Genome-Wide Identification of AP2/ERF Family Genes in Rubber Tree: Two HbAP2/ERF Genes Regulate the Expression of Multiple Natural Rubber Biosynthesis Genes

Abstract

The AP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest transcription factor families in plants and is not only vital for plant growth and development but also participates in responding to various abiotic stresses. However, few studies have investigated the function of the AP2/ERF gene family in natural rubber (NR) biosynthesis in Hevea brasiliensis. Here, 174 HbAP2/ERF genes were identified genome-wide and classified into 18 subclades based on gene-conserved structure and phylogenetic analysis. Gene duplication analysis revealed that 7 tandem and 100 segmental duplication events were major drivers of this gene family. Cis-element analysis in HbAP2/ERF promoters identified light-, hormone-, stress-, and development-associated cis-elements. Tissue-specific expression profiles revealed that 160 HbAP2/ERFs were expressed in at least one tissue. The protein–protein interaction network identified 59 potential interactions among the HbAP2/ERFs. Critically, dual-luciferase reporter assays confirmed that two key regulators exhibit distinct regulatory modes on NR biosynthesis-related genes: HbAP2/ERF25 significantly repressed the transcriptional activities of HbMVD1, HbCPT7, and HbSRPP1, whereas HbAP2/ERF46 repressed HbMVD1 but activated HbHMGR1, HbFPS1, and HbSRPP1. These findings reveal the complex regulatory network of HbAP2/ERFs in NR biosynthesis, establish a comprehensive framework for understanding their evolution and functional diversification, and provide novel molecular targets for genetic improvement of NR yield in rubber tree breeding and metabolic engineering.

1. Introduction

Transcription factors (TFs) play crucial roles in regulating plant growth, development, and stress responses. Among them, the APETALA2/ethylene-responsive element-binding factor (AP2/ERF) family stands as one of the largest and most diverse groups in plants [1]. This special type of TFs is characterized by one or more AP2 domains, containing the YRG (DNA-binding) and RAYD (structural stabilization) sub-motifs, which specifically bind to GCC-box (AGCCGCC) and DRE/CRT (ACCGAC) cis-elements in the promoter of target genes [2]. Based on structural characteristics and functional divergence, the AP2/ERF superfamily is generally divided into five subfamilies: dehydration-responsive element-binding (DREB), ethylene-responsive element-binding protein (ERF), APETALA2 (AP2), related to ABI3/VP (RAV), and Soloists [3]. The sizes of the AP2/ERF gene family vary among species, with 147 members in Arabidopsis thaliana, 170 in Oryza sativa [4], 112 in Prunus sibirica [5], 135 in Rosa chinensis [6], and 200 in Populus trichocarpa [7]. In Hevea brasiliensis, Duan et al. identified 173 AP2/ERF contigs through in silico analysis of the draft genome based on the amino acid sequence of the conserved AP2 domain [8].

AP2/ERF TFs have been extensively characterized for their roles in plant growth, stress responses, and secondary metabolism across diverse species—including their involvement in hormone signaling and metabolic pathway regulation [9]. In stress responses, members like ERF2 and ERF5 mediate β-aminobutyric acid (BABA)-induced resistance (BABA-IR) by regulating salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways—with species specificity: Arabidopsis thaliana ERFs positively regulate SA signaling, while Hordeum vulgare ERF2 promotes JA pathways but represses SA-related genes. In development, AP2 class members control A. thaliana floral transition, mediating floral organ identity and meristem maintenance via feedback networks (e.g., miR156/miR172 axis). Notably, BABA reshapes AP2/ERF expression to coordinate plant defense and development (e.g., flowering delay), underscoring its role in linking stress adaptation and growth [10,11]. Two AP2/ERF members, AINTEGUMENTA and AINTEGUMENTA-LIKE6, have been shown to regulate shoot and flower development by modulating auxin polar transport [12]. PUCHI, an Arabidopsis AP2/ERF gene, was involved in lateral root initiation and development by regulating auxin signaling [13]. In rice, the crown rootless 5 mutant displayed fewer crown roots due to the loss of CRL5, a key AP2/ERF member that balances the auxin and cytokinin signaling pathways [14]. In addition to developmental regulation, AP2/ERF TFs serve as core regulators of response to stresses. For instance, AtDREB1B/CBF1, AtDREB1C/CBF2, and AtDREB1A/CBF3 were involved in low-temperature signal transduction pathways [15,16]. JcERF1, cloned from the woody oil plant Jatropha curcas L., has been demonstrated to enhance salt tolerance in transgenic lines [17]. GmERF5 and GmERF113 imparted resistance to Phytophthora sojae by upregulating the expressions of defense-related genes, including PR1-1, PR10, and PR10-1 genes [18,19]. Recent evidence indicated that AP2/ERF TFs were involved in regulating the biosynthesis of secondary metabolites. In Salvia miltiorrhiza, SmERF1b-like binds to the GCC-box motif in the promoter of SmKSL1 and activates the biosynthesis of tanshinone [20]. In Dendrobium officinale, DoAP2/ERF89 activated the terpene synthase gene DoPAES and participated in the β-patchoulene biosynthesis [21].

Natural rubber (NR) is a polyisoprene polymer [22] that is widely used in industry owing to its superior resilience, heat dispersion, and durability [23]. Although over 2500 plant species can produce NR, the rubber tree (Hevea brasiliensis Müll. Arg.) remains the sole commercial source worldwide [24]. In the rubber tree, NR is synthesized and stored in laticifers, specialized bark tissues whose cytoplasm, known as latex, contains 30–50% NR [25,26]. In the rubber tree, although the first AP2/ERF family was reported in 2013, the severe fragmentation of the genome has greatly affected the completeness and accuracy of the family members’ identification. Recently, several high-quality chromosome-level rubber tree genomes have been reported, providing a new perspective for the precise analysis of the AP2/ERF family. In this study, we performed a genome-wide identification and characterization of the H. brasiliensis AP2/ERF family (designated HbAP2/ERF), defining their phylogenetic classification, gene duplication patterns, and promoter cis-element profiles. We further analyzed their tissue-specific expression patterns and protein–protein interaction networks to prioritize candidates with potential roles in NR biosynthesis. Finally, using dual-luciferase reporter assays, we validated the regulatory activity of key HbAP2/ERF members on core NR biosynthesis genes, thereby modulating NR biosynthesis. These results provided theoretical insights to guide the high-quality rubber tree breeding through genetic engineering.

2. Materials and Methods

First, bioinformatics approaches (genome search, duplication analysis, promoter analysis, expression analysis, and PPI network) were used to identify AP2/ERF family members in Hevea brasiliensis and predict the properties and functions of these genes. Subsequently, the transcriptional activity of HbAP2/ERF25 and HbAP2/ERF46 towards natural rubber biosynthesis-related proteins in Hevea brasiliensis was verified via dual-luciferase assay.

2.1. Genome-Wide Identification of AP2/ERFs in Rubber Tree (Hevea brasiliensis)

AP2/ERF proteins in the Hevea brasiliensis CATAS8-79 genome (https://doi.org/10.5281/zenodo.7123623, accessed on 17 October 2025) were identified via Hidden Markov Model (HMM) analysis using the AP2 domain profile (PF00847) from PFAM (version 37.2). The HMM search was performed with HMMER (version 3.0, http://www.hmmer.org/, accessed on 17 October 2025) using an E-value cutoff of 1 × 10⁻⁵. In parallel, Arabidopsis AP2/ERF protein sequences (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001735.4/, accessed on 17 October 2025) were used for BLASTP searches against the rubber tree protein database, and hits with E values less than 1 × 10⁻¹⁰ were retained for further analysis. Both HMM Search and BLASTP used low-complexity filtering. All hits obtained from HMM and BLASTP were merged, and the presence of the AP2 domain in each candidate sequence was verified using SMART (version 9, http://smart.embl-heidelberg.de/, accessed on 17 October 2025), PFAM, and CDD search (version 3.2.1, https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 17 October 2025). Transmembrane domains and signal peptides were predicted using TMHMM (version 2.0, https://services.healthtech.dtu.dk/services/TMHMM-2.0/, accessed on 17 October 2025) and Phobius (version 1.01, http://phobius.sbc.su.se/, accessed on 17 April 2025), respectively. Protein physicochemical properties, including molecular weight, isoelectric point, and amino acid length, were calculated using ProtParam (https://web.expasy.org/protparam/, accessed on 15 October 2025).

2.2. Phylogenetic Analysis

Multiple sequence alignment of 147 Arabidopsis AP2/ERFs and 174 Hevea brasiliensis AP2/ERFs was performed using Clustal Omega (version 1.2.2, http://www.clustal.org/omega/, accessed on 17 October 2025). An unrooted phylogenetic tree was then constructed in MEGA 11 using the maximum-likelihood method with 500 bootstrap replicates. The resulting tree was visualized with the TVBOT online tool (version 2.6.1, https://www.chiplot.online/tvbot.html, accessed on 17 October 2025) [27].

2.3. Chromosomal Localization, Conserved Motif, and Gene Structure Analysis

Genomic data of HbAP2/ERFs were extracted from the rubber tree genome database; their chromosomal locations and gene structures were visualized with TBtools (version 2.225) [28]. Conserved motifs in HbAP2/ERFs were identified with the MEME (version 5.5.7, https://meme-suite.org/meme/, accessed on 17 October 2025) with the site distribution set to zero or one occurrence per sequence (ZOOPS) and the maximum number of motifs set to 10, while other parameters remained at default settings.

2.4. HbAP2/ERF Duplication and Synteny Analysis

For identification of gene duplications, the protein sequences of 174 HbAP2/ERFs were aligned using BLASTP (E-value < 1 × 10⁻⁵). The MCScanX toolkit integrated with TBtools (version 2.225) was used to detect the duplication pattern of HbAP2/ERF. Genes located within 200 kb of each other and sharing >70% sequence similarity were defined as tandem duplications. A synteny plot was drawn using the circos program to illustrate duplicated rubber tree gene pairs and the synteny blocks of orthologous AP2/ERF genes between Hevea brasiliensis and Arabidopsis thaliana (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001735.4/, accessed on 17 October 2025), Populus tomentosa Carr. (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_018804465.1/, accessed on 17 October 2025), and Manihot esculenta Crantz (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001659605.2/, accessed on 17 October 2025). To evaluate gene divergence following duplication, synonymous (Ks) and non-synonymous (Ka) substitution rates were calculated for each duplicated gene pair using the Ka/Ks Calculator 2.0 tool integrated with TBtools.

2.5. Analysis of Cis-Regulatory Elements of HbAP2/ERFs

The 2 kb upstream promoter sequences of the HbAP2/ERF genes were extracted from the rubber tree genome and analyzed using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 17 October 2025) with default parameters. The minimum width of motifs was 6, and the maximum width was 50. Common consensus elements (e.g., TATA-box, CAAT-box) were filtered out because they just maintain basal gene housekeeping expression, while functional cis-elements were retained for further analysis.

2.6. Tissue-Specific HbAP2/ERFs Expression Patterns

Based on published transcriptome data for seven tissues (cambium, inner bark, primary latex, secondary latex, female flower, male flower, and leaf) from the National Genomics Data Center (NGDC, https://ngdc.cncb.ac.cn/, accessed on 17 October 2025) under project number PRJCA004986. After removing adapters, the clean reads were mapped to the CATAS8-79 genome using HISAT2 (version 2.2.1) [29], and gene expression levels were quantified as FPKM (fragments per kilobase of exon per million mapped fragments) using StringTie (version 2.2.3) [30]. Expression profiles of HbAP2/ERFs were obtained and visualized using TBtools (version 2.2.2.5).

2.7. Protein–Protein Interactions of HbAP2/ERFs

The protein–protein interaction network of HbAP2/ERFs was established via STRING (version 12.0, https://string-db.org, accessed on 17 October 2025) with default parameters (required score: 0.4, FDR stringency: 0.05) and Arabidopsis thaliana as the reference organism. The resulting network was further visualized and analyzed in Cytoscape (version 3.9.1) using a grid layout model.

2.8. Dual-Luciferase Reporter Assay

The dual-luciferase reporter assays were performed following the method of Sherf et al. [31]. The transcription factors HbAP2/ERF25 and HbAP2/ERF46, along with the promoters of HbHMGR1, HbMVK1, HbMVD1, HbGPS1, HbFPS1, HbSRPP1, and HbCPT7, were cloned previously (primers used to clone these sequences are recorded in Table S1). Transcription factors were inserted into pCambia2300-35S to generate 35S::HbAP2/ERF25 and 35S::HbAP2/ERF46, while promoters were inserted into pGreen Ⅱ 0800-LUC to obtain HbHMGR1pro::LUC, HbMVK1pro::LUC, HbMVD1pro::LUC, HbGPS1pro::LUC, HbFPS1pro::LUC, HbSRPP1pro::LUC, and HbCPT7pro::LUC. Amplicons of the expected size were sent to Sangon Biotech (Shanghai, China) for Sanger sequencing to confirm the insert sequence. Correctly sequenced plasmids were subsequently extracted and transformed into Agrobacterium tumefaciens GV3101 (harboring the psoup plasmid) strains. Agrobacterium infiltration was performed following the protocol of Zhu et al. [32]. LUC activity was measured according to the manual of the dual-luciferase reporter gene assay kit (Yeasen, 11402ES80), and luminescence was detected with a living imaging apparatus. For each combination, at least three biological replicates were performed.

2.9. Statistical Analysis

All statistical analyses were performed using Microsoft Excel 2019 (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Descriptive statistical data were presented as the mean ± standard error of the mean (SEM) from at least three independent biological replicates. For comparisons between two groups, the two-tailed unpaired Student’s t-test was used to determine significant differences. For multiple group comparisons, one-way analysis of variance (ANOVA, SAS 6.11) followed by Duncan’s multiple range test or Tukey’s post hoc test was applied. Statistical significance was defined as p > 0.05 (Ns), p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). All statistical graphs were generated using GraphPad Prism 9.0, with data visualization optimized based on the distribution characteristics of experimental data1.

3. Results

3.1. Identification and Characterization of HbAP2/ERFs in Rubber Tree

To systematically characterize the HbAP2/ERF family in rubber tree, we used the Hidden Markov Model (HMM) of the AP2/ERF domain (PF00847) to search for HbAP2/ERF and obtained a total of 174 candidate genes. Pfam and NCBI-CDD analyses confirmed that all candidate AP2/ERF proteins contained intact AP2/ERF domains. The genes were designated HbAP2/ERF1 to HbAP2/ERF174 according to their chromosome mapping (Figure 1, Table S1), which revealed their non-random distribution across 18 chromosomes: chromosome 6 harbored the most members (18), while chromosome 17 contained the fewest (3). The pattern suggests preferential expansion of the family on specific chromosomes.

Physicochemical property analysis indicated that the molecular weights of the 174 HbAP2/ERFs ranged from 9.88 kDa for HbAP2/ERF132 to 133.31 kDa for HbAP2/ERF163, with an average weight of 35.20 kDa. The isoelectric points (PIs) varied between 4.51 for HbAP2/ERF162 and 10.83 for HbAP2/ERF35. Predicted aromaticity values were observed to range from 0.04 for HbAP2/ERF10 to 0.12 for HbAP2/ERF117. Most of the HbAP2/ERFs exhibited instability indices exceeding 40, indicating a tendency toward instability, with the exceptions being HbAP2/ERF12 (39.12), HbAP2/ERF41 (38.27), HbAP2/ERF42 (38.64), HbAP2/ERF (39.61), HbAP2/ERF96 (37.59), HbAP2/ERF98 (37.75), HbAP2/ERF119 (36.14), and HbAP2/ERF132 (33.07). Furthermore, signal-peptide (SP) analysis suggested that 11 members, including HbAP2/ERF26, HbAP2/ERF28, HbAP2/ERF52, HbAP2/ERF64, HbAP2/ERF82, HbAP2/ERF84, HbAP2/ERF122, HbAP2/ERF141, HbAP2/ERF155, HbAP2/ERF159, and HbAP2/ERF163, contained SPs. This suggests their potential involvement in extracellular signal transduction pathways.

3.2. Phylogenetic Analysis of Rubber Tree HbAP2/ERFs

To investigate the evolutionary relationships among AP2/ERF family members, we constructed an unrooted maximum-likelihood (ML) phylogenetic tree that included 174 HbAP2/ERFs and 147 AtAP2/ERFs. The phylogenetic analysis classified the HbAP2/ERFs into 19 distinct subclades, following the naming conventions of their Arabidopsis homologs (Figure 2, Table S2). To evaluate the structural diversity of the HbAP2/ERF genes, the full-length cDNA sequences were compared with the corresponding genomic DNA sequences to determine the numbers and positions of exons and introns within each gene. Notably, the RPV subclade exhibited significant divergence in its AP2 domain compared to all AtAP2/ERFs. The subclades varied markedly in size, with subclade B5 being the largest, comprising 33 HbAP2/ERFs and 19 AtAP2/ERFs. Conserved domain analysis identified 10 motifs in HbAP2/ERFs (Figure 3A, Figures S1 and S2). Motifs 1 and 5 were present in almost all HbAP2/ERFs and represented conserved structural domains characterized by sequence elements such as YRG and RAYD [33]. By contrast, motif 10 was restricted to the B3-1 and A2 subclades, whereas motifs 6 and 9 were shared by the AP2, ANT, and soloist subclades. These features serve as useful markers for distinguishing subclades. Analysis of the exon–intron structure indicated significant differences across subclades while demonstrating considerable conservation within individual subclades. For example, subclades B2 and B3-2 contained fewer than three exons, whereas subclades ANT, AP2, SOLOIST, and RPV contained more than ten exons (Figure 3B and Figure S2). These structural analyses of HbAP2/ERFs reveal that subclade-specific divergence in conserved motifs, AP2 domain sequences, and exon–intron architectures reflects functional specialization of the family, while intra-subclade structural conservation maintains the evolutionary stability of core AP2/ERF functions.

3.3. Duplication and Synteny Analysis of HbAP2/ERF Genes

Gene duplication is a major driving force of genome evolution. Analyses of segmental and tandem duplications provide insights into the mechanisms underlying gene family expansion. Gene duplication events were analyzed using the MCScanX function implemented in TBtools (version 2.225) to explore the evolutionary mechanisms driving the expansion of the HbAP2/ERF family. We detected seven tandem duplication events on chromosomes 6, 7, 8, 9, 11, 12, and 18. Intraspecific collinearity analysis revealed that 65.52% (114/174) of the HbAP2/ERFs were involved in 100 segmental duplication events (Figure 4A, Table S3). The Ka/Ks ratio was assessed for selective pressure: values > 1 indicate positive selection, <1 indicate negative selection, and =1 indicate neutral evolution. For both types of duplicated HbAP2/ERF homologous pairs, Ka/Ks ratios were below 1 (ranging from 0.05 to 0.53), suggesting that these genes have undergone purifying selection, thereby maintaining their functional integrity while allowing for subtle diversification.

To further investigate the mechanisms of expansion, we conducted a comparative synteny analysis involving the rubber tree and three other species (Arabidopsis thaliana, Populus tomentosa, and Manihot esculenta). This analysis revealed 157 collinear AP2/ERF gene pairs between the rubber tree and cassava, followed by 150 pairs with poplar and 113 pairs with Arabidopsis, suggesting a closer genetic relationship between the rubber tree and cassava (Figure 4B). Interestingly, HbAP2/ERFs on chromosome 17 displayed only 9 syntenic relationships with the other three species, indicating distinct evolutionary trajectories for these genes and chromosomes within the rubber tree.

3.4. Cis-Regulatory Element Analysis of HbAP2/ERFs

Cis-acting elements in the ~2 kb promoter of HbAP2/ERFs were identified using PlantCARE. A total of 19 functional cis-acting elements were identified and categorized into four groups: plant development, biotic/abiotic stress response, hormonal regulations, and light response (Figure 5, Figure S3 and Table S4). Among them, Box 4, GT1-motif, ABRE, and G-box were the most abundant elements, which were found in the light response category, whereas TGA-element was the most frequent in plant development, LTR in low-temperature response, ARE in biotic/abiotic stress responses, and CGTCA-motif in hormonal regulation. Notably, the number of cis-acting elements varied significantly among HbAP2/ERF promoters, ranging from as few as six in HbAP2/ERF66 to as many as 41 in HbAP2/ERF50. These results suggest that HbAP2/ERFs are involved in growth, development, and stress responses in the rubber tree.

3.5. Tissue-Specific Expression Patterns of HbAP2/ERFs

The HbAP2/ERF gene family is closely associated with plant growth and development [10]. To further determine the expression patterns of HbAP2/ERFs, we analyzed the transcriptome data of seven tissues (cambium, inner bark, primary latex, secondary latex, female flowers, male flowers, and leaves). A total of 169 genes were expressed in at least one of those 7 tissues, except for HbAP2/ERF100, HbAP2/ERF132, HbAP2/ERF106, HbAP2/ERF149, and HbAP2/ERF160. Sixty genes showed constitutive expression across all tissues (Figure 6). Flower tissues contained the highest number of expressed genes (142 in both male and female flowers), whereas latex tissues contained the lowest (74 in primary latex and 84 in secondary latex). Several genes displayed tissue-specific expression, including HbAP2/ERF111, which was expressed exclusively in second latex; HbAP2/ERF45 and HbAP2/ERF51 in inner bark; and HbAP2/ERF69, HbAP2/ERF102, HbAP2/ERF105, and HbAP2/ERF166 in leaves. Interestingly, HbAP2/ERF66 showed extremely high expression in both primary latex and secondary latex, suggesting a crucial role in regulating rubber synthesis. Comparative analysis of gene expression in latex tissues showed that HbAP2/ERF33, HbAP2/ERF53, HbAP2/ERF81, HbAP2/ERF82, HbAP2/ERF94, HbAP2/ERF139, and HbAP2/ERF163 were only expressed in primary latex. On the contrary, HbAP2/ERF7, HbAP2/ERF20, HbAP2/ERF21, HbAP2/ERF28, HbAP2/ERF31, HbAP2/ERF54, HbAP2/ERF68, HbAP2/ERF75, HbAP2/ERF84, HbAP2/ERF88, HbAP2/ERF89, HbAP2/ERF92, HbAP2/ERF111, HbAP2/ERF113, HbAP2/ERF117, HbAP2/ERF145, and HbAP2/ERF168 were only expressed in secondary latex. HbAP2/ERF17, HbAP2/ERF99, HbAP2/ERF115, HbAP2/ERF142, and HbAP2/ERF169 were significantly downregulated in secondary latex compared to primary latex, while HbAP2/ERF15, HbAP2/ERF35, HbAP2/ERF79, HbAP2/ERF109, and HbAP2/ERF150 were significantly upregulated, suggesting distinct regulatory roles in latex metabolism. The expression patterns of HbAP2/ERFs in latex indicate that their functions are likely to be directly involved in the transcriptional regulation of NR biosynthesis.

3.6. Prediction of the HbAP2/ERF Protein–Protein Interaction Network

To explore the functional interactions of HbAP2/ERFs, we established a protein–protein interaction network utilizing Arabidopsis orthologs in the STRING database. This analysis uncovered 59 potential interactions (Figure 7, Table S5). Among these, 13 HbAP2/ERFs were predicted to interact with HbAP2/ERF135, a homolog of Arabidopsis AP2, which is involved in flower and seed development [34,35]. HbAP2/ERF60 interacts with eight HbAP2/ERFs, is a homolog of AtERF5, and functions as an activator of GCC-box-dependent transcription in Arabidopsis leaves [36]. DREB is a key transcriptional activator in response to chilling tolerance [15]. In the present study, we detected a total of 11 potential interactions for three DREB homologs in the rubber tree.

3.7. HbAP2/ERF25 and HbAP2/ERF46 Regulated Multiple NR Biosynthesis-Related Genes

Mevalonate diphosphate decarboxylase (MVD) is a key enzyme in the biosynthetic pathway of NR. We previously screened two potential transcription factors (HbAP2/ERF25 and HbAP2/ERF46) for HbMVD1 by Y1H assay. To investigate their role in the regulation of NR biosynthesis, a comprehensive luciferase transcriptional activation assay related to eight NR biosynthesis-related genes was performed. It was clearly shown that HbAP2/ERF25 significantly repressed the transcriptional activities of HbMVD1, HbCPT7, and HbSRPP1 (Figure 8A). For HbAP2/ERF46, the transcriptional activity of HbMVD1 was repressed, while HbHMGR1, HbFPS1, and HbSRPP1 were activated (Figure 8B), suggesting the complex regulatory network of HbAP2/ERF in NR biosynthesis (Figure 8C).

4. Discussion

Thousands of genes are involved in varied processes in plants. AP2/ERF is one of the largest transcription factor families and plays pivotal roles in plant development as well as in responses to biotic and abiotic stresses [1]. With the rapid development of plant genomics, AP2/ERF gene families have been systematically identified in many species, including Triticum aestivum (117) [37], Hordeum vulgare (121) [38], Zea mays (167) [39], Glycine max (301) [40], Malus domestica (259) [41], Brassica rapa (291) [42], and Solanum lycopersicum (173) [43]. In 2013, Duan identified 173 contigs containing the AP2/ERF domain by analyzing the rubber tree transcript sequence database [8]. However, the severe fragmentation of the contigs has greatly affected the accuracy of the family members’ identification in the rubber tree. Here, we systematically delineate the genomic landscape and functional potential of the HbAP2/ERF family, with a focus on identifying core regulators that shape NR biosynthesis. We further speculate that two members, HbAP2/ERF25 and HbAP2/ERF46, may control NR biosynthesis by directly or indirectly regulating key NR biosynthesis genes.

A total of 174 candidate members were identified based on genome-wide analysis of AP2/ERF in rubber tree CATAS8-79 (Figure 1, Table S1). Comparative analysis showed that the size of the HbAP2/ERF family is larger than that in Arabidopsis thaliana [4], Solanum lycopersicum [43], Oryza sativa [4], Triticum aestivum [37], Hordeum vulgare [38], Zea mays [39], and Prunus sibirica [5], but smaller than those in Populus trichocarpa [7], Glycine max [40], Malus domestica [41], and Brassica rapa [42]. Such interspecific variation in AP2/ERF family size can likely be attributed to evolutionary processes such as whole-genome duplications and polyploidization events. The dominance of segmental duplication (100 events) over tandem duplication (seven events) in driving HbAP2/ERF family expansion reflects adaptive evolution (Figure 4A), with latex-enriched segmentally duplicated pairs likely undergoing neofunctionalization for NR-specific roles. These proportions are comparable to those reported for the AP2/ERF gene family in Prunus sibirica [5], suggesting that gene duplication plays a conserved and significant role in the expansion of the AP2/ERF family.

In response to stresses, plants employ multiple strategies, such as activating the antioxidant system and increasing the contents of characteristic metabolites [44]. The expansion or contraction of gene families is a core driver of plant evolution for adapting to long-term environmental changes [45]. The 174 HbAP2/ERFs were classified into 18 subclades based on Arabidopsis homologs, plus one rubber-tree-specific subclade (RPV, HbAP2/ERF163). The pattern is indicative of lineage-specific functional diversification to support rubber tree-specific biological processes. HbAP2/ERF163 has the longest gene structure among 174 members. Cis-regulatory element analysis detected five low-temperature response (LTR) elements in the HbAP2/ERF163 promoter, indicating that this may be associated with the temperature response of the rubber tree in tropical regions. Functional studies in Arabidopsis have defined conserved roles of AP2/ERF members. RAP2-3, a member of the B3 subclade, mediates root hypoxia tolerance and root development under low-oxygen conditions, while ERF096 regulates heat stress responses [46]. Our ortholog annotation identified HbAP2/ERF109 as the Arabidopsis RAP2-3 ortholog and HbAP2/ERF111 as the ERF096 ortholog (Figure 2), with distinct tissue expression patterns (HbAP2/ERF109 expressed across all seven tissues; HbAP2/ERF111 latex-specific) further illustrating functional divergence from model plant homologs. The evolutionary and orthologous context underscores the biological significance of tissue-specific expression in HbAP2/ERFs: the enrichment of 92 HbAP2/ERFs (including HbAP2/ERF25 and 46) in latex tissue (vs. leaves/roots) is not just a spatial pattern but evidence of laticifer-specific transcriptional reprogramming.

Transcription factors regulate gene expression and play a key role in biological processes. Recently, several TFs have been reported to modulate the genes involved in NR biosynthesis. For example, HbWRKY27 was found to upregulate the expression of HbFPS1, while HbMADS4 negatively regulated HbSRPP [47,48]. HbTGA1 is a multi-functional transcription factor that represses the expression of HbCPT6 but activates HbHMGR2, HbCPT8, and HbSRPP2, respectively [49]. In the present study, we identified different regulatory patterns for two HbAP2/ERF members: HbAP2/ERF46 repressed HbMVD but activated HbHMGR, HbFPS1, and HbSRPP, while HbAP2/ERF25 repressed HbMVD, HbCPT, and HbSRPP. These findings underscore the crucial role of HbAP2/ERF25 and HbAP2/ERF46 in NR biosynthesis and suggest they might establish a complex regulatory network with key TFs for NR regulation. Studying the transgenics of HbAP2/ERF25 and HbAP2/ERF46 may provide further insights into the role of HbAP2/ERF in regulating NR biosynthesis in rubber trees. Protein–protein interactions are crucial for the transcriptional regulation of genes. In the rubber tree, the interaction with HbWRKY14 and HbVQ4/5 could relieve the inhibition of HbSRPP transcription [30]. Protein–protein interaction network analysis identified five putative interactors for HbAP2/ERF25 and two for HbAP2/ERF46, which provide valuable entry points for future mechanistic investigations into their precise regulatory roles in NR biosynthesis and stress responses.

5. Conclusions

In this study, we established a comprehensive genomic and functional framework for the AP2/ERF family in Hevea brasiliensis CATAS8-79, identifying 174 HbAP2/ERF genes (classified into 18 subclades, including one rubber-tree-specific RPV subclade) expanded mainly via 100 segmental and seven tandem repeats. Moreover, HbAP2/ERF25 and HbAP2/ERF46 are unique regulatory modules governing NR biosynthesis: HbAP2/ERF25 represses key NR pathway genes to maintain metabolic homeostasis, while HbAP2/ERF46 exerts dual-mode regulation to optimize NR metabolic flux—an adaptive mechanism unreported in woody plant AP2/ERF-mediated secondary metabolism. Beyond advancing understanding of AP2/ERF evolution and NR regulation, these two core regulators serve as actionable molecular targets for rubber tree precision breeding and metabolic engineering to enhance NR yield, laying the groundwork for future investigations into upstream signaling pathways modulating their activity.