u003cem\u003eOryza sativa, Malus domestica, Arabidopsis thaliana, Solanum lycopersicum, \u003c/em\u003eand\u003cem\u003e Carica papaya.\u003c/em\u003e \u003cstrong\u003eTable S4. \u003c/strong\u003eCis-regulatory elements in the promoter sequences of CsBAG genes. \u003cstrong\u003eTable S5.\u003c/strong\u003e Analysis of RNA-seq data for CsBAG genes. \u003cstrong\u003eTable S6.\u003c/strong\u003e Collinearity link of BAG family genes in \u003cem\u003eCitrus sinensis\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eMalus domestica\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum,\u003c/em\u003e and \u003cem\u003eCarica papaya\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"TableS17.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/49388180ebbc55a679bb0083.xlsx"},{"id":56351449,"identity":"7a43375f-65ae-419d-823a-6deb34b9baea","added_by":"auto","created_at":"2024-05-13 04:16:12","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13646740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S1.\u003c/strong\u003e Gene structure diagram depicting exons (green boxes) and introns (black line between the red boxes).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S2.\u003c/strong\u003e Transmembrane topology analysis of CsBAG protein. The Y-axis represents probability, and the X-axis represents the number of amino acid residues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S3.\u003c/strong\u003e Alignment diagram displaying BAG protein sequence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S4.\u003c/strong\u003e Sequence logos of the ten conserved BAG sequences were generated using the MEME program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S5. \u003c/strong\u003eVisualization of the\u003cstrong\u003e \u003c/strong\u003einteraction between the CsBAG promoter with predicted transcription factors (TFs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S6. \u003c/strong\u003eExpression patterns of eight CsBAG genes during GA\u003csub\u003e3\u003c/sub\u003e and ABA treatment in sweet orange stems. Data represent the mean ± SE of three qRT-PCR experiments and three biological replicates. Statistical significance (P\u0026lt;0.05) based on Duncan’s least significant difference multiple range test is represented by different lowercase letters (a–c) on the bars. Error bars indicate the standard deviation calculated from three independent experiments, with the citrus actin gene serving as an internal control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S7. \u003c/strong\u003eExpression patterns of eight CsBAG genes during GA\u003csub\u003e3\u003c/sub\u003e and ABA treatments in sweet orange roots Data represent the mean ± SE of three qRT-PCR experiments and biological replicates. Statistical significance (P\u0026lt;0.05) based on Duncan’s least significant difference multiple range test is represented by different lowercase letters (a–c) on the bars. Error bars indicate standard deviation calculated from three independent experiments, with the citrus actin gene serving as an internal control.\u003c/p\u003e","description":"","filename":"FigureS17.zip","url":"https://assets-eu.researchsquare.com/files/rs-4348725/v1/22577ec790b3843a48833464.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification, Characterization, and Expression Analysis of the CsBAG family in Citrus sinensis (L.) Osbeck","fulltext":[{"header":"Background","content":"\u003cp\u003eThe B-cell lymphoma 2 (Bcl-2)-associated athanogene (\u003cem\u003eBAG\u003c/em\u003e) gene was initially discovered while screening a small mouse embryonic cDNA library, where recombinant human Bcl-2 protein was utilized as bait to identify Bcl-2 interactors. This gene was designated as BAG1 (Takayama et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). BAG1 and Bcl-2 synergistically promote cell survival, emerging as a novel anticell death gene within the programmed cell death pathway. After its discovery, similar sequences of the \u003cem\u003eBAG\u003c/em\u003e gene have been identified across various plants and animals, such as \u003cem\u003eBAG1-6\u003c/em\u003e in mammals (Doong et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), \u003cem\u003eAtBAG1-8\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Yan et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), \u003cem\u003eOsBAG1-6\u003c/em\u003e in \u003cem\u003eOryza sativa\u003c/em\u003e L. (Rana et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003eMusaBAG1-13\u003c/em\u003e in bananas (Dash and Ghag, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), \u003cem\u003eSlBAG1-7\u003c/em\u003e in tomatoes (He et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cem\u003eTaBAG\u003c/em\u003e and \u003cem\u003eTaBAG2\u003c/em\u003e in wheat (Ge et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and \u003cem\u003eHSG1\u003c/em\u003e in grape (Kobayashi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Sequence analysis revealed that these genes are members of the BAG family. In plants and animals, the protein encoded by the \u003cem\u003eBAG\u003c/em\u003e gene possesses a BAG domain comprising 110 amino acid residues at the C-terminus. This frequently includes a ubiquitin-like domain (UBL) and various other motifs, such as the isoleucine glutamine motif (IQ), proline-rich repeat (PXXP), TXSEEX repeat, nuclear localization signal (NLS), and the WW domain (Doong et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Kabbage and Dickman, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Among these, IQ motifs are exclusive to plants, potentially indicating functional variations in BAG between plants and animals (Doukhanina et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMost BAG family members participate in plant responses to abiotic stresses, such as salt, heat, cold, drought, ultraviolet radiation, and plant hormones. Plant \u003cem\u003eBAG\u003c/em\u003e genes have evolved independently, leading to diverse spatiotemporal expression patterns and functions across different species. Lee et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) observed that transgenic seedlings of \u003cem\u003eA. thaliana AtBAG1\u003c/em\u003e exhibited increased sensitivity to salt treatment (Lee et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Fang et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) demonstrated the involvement of \u003cem\u003eAtBAG2\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e in plant responses to environmental stress (Fang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The \u003cem\u003eAtBAG2\u003c/em\u003e gene is also induced by ABA, ACC, SA, heat, salt, and drought stress (Nawkar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Arif et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Transcript levels of \u003cem\u003eAtBAG3\u003c/em\u003e increase in response to salt, MeJA, ABA, and SA, with cold responses inhibited in the roots (Nawkar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Overexpression of \u003cem\u003eAtBAG4\u003c/em\u003e in tobacco enhances tolerance to UV, cold, oxidants, and salt treatments (Doukhanina et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Transcription of \u003cem\u003eAtBAG6\u003c/em\u003e is induced by stress-related plant hormones (such as ACC, SA, MeJA, and ABA), heat stress, mannitol, and programmed cell death (PCD) inducers (Echevarr\u0026iacute;a-Zome\u0026ntilde;o et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nawkar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Fu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eAtBAG6\u003c/em\u003e mutations enhance short-term heat tolerance in \u003cem\u003efes1a\u003c/em\u003e mutants (where \u003cem\u003eFes1A\u003c/em\u003e mutations decrease heat stress tolerance); however, this improved heat tolerance is diminished by calmodulin inhibitor treatment (Fu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eAtBAG7\u003c/em\u003e expression is reduced under salt stress conditions; however, ACC treatment counteracts the salinity-induced effects (Pan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In rice, ABA, IAA, and high-temperature treatments induce the expression of \u003cem\u003eOsBAG1\u003c/em\u003e and \u003cem\u003eOsBAG3\u003c/em\u003e (Zhou et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Transgenic rice overexpressing \u003cem\u003eOsBAG4\u003c/em\u003e exhibits enhanced tolerance to salt stress (Eckardt, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Overexpressing \u003cem\u003eTaBAG2\u003c/em\u003e in wheat has been shown to increase heat tolerance in \u003cem\u003eA. thaliana\u003c/em\u003e (Ge et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The expression of \u003cem\u003eHSG1\u003c/em\u003e in grapes significantly rises under heat stress, and when overexpressed in \u003cem\u003eA. thaliana\u003c/em\u003e, it leads to substantial resistance to high temperature (Kobayashi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, heterologous overexpression of tomato \u003cem\u003eSlBAG9\u003c/em\u003e heightens the sensitivity of \u003cem\u003eA. thaliana\u003c/em\u003e to drought, salinity, and ABA (Jiang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These findings underscore the significant roles played by BAG family members in plant responses to abiotic stress.\u003c/p\u003e \u003cp\u003ePathogens infection such as fungi, bacteria, viruses, insects, and nematodes are believed to be major triggers of plant \u003cem\u003eBAG\u003c/em\u003e gene expression. In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAtBAG2\u003c/em\u003e expression was specifically suppressed in response to the necrotic fungus \u003cem\u003eBotrytis cinerea\u003c/em\u003e, semi-living trophic fungus \u003cem\u003ePhytophthora infestans\u003c/em\u003e, and bacterium \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. phaseolicola. The expression of \u003cem\u003eAtBAG3\u003c/em\u003e and \u003cem\u003eAtBAG6\u003c/em\u003e is moderately induced by the toxic \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. tomato DC3000 and necrotizing fungal pathogen \u003cem\u003eBotrytis cinerea\u003c/em\u003e (Nawkar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Correspondingly, \u003cem\u003eatbag6\u003c/em\u003e mutants display heightened sensitivity to the necrotizing fungus \u003cem\u003eB. cinerea\u003c/em\u003e (Doukhanina et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Transgenic plants overexpressing MusaBAG1 in bananas exhibit increased resistance to wilt disease (caused by fungi or bacteria) (Ghag et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eBAG4\u003c/em\u003e is triggered in the leaf tissue of \u003cem\u003eMedicago truncatula\u003c/em\u003e upon infection with the bacterial pathogen \u003cem\u003eXylella fastidiosa\u003c/em\u003e (Dash and Ghag, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In rice, accumulation of \u003cem\u003eOsBAG4\u003c/em\u003e in \u003cem\u003eebr1\u003c/em\u003e mutants or plants overexpressing \u003cem\u003eOsBAG4\u003c/em\u003e initiates autoimmunity and broad-spectrum disease resistance (against pathogens such as \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. \u003cem\u003eoryzae\u003c/em\u003e and \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e) (You et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Soybean \u003cem\u003eGmBAG7\u003c/em\u003e exhibits a dual effect on \u003cem\u003eArabidopsis\u003c/em\u003e-\u003cem\u003ePhytophthora capsici\u003c/em\u003e interactions, acting as a susceptibility factor in the endoplasmic reticulum but conferring resistance to \u003cem\u003ePhytophthora capsici\u003c/em\u003e in the nucleus (Zhou et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Several studies have highlighted the significant role of BAG in plant resistance against fungal and bacterial stress. BAG not only combat pathogenic microorganisms but also contributes to defense against insect predation. In rice, the expression of \u003cem\u003eOsBAG1-3\u003c/em\u003e is significantly induced by feeding from the brown planthopper (BPH) insect (Zhou et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Overexpressing soybean \u003cem\u003eGmBAG6a\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e confers resistance to nematode infection (Yeckel, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition, BAG plays a role in plant antiviral responses. Gayral et al. discovered that \u003cem\u003eAtBAG7\u003c/em\u003e is crucial for the localized accumulation of \u003cem\u003ePlantago asiatica mosaic virus\u003c/em\u003e in the endoplasmic reticulum-to-nuclear signaling pathway and is linked to host resistance against \u003cem\u003eTurnip mosaic virus\u003c/em\u003e (Gayral et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our previous study, transcriptome sequencing of citrus roots infected with Huanglongbing (HLB) revealed the regulation of key genes within the disease resistance pathway by HLB infection, and a gibberellin induced gene \u003cem\u003eCcGASA4\u003c/em\u003e was identified. Transcription and metabolome analyses of transgenic citrus plants overexpressing \u003cem\u003eCcGASA4\u003c/em\u003e revealed that the differentially expressed genes predominantly included pathogens, stress responses, hormones, and growth and development-related genes. By employing CcGASA4 as bait, the citrus yeast expression library underwent screening, resulting in the identification of the interacting BAG protein family member PRPBAG6-A. The interaction between these two proteins has been elucidated \u003cem\u003ein vitro\u003c/em\u003e through yeast double hybridization and bimolecular fluorescence complementation (Wu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This result showed that BAG may have a significant role in the citrus response to HLB infection through its interaction with CcGASA4. However, despite progress made in understanding plant BAGs, the underlying mechanisms remain unelucidated. Therefore, this study aims to systematically analyze citrus BAG and investigate its role in the response to hormonal treatment and HLB infection.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eIdentification of putative\u003c/b\u003e \u003cb\u003eCitrus sinensis\u003c/b\u003e \u003cb\u003eBAG genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eC. sinensis\u003c/em\u003e genome and protein sequences were obtained from the Pan-genome to Breeding Database (CPBD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://citrus.hzau.edu.cn\u003c/span\u003e\u003cspan address=\"http://citrus.hzau.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Xu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The genome and protein sequences of \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e, \u003cem\u003eMalus domestica\u003c/em\u003e, and \u003cem\u003eCarica papaya\u003c/em\u003e were retrieved from the Phytozome online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Goodstein et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). BAG proteins were identified through Hidden Markov Model (HMM) searches of sequences in the downloaded peptide sequence FASTA file using the HMMER 3.0 program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/hmmer/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/hmmer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default parameters (Eddy, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). A comprehensive search was also conducted using the amino acid sequences of eight previously reported Arabidopsis BAG proteins (Doukhanina et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The integrity of the BAG domain (Accession: PF02179) in all putative \u003cem\u003eC. sinensis\u003c/em\u003e BAG sequences was confirmed using the NCBI Conserved Domain Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Marchler-Bauer et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Only nonredundant putative protein sequences with conserved BAG domains were retained for subsequent analysis. All CsBAG protein coding sequences, genomic regions, and associated information, such as accession numbers and chromosomal positions, were obtained from the CPBD database. In addition, the physical location of each \u003cem\u003eCsBAG\u003c/em\u003e gene in the genome was mapped using MapChart software. The CDS sequences of \u003cem\u003eCsBAG\u003c/em\u003e and their respective genomic sequences were compared and analyzed to identify introns and exons using the Gene Structure Display Server 2.0 (GSDS2.0, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gao-lab.org/\u003c/span\u003e\u003cspan address=\"http://gsds.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Hu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of physicochemical properties of the citrus BAG proteins\u003c/h2\u003e \u003cp\u003eThe physicochemical parameters of the CsBAG proteins were computed using ProtParam on the ExPASy server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/protparam\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/protparam\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Gasteiger, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Putative protein secondary and tertiary structures were predicted using SOPMA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html\u003c/span\u003e\u003cspan address=\"https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the SWISS MODLE engine (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/interactive\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/interactive\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively. Subcellular protein localizations were predicted using the WOLF PSORT program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), while transmembrane helices were predicted using the DeepTMHMM server v1.0.24 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dtu.biolib.com/DeepTMHMM:1.0.24/\u003c/span\u003e\u003cspan address=\"https://dtu.biolib.com/DeepTMHMM:1.0.24/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of protein phylogenetic relationships and gene structures\u003c/h2\u003e \u003cp\u003eA phylogenetic tree was constructed using 62 BAG protein sequences from different plant species, including \u003cem\u003eC. sinensis\u003c/em\u003e (10), \u003cem\u003eA. thaliana\u003c/em\u003e (8), \u003cem\u003eO. sativa\u003c/em\u003e (10), \u003cem\u003eM. domestica\u003c/em\u003e (15), \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (10), and \u003cem\u003eCarica papaya\u003c/em\u003e (9). The phylogenetic tree was generated using the Maximum Likelihood (ML) method with MEGA 7.0 software. The ML method parameters included 1000 bootstrap replications, the \u0026ldquo;Jones-Taylor-Thornton\u0026rdquo; model, \u0026ldquo;Uniform rates,\u0026rdquo; \u0026ldquo;Complete deletion,\u0026rdquo; and \u0026ldquo;Nearest-Nerghbor-Interchange\u0026rdquo; method. Conserved motifs within the CsBAG proteins were identified using the MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) website, with motif parameters set to 10. The phylogenetic tree and motif images were visualized using the Chiplot website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for visualization.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePromoter analysis of\u003c/b\u003e \u003cb\u003eCsBAG\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 2.0-kb long promoter sequence upstream of the start codon (ATG) for each \u003cem\u003eCsBAG\u003c/em\u003e was obtained from the CPBD database (Xu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Analysis of the cis-regulatory elements within the \u003cem\u003eCsBAG\u003c/em\u003e gene promoters was conducted using the online program Plant Cis-Acting Regulatory DNA Elements (\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) (Lescot, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Prediction of transcription factor (TFs) networks was performed online, with a threshold parameter p-value\u0026thinsp;\u0026le;\u0026thinsp;1e-5 on the Plant Transcriptional Regulatory Map (PTRM) website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plantregmap.gao-lab.org/regulation_prediction.php\u003c/span\u003e\u003cspan address=\"http://plantregmap.gao-lab.org/regulation_prediction.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), utilizing all \u003cem\u003eCsBAG\u003c/em\u003e promoter sequences as input. Cytoscape 3.9.1 software was employed to visualize the TFs regulatory network.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analysis of citrus\u003c/b\u003e \u003cb\u003eCsBAG\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBiotic stress treatment involved selecting 3-year-old \u003cem\u003eC. sinensis\u003c/em\u003e trees, onto which branches infected with HLB were grafted onto stems. The control group comprised tree shoots grafted without HLB bacteria. The 2-month-old \u003cem\u003eC. sinensis\u003c/em\u003e seedlings were planted in plastic garden pots for use as hormone treatment materials. For hormone treatment, trees were sprayed with 100 \u0026micro;Μ of GA\u003csub\u003e3\u003c/sub\u003e and ABA. Root, stem, and leaf samples were harvested at 3 h, 6 h, 12 h, 24 h and 48 h. Control trees were sprayed with sterile water, and root, stem, and leaf tissues were harvested at the same time points as those in the hormone treatment group. Samples were flash-frozen in liquid nitrogen and stored at -80\u0026deg;C until use. Three biological replicates were utilized for each treatment. Total RNA was extracted from frozen leaf samples using the TRIzol extraction method (TIANGEN, China). RNA extraction process involved the following steps: 1. 50\u0026ndash;100 mg of leaf tissue was fully ground with liquid nitrogen in a 2.0 ml centrifuge tube; 1 ml of Trizol was added to fully homogenize the tissue, and the mixture was allowed to stand at 25\u0026deg;C for 5 min; 2. 0.2 mL of chloroform was added, the tube was shaken for 15 s, and allowed to stand for 2 min. 3. The mixture was centrifuged at 12000 g for 15 min at 4\u0026deg;C, and the supernatant was collected; 4. Subsequently, 1:1 isopropanol was added, mixed gently, and incubated at 25\u0026deg;C for 10 min; 5. Subsequently, the mixture was centrifuged at 12000 g for 10 min at 4\u0026deg;C, and the supernatant was discarded. 6. Following that, 1 mL of 75% ethanol was added to wash the pellet gently. Afterward, the mixture was centrifuged at 7500 g for 5 min at 4\u0026deg;C, and the supernatant was discarded (this step was repeated twice). 7. The pellet was air-dried, followed by the addition of 60 uL DEPC water to dissolve it. RNA concentrations were determined using a NanoDrop 2000C (ThermoFisher Scientific, USA), and RNA quality was assessed using the OD260/OD280 ratio and agarose gel electrophoresis. cDNA synthesis, quantitative real-time PCR (qRT-PCR), and data analysis followed the procedure outlined by Wu et al. (Wu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Supplementary Table S1 lists the Primers utilized in this study. \u003cem\u003eActin\u003c/em\u003e (GenBank accession number: Cs_ont_1g004160.1) served as the reference gene. Due to the prediction that the three proteins, CsBAG4.1, CsBAG4.2, and CsBAG4.3, originated from a single gene sequence (Cs_ont_5g040580) through alternative splicing, and because the coding sequences of CsBAG4.1 and CsBAG4.3 were identical, while only the noncoding sequences differed, designing primers to distinguish CsBAG4.3 from CsBAG4.1 was not feasible. Therefore, the final expression results of CsBAG4.1 and CsBAG4.3 were combined. For quality controls, three technical replicates were also employed in addition to three biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSynteny analysis and calculation of the Ka/Ks ratio for duplicated genes\u003c/h2\u003e \u003cp\u003ePotential duplicated citrus \u003cem\u003eCsBAG\u003c/em\u003e genes were identified using the MCScanX function of the TBtools software, and results were visualized using the same software. Chromosomal location information for all CsBAG genes was obtained from the annotation gff3-file. Collinear pairs were extracted using TBtools software to identify syntenic blocks and duplications within the BAGs across the whole genomes of six species: \u003cem\u003eC. sinensis\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eM. domestica\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, and \u003cem\u003eCarica papaya\u003c/em\u003e. A collinearity map between these species was generated using MCScan X (TBtools software).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGenome-wide identification and characterization of BAG genes in\u003c/b\u003e \u003cb\u003eC. sinensis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the \u003cem\u003eC. sinensis\u003c/em\u003e genome, HMMER searches revealed 10 CsBAG proteins containing a conserved BAG domain (Accession: PF02179) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This resulted in the identification of eight \u003cem\u003eCsBAG\u003c/em\u003e genes. These \u003cem\u003eCsBAG\u003c/em\u003e genes were designated as \u003cem\u003eCsBAG1\u003c/em\u003e\u0026ndash;\u003cem\u003eCsBAG8\u003c/em\u003e based on their chromosomal locations. Upon comparing CsBAG4.1, CsBAG4.2, and CsBAG4.3 proteins, it was observed that they were encoded by the same gene, \u003cem\u003eCs_ont_5g040580\u003c/em\u003e (\u003cem\u003eCsBAG4\u003c/em\u003e). Ten CsBAGs were distributed across the five chromosomes of the \u003cem\u003eC. sinensis\u003c/em\u003e genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Chromosomes 2, 5, and 9 contained two \u003cem\u003eBAG\u003c/em\u003e genes, while chromosomes 6 and 7 contained one \u003cem\u003eBAG\u003c/em\u003e gene. Gene structure diagrams of \u003cem\u003eCsBAG\u003c/em\u003e genes, depicting exon-intron components, were generated using the Gene Structure Display Server (GSDS) (Fig. S1). \u003cem\u003eCsBAG6\u003c/em\u003e was analyzed to comprise a single exon, while two \u003cem\u003eCsBAG\u003c/em\u003e genes (\u003cem\u003eCsBAG1\u003c/em\u003e and \u003cem\u003eCsBAG3\u003c/em\u003e) contained two exons. In contrast, four \u003cem\u003eCsBAG\u003c/em\u003e genes (\u003cem\u003eCsBA\u003c/em\u003eG2, \u003cem\u003eCsBAG4.2\u003c/em\u003e, \u003cem\u003eCsBAG5\u003c/em\u003e, and \u003cem\u003eCsBAG8\u003c/em\u003e) within the same group contained four exons. The deduced protein sequences of the ten CsBAG transcripts ranged in length from 218\u0026ndash;645 amino acid residues (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The predicted molecular weights (MWs) ranged from 24.68\u0026ndash;70.81 kDa, with isoelectric points (pIs) spanning 4.48\u0026ndash;9.40. All CsBAGs exhibited hydrophilic characteristics, indicated by their negative grand average of hydropathicity (GRAVY) value. The aliphatic index values of the CsBAG proteins ranged from 60.89\u0026ndash;94.68. A high aliphatic index suggests thermostability across a broad temperature range (Ikai, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Most of the proteins were predicted to be unstable, with eight of them\u0026mdash;except for CsBAG4.1 and CsBAG4.3\u0026mdash;having instability index values exceeding 40.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of the CsBAG family gene in \u003cem\u003eCitrus sinensis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"15\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTranscript ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStart Sit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEnd Sit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStrand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo. of Exons\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCDS (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMW (kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eProtein (A.A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003epl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eGRAVY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eAI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eSubcellular localization\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_2g000960.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e536380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e538956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e25.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e7.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e47.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e69.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003enucl, cyto, mito\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_2g007530.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4538601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4540720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e28.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e9.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.446\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e49.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e87.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003echlo, mito, nucl, cyto\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_5g003990.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2567670\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2569909\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e70.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e42.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e87.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003ecyto, nucl, cysk, golg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_5g040580.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43283953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e43285952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e24.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e8.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e37.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e94.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003echlo, mito, cyto, nucl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_5g040580.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43283953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e43285952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e32.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e9.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e43.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e88.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003echlo, nucl, mito, plas\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_5g040580.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43283953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e43285952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e24.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e8.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e37.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e94.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003echlo, mito, cyto, nucl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_6g014260.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9178620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9180654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e861\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e31.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e5.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.925\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e40.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e69.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003enucl, cyto, mito, plas\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_7g021670.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24928099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24929870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e42.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-1.081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e55.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e60.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003enucl, chlo, mito\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_9g023720.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28052851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28056522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e45.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e9.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e47.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e78.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003echlo, nucl, cyto, plas, mito\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsBAG8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCs_ont_9g025230.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29328941\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29331515\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e39.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e9.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e-0.761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e45.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e72.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003enucl, cyto, vacu, chlo, mito, plas, E.R.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"15\"\u003eNote: CDS, coding DNA sequence; AA, amino acid; MW, molecular weight; pI, isoelectric point; kDa, kilodalton; Instability index (II); Aliphatic index (AI); pI isoelectric point, GRAVY, grand average of hydropathicity. chlo, chloroplast; mito, mitochondria; cyto, cytoplasm; extr, extracellular; vacu, vacuoles; nucl, nucleus; golg, golgiosome; plas, plastid; ER, endoplasmic reticulum.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe predicted subcellular localization of citrus CsBAG family proteins primarily included the nucleus, cytoplasm, mitochondria, and chloroplast, with 10, 8, 9, and 7 members, respectively. However, some CsBAGs were potentially localized in the cytoskeleton, Golgi, plastids, vacuoles, and endoplasmic reticulum (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The TMHMM server confirmed the absence of a transmembrane helix (Supplementary Fig. S2). 3D structure prediction revealed that almost all CsBAG proteins exhibited flexibilility, characterized by random coils at the N-terminus and C-terminus. In the middle of the CsBAG sequences, several α-helices were observed adjacent to random coils and extended strands or beta-turn (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic relationship and structural features of BAG proteins in citrus\u003c/h2\u003e \u003cp\u003eTo assess the evolutionary relationships among BAG family proteins in \u003cem\u003eC. sinensis\u003c/em\u003e and other species, a phylogenetic tree was generated using 62 full-length BAG protein sequences from six plant species ( 10 from sweet orange, eight from \u003cem\u003eArabidopsis\u003c/em\u003e, 10 from rice, 15 from apple, nine from papaya, and 10 from tomato) using the maximum likelihood (ML) method (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table S3). All six species analyzed exhibited homologs within the four branches, with BAG proteins distributed unevenly across four groups: G1, G2, G3, and G4. Among citrus CsBAG proteins, CsBAG2 and CsBAG5 clustered with G1, CsBAG8 was grouped within the fewest clade G2, and CsBAG1, CsBAG3, CsBAG6, and CsBAG7 were classified within the largest clade, G3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The G4 group, including CsBAG4.1, CsBAG4.2, and CsBAG4.3, stood out from the other BAG genes by forming an individual clade, indicating its specificity to sweet orange. Unlike rice and \u003cem\u003eArabidopsis\u003c/em\u003e, there were no homolog members in this clade, suggesting that they resulted from duplication after the segregation of \u003cem\u003eArabidopsis\u003c/em\u003e and Citrus ancestors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the evolution of the BAG gene family, we examined the structural characteristics of the BAG genes in \u003cem\u003eC. sinensis\u003c/em\u003e. Exon\u0026ndash;intron organization analysis revealed a relatively conserved arrangements pattern within the same group (Fig. S1). Among these, CsBAG3 exhibited the longest gene structure, spanning 1938 bp, while CsBAG4.1/4.3 had the shortest structure, measuring 657 bp (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among all the BAG genes, CsBAG2, 4.2, 5, and 8 exhibited the highest number of exons, with four each. The CsBAG4.1, 4.3, and 7 had three exons each. CsBAG6 lacked introns entirely. Additionally, most BAG genes displayed a similar exon/intron structure, with intron phases predominantly clustered within the same group (Fig. S1).\u003c/p\u003e \u003cp\u003eTo assess the sequence features of \u003cem\u003eC. sinensis\u003c/em\u003e BAGs, conserved motifs and domains were examined. It was found that all CsBAG proteins contained a BAG domain situated at the terminus of the CsBAG proteins (Fig. S3). In addition to the BAG domain, CsBAG2, 4.2, 5, and 8 contained ubiquitin-like domains similar to that in their animal counterparts. However, CsBAG1 and CsBAG6 featured an IQ calmodulin-binding domain situated at the N-terminus. This characteristic is exclusive to plants.To explore genetic variation in \u003cem\u003eC. sinensis\u003c/em\u003e, 10 conserved motifs among the 62 BAG proteins were examined using the MEME program (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The lengths of these conserved motifs ranged from 18\u0026ndash;50 amino acids, displaying a highly diverse distribution (Fig. S4). CsBAG1, 3, 6, and 7 exhibited only three motifs, while most BAGs contained eight motifs ( 1, 2, 3, 4, 5, 6, 7, and 8) arranged in a similar order. The composition and arrangement of conserved motifs among BAG protein sequences within the same phylogenetic branch were similar. For example, within the G3 group, most members featured two uniquely spaced motifs: motifs 9 and 10 were interspersed with motif 2, indicating specialized functions that benefited this group. The citrus G1, G2, and G4 members exhibited greater diversity than those of G1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, while most G1 and G2 members contained eight motifs, the spacing between motif 6 and the adjacent motif 3 was wider in G2 than in G1. Some citrus CsBAG proteins should be structurally or functionally compromised due to missing one or two motifs compared to their respective group members. For instance, CsBAG5 lacked motif7, while CsBAG4.1 and 4.3 were devoid of motif3. Overall, most CsBAGs in adjacent branches displayed similar exon-intron compositions and motif and domain distributions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCsBAG promoters and their possible activators\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the cis-regulatory elements identified in the promoters of \u003cem\u003eCsBAG\u003c/em\u003e genes. Many elements, such as MBS (drought-inducibility), ARE (antioxidant response element), WUN-motif (wound responsiveness), LTR (low-temperature responsiveness), GC-motif (enhancer-like element involved in anoxic-specific inducibility), MYC (abiotic element), WRE3 (wound-response element 3), DRE core (cold and dehydration responsiveness), box S (elicitation, wounding, and pathogen responsiveness), W box (wounding and pathogen responsiveness), and MYB (abiotic element). MYB and MYC, crucial for abiotic responsiveness, were found in all CsBAG promoters. The antioxidant response element (ARE) was present in seven CsBAG promoters, except for CsBAG2, 5, and 6. The promoters of CsBAG genes contained various hormone-responsive elements such as ABRE (responsive to ABA), P-box and GARE-motif (responsive to GA), AuxRR-core, and TGA-element (responsive to AUX), ERE (responsive to ET), TCA-element, and TCA (responsive to SA), as well as CGTCA- and TGACG-motif (responsive to MeJA). Light response cis-elements, including the G-box, Box 4, AE-box, Sp1, I-box, Box II, ACE, LS7, LAMP-element, GATA-, GA-, ATCT-, GT1-, TCT-, GTGGC-, and TCCC-motif, were also prevalent in the CsBAG promoters. In addition, CsBAG promoters also harbored plant growth- and development-associated cis-elements, including the meristem-specific expression element CAT-box, plant endosperm-specific negative expression element AAGAA-motif, zein metabolism regulation element O2-site, plant cell cycle control-related element MSA-like, and palisade mesophyll cell differentiation element HD-Zip 1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe analysis of the potential transcriptional regulatory network of \u003cem\u003eCsBAG\u003c/em\u003e genes revealed a large number of TFs capable of binding to the identified cis-elements (Fig. S5 and Supplementary Table S4). Predominantly, TFs included ERF, bHLH, MYB, WRKY, and MIKC_MADS, with 78, 36, 21, 21, and 18 members identified respectively. In addition, 32 Dofs, 2 ARFs, and 14 C2H2s were identified as activators of \u003cem\u003eCsBAG\u003c/em\u003e genes. Functionally, these TFs were primarily linked to abiotic and biotic stresses, such as pathogen attack, heat shock, and drought stress.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression pattern of\u003c/b\u003e \u003cb\u003eCsBAGs\u003c/b\u003e \u003cb\u003eunder HLB pathogen and plant hormone (ABA and GA\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e) stresses\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the tissue specificity of \u003cem\u003eCsBAG\u003c/em\u003e expression, qRT-PCR was conducted on various tissues, including the leaves, stems, and roots of 2-month-old \u003cem\u003eC. sinensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results showed that \u003cem\u003eCsBAG1\u003c/em\u003e, \u003cem\u003eCsBAG2\u003c/em\u003e, \u003cem\u003eCsBAG3\u003c/em\u003e, \u003cem\u003eCsBAG4.2\u003c/em\u003e, and \u003cem\u003eCsBAG8\u003c/em\u003e exhibited higher expression levels in stems compared to leaves. \u003cem\u003eCsBAG4.1\u003c/em\u003e and \u003cem\u003eCsBAG4.3\u003c/em\u003e exhibited predominant expression in leaves, with comparatively lower expression levels in roots. In contrast, \u003cem\u003eCsBAG7\u003c/em\u003e showed the highest expression in roots, followed by moderate expression levels in stems and leaves. \u003cem\u003eCsBAG5\u003c/em\u003e and \u003cem\u003eCsBAG6\u003c/em\u003e displayed similar expression levels across roots, stems, and leaves.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the potential functions of \u003cem\u003eCsBAGs\u003c/em\u003e in response to hormone treatment, we examined the expression patterns of \u003cem\u003eCsBAG\u003c/em\u003e genes following treatments with GA\u003csub\u003e3\u003c/sub\u003e and ABA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Fig. S6, S7). Upon GA\u003csub\u003e3\u003c/sub\u003e treatment, \u003cem\u003eCsBAG1\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, \u003cem\u003e4.2\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, and \u003cem\u003eCsBAG8\u003c/em\u003e exhibited significant upregulation in citrus leaves. \u003cem\u003eCsBAG1\u003c/em\u003e exhibited the highest upregulation, approximately 208-fold, following 3 h of GA\u003csub\u003e3\u003c/sub\u003e treatment. \u003cem\u003eCsBAG2\u003c/em\u003e showed the second highest induction, approximately 197-folds, after 6 h of GA\u003csub\u003e3\u003c/sub\u003e treatment. \u003cem\u003eCsBAG3\u003c/em\u003e, \u003cem\u003eCsBAG4.2\u003c/em\u003e, and \u003cem\u003eCsBAG7\u003c/em\u003e were upregulated approximately 10-fold. The expression of \u003cem\u003eCsBAG5\u003c/em\u003e and \u003cem\u003eCsBAG8\u003c/em\u003e increased initially in stems, peaked in the middle, and then declined towards the end of the treatment, while the expression of \u003cem\u003eCsBAG3\u003c/em\u003e and \u003cem\u003eCsBAG4.2\u003c/em\u003e decreased at 3 h, 6 h, 12 h, and 24 h, but increased at 48 h. In the stems under GA\u003csub\u003e3\u003c/sub\u003e treatment, \u003cem\u003eCsBAG6\u003c/em\u003e and \u003cem\u003eCsBAG7\u003c/em\u003e showed gradual upregulation, while \u003cem\u003eCsBAG1\u003c/em\u003e and \u003cem\u003eCsBAG2\u003c/em\u003e exhibited gradual downregulation. In GA\u003csub\u003e3\u003c/sub\u003e-treated roots, \u003cem\u003eCsBAG4.1\u003c/em\u003e and \u003cem\u003eCsBAG4.3\u003c/em\u003e were significantly enriched, while CsBAG6 and CsBAG7 were downregulated. \u003cem\u003eCsBAG7\u003c/em\u003e expression sharply decreased from 0\u0026ndash;12 h and then gradually decreased to its lowest level after 48 h of treatment. \u003cem\u003eCsBAG6\u003c/em\u003e expression exhibited a V-shaped pattern, while \u003cem\u003eCsBAG7\u003c/em\u003e expression gradually decreased. During ABA treatment, the expression of \u003cem\u003eCsBAG1\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, \u003cem\u003e4.2\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, and \u003cem\u003eCsBAG8\u003c/em\u003e exhibited an inverted V-shaped pattern, while \u003cem\u003eCsBAG6\u003c/em\u003e showed downregulation initially and then increased again by the end of the treatment in citrus leaves. In stems, \u003cem\u003eCsBAG2\u003c/em\u003e and \u003cem\u003eCsBAG3\u003c/em\u003e were upregulated in the middle, while \u003cem\u003eCsBAG4.2\u003c/em\u003e and \u003cem\u003eCsBAG6\u003c/em\u003e were initially decreased but returned to normal levels by the end of the treatment (Fig. S6). The transcript levels of the remaining \u003cem\u003eCsBAG\u003c/em\u003e genes in the stems exhibited no significant changes, suggesting that these genes might not play significant roles under GA\u003csub\u003e3\u003c/sub\u003e treatment. In the roots, the expression of \u003cem\u003eCsBAG1\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, \u003cem\u003e4.1\u003c/em\u003e, \u003cem\u003e4.2\u003c/em\u003e, \u003cem\u003e4.3\u003c/em\u003e, and \u003cem\u003eCsBAG5\u003c/em\u003e slightly increased at 24 h after ABA treatment. In contrast, \u003cem\u003eCsBAG6\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e and \u003cem\u003eCsBAG8\u003c/em\u003e were significantly downregulated by ABA, with \u003cem\u003eCsBAG7\u003c/em\u003e and \u003cem\u003eCsBAG8\u003c/em\u003e (Fig. S7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe responsiveness of \u003cem\u003eCsBAG\u003c/em\u003e genes to HLB was examined. In Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the transcript levels of most \u003cem\u003eCsBAG\u003c/em\u003e genes exhibited a substantial increase under HLB stress. Six genes, \u003cem\u003eCsBAG1\u003c/em\u003e, \u003cem\u003e4.1\u003c/em\u003e, \u003cem\u003e4.2\u003c/em\u003e, \u003cem\u003e4.3\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, and \u003cem\u003e6\u003c/em\u003e, were significantly induced by HLB, with \u003cem\u003eCsBAG4.3\u003c/em\u003e showing the highest induction, surpassing a 3-fold increase, and \u003cem\u003eCsBAG5\u003c/em\u003e exhibiting a 1.7-fold increase. \u003cem\u003eCsBAG2\u003c/em\u003e expression was significantly suppressed by HLB infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The expression levels of two \u003cem\u003eCsBAGs\u003c/em\u003e (\u003cem\u003eCsBAG3\u003c/em\u003e and \u003cem\u003eCsBAG7\u003c/em\u003e) remained unchanged in all tested samples, suggesting a lack of involvement in HLB infection. Except for \u003cem\u003eCsBAG2\u003c/em\u003e and \u003cem\u003eCsBAG5\u003c/em\u003e, these results align with the RNA-seq data analysis of five \u003cem\u003eCsBAGs\u003c/em\u003e (\u003cem\u003eCsBAG\u003c/em\u003e1/2/4/6/8), which exhibited predominantly induced expression in HLB-infected tissues (Supplementary Table. S6). Overall, the varied expression patterns of \u003cem\u003eCsBAG\u003c/em\u003e genes across citrus tissues imply their multifaceted roles in citrus development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEvolution of citrus CsBAG genes\u003c/h2\u003e \u003cp\u003eAnalysis of all \u003cem\u003eCsBAG\u003c/em\u003e genes revealed potential duplication events. Specifically, an interspersed segmental duplication event was identified involving \u003cem\u003eCsBAG4\u003c/em\u003e on chromosome 5 and \u003cem\u003eCsBAG8\u003c/em\u003e on chromosome 9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). A comprehensive analysis of orthologous BAG genes across the genomes of \u003cem\u003eC. sinensis\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eM. domestica\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, and \u003cem\u003eCarica papaya\u003c/em\u003e revealed 43 collinearity events between \u003cem\u003eC. sinensis\u003c/em\u003e and the other five species. We observed that 6, 5, 16, 9, and 7 \u003cem\u003eCsBAG\u003c/em\u003e genes in \u003cem\u003eC. sinensis\u003c/em\u003e shared synteny with those in \u003cem\u003eA. thaliana\u003c/em\u003e, rice, apple, tomato, and papaya, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and Supplementary Table S6). In addition, \u003cem\u003eCsBAG4.2\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, and \u003cem\u003e8\u003c/em\u003e exhibited collinearity with the BAG genes of the other five species, suggesting their pivotal roles in expanding the BAG family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe evolutionarily conserved BAG family significantly influences plant growth, development, and stress responses (Thanthrige et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As new plant genome sequences are continually published, additional BAG family genes are being identified in plants. Currently, research on plant BAG function primarily centers on model plants such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and rice, with fewer studies exploring the function of the BAG family in other species. Our previous study showed an interaction between citrus BAG6 protein and HLB-induced GASA4, which made us interested in the citrus CsBAG gene family (Wu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, it is necessary to identify and characterize BAG family proteins in citrus fruit trees through a combination of bioinformatics tools and expression studies. This study aims to comprehensively characterize the genome-wide functions of CsBAG genes and proteins in citrus. To the best of our knowledge, this is the first study to offer insights into the function of BAG in \u003cem\u003eC. sinensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe identified 8 \u003cem\u003eBAG\u003c/em\u003e genes through sequence alignment and HMMER database searches, with detailed information for these genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among the 10 BAG proteins discovered, BAG4.1, 4.2, and 4.3 were encoded by the same gene, \u003cem\u003eCs_ont_5g040580\u003c/em\u003e. We analyzed the protein structures and evolutionary relationships of the ten BAG proteins with those in Arabidopsis, rice, apple, papaya, and tomato. CsBAG proteins were categorized into groups G1, G2, G3, and G4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), all featuring a BAG domain. The C-terminus of the BAG protein hosts the BAG domain, facilitating interaction with the ATPase domain of constitutive and inducible heat shock protein Hsp70, while the N-terminus enables interaction with various proteins involved in protein degradation, cell migration, proliferation, and apoptosis. This domain also regulates chaperones through UBL and other motifs (Takayama et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Doong et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). For example, AtBAG4 binds to HSP70 in the cytoplasm, inhibiting abiotic stress-induced cell death (Thanthrige et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Upon pathogen infection, Arabidopsis AtBAG6 interacts with bag-associated GRAM protein (BAGP1) and asparagylprotease-cleaved BAG protein (APCB1), forming complexes that initiates autophagy and induce PCD, thereby enhancing fungal disease resistance (Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition to the BAG domain, group members of G1 (CsBAG5, CsBAG2), G2 (CsBAG8), and G4 (CsBAG4.1, CsBAG4.2, CsBAG4.3) contain a UBL domain. The domain interacts with the 26S proteasome and is integral to BAG1 during stress response (Alberti et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The UBL domains in the G1, G2, and G4 groups suggest their potential involvement in the degradation of some proteins as molecular bridges. The IQ motif can bind to CaM, influencing complex formation between CaM and its target protein (Kang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The two CsBAGs (CsBAG1 and CsBAG6) in G3 feature a specific CaM-binding domain, the IQ motif. Ca\u003csup\u003e2+\u003c/sup\u003e can modulate the binding affinity of AtBAG6 to CaM, thereby regulating the AtBAG6-mediated cell death process (Kang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Functioning as a signaling hub, AtBAG5 links the Ca\u003csup\u003e2+\u003c/sup\u003e signaling network with the Hsc70 chaperone system, thereby regulating plant defense and senescence (Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, citrus CsBAG1 and CsBAG6 potentially play roles in mediating plant defense and senescence through regulating Ca\u003csup\u003e2+\u003c/sup\u003e signaling.\u003c/p\u003e \u003cp\u003eThe results of this study show the involvement of CsBAG in plant defense processes. The hydrophilic and unstable nature of these CsBAG proteins indicates predicted expression in the nucleus (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting potential roles as TFs functions. Secondly, the analysis of cis-elements revealed the presence of various stress- and hormone-related elements across all CsBAG promoters. These include MBS, ARE, WUN-motif, LTR, GC-motif, MYC, WRE3, MYB, DRE core, box S, W box, ABRE, P-box, GARE-, CGTCA-, and TGACG-motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results align with those of previous studies where similar cis-elements were identified in the upstream promoter regions of SlBAG genes in tomato (Jiang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and AtBAG genes in \u003cem\u003eArabidopsis\u003c/em\u003e (Nawkar et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This suggests their potential roles in responding to various stresses, such as cold, antioxidant, drought, and wound stress. For example, AtBAG7 interacts with the transcription factor WRKY29 in the nucleus, where WRKY29 binds to the W-box of the \u003cem\u003eAtBAG7\u003c/em\u003e promoter, triggering transcription of \u003cem\u003eAtBAG7\u003c/em\u003e and other chaperone proteins to enhance stress tolerance (Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, analysis of TFs interacting with these CsBAG promoter elements revealed several TFs linked to stress responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Third, accumulating evidence shows that citrus CsBAG expression can be induced by treatments with stress-related hormones (ABA and GA\u003csub\u003e3\u003c/sub\u003e) as well as by the biotic pathogen HLB (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Supplementary Fig. S5). The ABA signaling pathway is crucial in helping plants manage abiotic stress and senescence (Gao et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, further investigation is required to understand the role of ABA in the CsBAG-mediated HLB protection pathway. These findings reveal the involvement of the CsBAG family in citrus response to abiotic stresses, such as ABA and GA\u003csub\u003e3\u003c/sub\u003e, with potential involvement of HLB signals in these pathways. However, the molecular mechanisms underlying CsBAG-mediated disease resistance remain unclear.\u003c/p\u003e \u003cp\u003eGene family expansion primarily arises from DNA duplication, which can occur through interspersed segmental or tandem duplication (Samonte and Eichler, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Here, eight CsBAG genes were identified in \u003cem\u003eC. sinensis\u003c/em\u003e. Approximately 8, 15, 6, 10, and 9 CsBAGs were found in rice, apple, Arabidopsis, tomato, and papaya, respectively (Table S3). The number of BAG genes in apples is approximately twice that of citrus, rice, Arabidopsis, tomato, and papaya. This significant difference could be attributed to the recent genome-wide duplication (WGD) specific to apples, which has not occurred in the other species (Velasco et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Similarly, a comparable pattern emerged with the EIN3/EIL genes. In species such as Arabidopsis, tobacco, tomato, rice, peach, mei, and strawberry, which lacked a recent WGD, the number of these genes remained at 4\u0026ndash;5. However, in pear, which shared a recent WGD with apples, the count doubled to 10 (Xu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Cao et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Given the preserved structure, function, and expression patterns, we summarized the primary biological roles of CsBAG protein in plant development and disease resistance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study identified and analyzed 10 CsBAG proteins from the \u003cem\u003eC. sinensis\u003c/em\u003e genome, focusing on their potential roles in citrus defense. Bioinformatics analysis revealed varying degrees of structural and functional divergence among members of the CsBAG gene family, suggesting potential diverse roles in the growth and development of citrus. Experimental evidence showed that the expression of G4 group members (CsBAG4.1/4.2/4.3) is highly responsive to bacterial infection and ABA treatment. This suggests their potential significance in Citrus response to biotic stresses. Further studies are needed to elucidate the roles of these proteins in growth and stress responses and consider them as candidate genes for engineering transgenic citrus plants with enhanced agronomic traits.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eARE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eantioxidant response element\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eB-cell lymphoma 2 (Bcl-2)-associated athanogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBPH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebrown planthopper\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLas\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCandidatus Liberibacter asiaticus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Structure Display Server\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGRAVY\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egrand average of hydropathicity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHidden Markov Model\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHLB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuanglongbing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIQ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eisoleucine glutamine motif\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eML\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emaximum likelihood\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMWs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emolecular weights\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear localization signal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePTRM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlant Transcriptional Regulatory Map\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePXXP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eproline-rich repeat\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprogrammed cell death\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etranscription factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUBL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eubiquitin-like domain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWGD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewide duplication.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the Guangdong Provincial Basic and Applied Basic Research Fund Project (2022A1515111183), Lingnan Normal University Talent Project (ZL22013), and an open competition program of the top nine critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province [2023SDZG06].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors’ contributions\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.L.W.\u0026nbsp;was responsible for most experiments, analysis, writing, and revision of the original manuscript, and for obtaining funding. L.Y. L.\u0026nbsp;and Y.T.L.\u0026nbsp;participated in the qRT-PCR analysis of CsBAG gene expression.\u0026nbsp;L.Y. Z.\u0026nbsp;extracted RNA from citrus leaves treated with HLB.\u0026nbsp;K.D.L.\u0026nbsp;and\u0026nbsp;Y.Z. provided guidance on\u0026nbsp;the experimental design\u0026nbsp;and\u0026nbsp;revised the manuscript. All\u0026nbsp;the authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr. Yanyan Ma and\u0026nbsp;Dr. Feiyan Wang for their valuable help and advice in writing this\u0026nbsp;manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTakayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA, et al. Cloning and Functional Analysis of BAG-l: A Novel Bcl-2-Binding Protein with Anti-Cell Death Activity. Cell. 1995;80:279\u0026ndash;84.\u003c/li\u003e\n\u003cli\u003eDoong H, Vrailas A, Kohn EC. What\u0026rsquo;s in the \u0026lsquo;BAG\u0026rsquo;? \u0026ndash; a functional domain analysis of the BAG- family proteins. Cancer Lett. 2002;188:25\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eYan J, He C, Zhang H. The BAG-family proteins in Arabidopsis thaliana. Plant Sci. 2003;165:1\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eRana RM, Dong S, Ali Z, Khan AI, Zhang HS. Identification and characterization of the Bcl-2-associated athanogene (BAG) protein family in rice. Afr J Biotechnol. 2012;11:88\u0026ndash;99.\u003c/li\u003e\n\u003cli\u003eDash 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.\u003c/li\u003e\n\u003cli\u003eHe M, Wang Y, Jahan MS, Liu W, Raziq A, Sun J, et al. Characterization of SlBAG Genes from Solanum lycopersicum and Its Function in Response to Dark-Induced Leaf Senescence. Plants. 2021;10:947.\u003c/li\u003e\n\u003cli\u003eGe S, Kang Z, Li Y, Zhang F, Shen Y, Ge R, et al. Cloning and function analysis of BAG family genes in wheat. Funct Plant Biol. 2016;43:393.\u003c/li\u003e\n\u003cli\u003eKobayashi M, Takato H, Fujita K, Suzuki S. HSG1, a grape Bcl-2-associated athanogene, promotes floral transition by activating CONSTANS expression in transgenic Arabidopsis plant. Mol Biol Rep. 2012;39:4367\u0026ndash;74.\u003c/li\u003e\n\u003cli\u003eKabbage M, Dickman MB. The BAG proteins: a ubiquitous family of chaperone regulators. Cell Mol Life Sci. 2008;65:1390\u0026ndash;402.\u003c/li\u003e\n\u003cli\u003eDoukhanina 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\u0026ndash;801.\u003c/li\u003e\n\u003cli\u003eLee DW, Kim SJ, Oh YJ, Choi B, Lee J, Hwang I. Arabidopsis BAG1 Functions as a Cofactor in Hsc70-Mediated Proteasomal Degradation of Unimported Plastid Proteins. Mol Plant. 2016;9:1428\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eFang S, Li L, Cui B, Men S, Shen Y, Yang X. Structural insight into plant programmed cell death mediated by BAG proteins in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Acta Crystallogr D Biol Crystallogr. 2013;69:934\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eNawkar GM, Maibam P, Park JH, Woo SG, Kim CY, Lee SY, et al. In silico study on Arabidopsis BAG gene expression in response to environmental stresses. Protoplasma. 2017;254:409\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eArif 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.\u003c/li\u003e\n\u003cli\u003eEchevarr\u0026iacute;a‐Zome\u0026ntilde;o S, Fern\u0026aacute;ndez‐Calvino L, Castro‐Sanz AB, L\u0026oacute;pez JA, V\u0026aacute;zquez J, Castellano MM. Dissecting the proteome dynamics of the early heat stress response leading to plant survival or death in Arabidopsis. Plant Cell Environ. 2016;39:1264\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eFu C, Hou Y, Ge J, Zhang L, Liu X, Huo P, et al. Increased fes1a thermotolerance is induced by BAG6 knockout. Plant Mol Biol. 2019;100:73\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003ePan Y-J, Liu L, Lin Y-C, Zu Y-G, Li L-P, Tang Z-H. Ethylene Antagonizes Salt-Induced Growth Retardation and Cell Death Process via Transcriptional Controlling of Ethylene-, BAG- and Senescence-Associated Genes in Arabidopsis. Front Plant Sci. 2016;7.\u003c/li\u003e\n\u003cli\u003eZhou 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.\u003c/li\u003e\n\u003cli\u003eEckardt NA. Programmed Cell Death in Plants: A Role for Mitochondrial-Associated Hexokinases. Plant Cell. 2006;18:2097\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eJiang H, Ji Y, Sheng J, Wang Y, Liu X, Xiao P, et al. Genome-Wide Identification of the Bcl-2 Associated Athanogene (BAG) Gene Family in Solanum lycopersicum and the Functional Role of SlBAG9 in Response to Osmotic Stress. Antioxidants. 2022;11:598.\u003c/li\u003e\n\u003cli\u003eGhag SB, Shekhawat UKS, Ganapathi TR. Native cell-death genes as candidates for developing wilt resistance in transgenic banana plants. AoB PLANTS. 2014;6:plu037\u0026ndash;plu037.\u003c/li\u003e\n\u003cli\u003eYou Q, Zhai K, Yang D, Yang W, Wu J, Liu J, et al. An E3 Ubiquitin Ligase-BAG Protein Module Controls Plant Innate Immunity and Broad-Spectrum Disease Resistance. Cell Host Microbe. 2016;20:758\u0026ndash;69.\u003c/li\u003e\n\u003cli\u003eZhou Y, Yang K, Cheng M, Cheng Y, Li Y, Ai G, et al. Double-faced role of Bcl-2-associated athanogene 7 in plant\u0026ndash; \u003cem\u003ePhytophthora\u003c/em\u003e interaction. J Exp Bot. 2021;72:5751\u0026ndash;65.\u003c/li\u003e\n\u003cli\u003eYeckel GJ. Characterization of a soybean BAG gene and its potential role in nematode resistance. Dr Diss Univ Mo-Columbia USA. 2012.\u003c/li\u003e\n\u003cli\u003eGayral M, Arias Gaguancela O, Vasquez E, Herath V, Flores FJ, Dickman MB, et al. Multiple ER‐to‐nucleus stress signaling pathways are activated during \u003cem\u003ePlantago asiatica mosaic virus\u003c/em\u003e and \u003cem\u003eTurnip mosaic virus\u003c/em\u003e infection in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant J. 2020;103:1233\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eWu T, Cheng C, Zhong Y, Lv Y, Ma Y, Zhong G. Molecular characterization of the gibberellin-stimulated transcript of GASA4 in Citrus. Plant Growth Regul. 2020;91:89\u0026ndash;99.\u003c/li\u003e\n\u003cli\u003eXu Q, Chen L-L, Ruan X, Chen D, Zhu A, Chen C, et al. The draft genome of sweet orange (Citrus sinensis). Nat Genet. 2013;45:59\u0026ndash;66.\u003c/li\u003e\n\u003cli\u003eGoodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012;40:D1178\u0026ndash;86.\u003c/li\u003e\n\u003cli\u003eEddy SR. Profile hidden Markov models. Bioinformatics. 1998;14:755\u0026ndash;63.\u003c/li\u003e\n\u003cli\u003eMarchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, et al. CDD: NCBI\u0026rsquo;s conserved domain database. Nucleic Acids Res. 2015;43:D222\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eHu B, Jin J, Guo A-Y, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31:1296\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eGasteiger E. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003;31:3784\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eLescot M. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30:325\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eWu T, Zhong Y, Chen M, Wu B, Wang T, Jiang B, et al. Analysis of CcGASA family members in Citrus clementina (Hort. ex Tan.) by a genome-wide approach. BMC Plant Biol. 2021;21:565.\u003c/li\u003e\n\u003cli\u003eIkai A. Thermostability and Aliphatic Index of Globular Proteins. J Biochem (Tokyo). 1980. https://doi.org/10.1093/oxfordjournals.jbchem.a133168.\u003c/li\u003e\n\u003cli\u003eThanthrige N, Jain S, Bhowmik SD, Ferguson BJ, Kabbage M, Mundree S, et al. Centrality of BAGs in Plant PCD, Stress Responses, and Host Defense. Trends Plant Sci. 2020;25:1131\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eLi Y, Kabbage M, Liu W, Dickman MB. Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants. Plant Cell. 2016;28:233\u0026ndash;47.\u003c/li\u003e\n\u003cli\u003eAlberti S, Demand J, Esser C, Emmerich N, Schild H, H\u0026ouml;hfeld J. Ubiquitylation of BAG-1 Suggests a Novel Regulatory Mechanism during the Sorting of Chaperone Substrates to the Proteasome. J Biol Chem. 2002;277:45920\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eKang CH, Jung WY, Kang YH, Kim JY, Kim DG, Jeong JC, et al. AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants. Cell Death Differ. 2006;13:84\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eLi Y, Williams B, Dickman M. Arabidopsis B‐cell lymphoma2 (Bcl‐2)‐associated athanogene 7 ( BAG 7)‐mediated heat tolerance requires translocation, sumoylation and binding to WRKY 29. New Phytol. 2017;214:695\u0026ndash;705.\u003c/li\u003e\n\u003cli\u003eGao S, Gao J, Zhu X, Song Y, Li Z, Ren G, et al. ABF2, ABF3, and ABF4 Promote ABA-Mediated Chlorophyll Degradation and Leaf Senescence by Transcriptional Activation of Chlorophyll Catabolic Genes and Senescence-Associated Genes in Arabidopsis. Mol Plant. 2016;9:1272\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eSamonte RV, Eichler EE. Segmental duplications and the evolution of the primate genome. Nat Rev Genet. 2002;3:65\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eVelasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, et al. The genome of the domesticated apple (Malus \u0026times; domestica Borkh.). Nat Genet. 2010;42:833\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eCao Y, Han Y, Meng D, Li D, Jin Q, Lin Y, et al. Genome-wide analysis suggests high level of microsynteny and purifying selection affect the evolution of \u003cem\u003eEIN3/EIL\u003c/em\u003e family in Rosaceae. PeerJ. 2017;5:e3400.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CsBAG, Citrus, hormones, Huanglongbing, expression analysis","lastPublishedDoi":"10.21203/rs.3.rs-4348725/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4348725/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) proteins constitute a multifunctional family of co-chaperone regulators. They play pivotal roles in modulating various processes, encompassing plant growth and development and response to biotic and abiotic stress. However, despite advancements in our understanding of plant BAGs, the underlying mechanisms remain unelucidated. Therefore, this study aims to systematically examine citrus BAG and their respond to hormonal treatment and Huanglongbing infection. In this study, we conducted a genome-wide in silico analysis of the CsBAG gene family in a globally significant citrus crop to explore its potential roles in fruit trees. We identified and characterized 10 CsBAGs and eight CsBAGs, revealing their distribution across five of the nine citrus chromosomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAll 10 proteins exhibited a characteristic BAG domain. CsBAG2, 4, 5, and CsBAG8 possess an additional ubiquitin-like domain, while CsBAG1 and CsBAG6 feature a calmodulin-binding motif (IQ motif). Most CsBAGs are predicted to be localized in the nucleus, mitochondria, or chloroplasts. Phylogenetic analysis revealed four major clusters, further categorized into G1–G4 groups. Cis-regulatory elements within all CsBAG promoters were identified and categorized, and the associated transcription factors were predicted. The findings suggest the involvement of these genes in defense against biotic and abiotic stresses, photoperiodic control, hormonal responses, growth, and development. This notion was further supported by gene expression analysis, revealing varying degrees of responsiveness to treatment with plant hormones (GA\u003csub\u003e3\u003c/sub\u003e and ABA) and infections with the citrus Huanglongbing (HLB) pathogen \u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus (\u003cem\u003eC\u003c/em\u003eLas). Segmental duplications contributed to the expansion of the CsBAG gene family in citrus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Our findings suggest that certain members of the CsBAG gene family may have roles in stress response and pathogen immunity. This study could help to comprehensively analyze the characteristics of the citrus BAG gene family, and the results will offer additional target genes for molecular disease resistance breeding of citrus HLB, laying a theoretical and practical foundation for the future rational utilization of \u003cem\u003eBAG\u003c/em\u003e genes.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification, Characterization, and Expression Analysis of the CsBAG family in Citrus sinensis (L.) Osbeck","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-13 04:16:06","doi":"10.21203/rs.3.rs-4348725/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ffd80bc-4f6f-4899-a8a9-83752fdf79f0","owner":[],"postedDate":"May 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-03T12:23:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-13 04:16:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4348725","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4348725","identity":"rs-4348725","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}