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 (CsBAG2, 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 GA3) 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 GA3 and ABA (Fig. 6; Fig. S6, S7). Upon GA3 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 GA3 treatment. CsBAG2 showed the second highest induction, approximately 197-folds, after 6 h of GA3 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 GA3 treatment, CsBAG6 and CsBAG7 showed gradual upregulation, while CsBAG1 and CsBAG2 exhibited gradual downregulation. In GA3-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 GA3 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 (CsBAG1/2/4/6/8), 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.