Genome-Wide Identification and Analyses of the AHL Gene Family in Pepper (Capsicum annuum L.)

1. Introduction

Capsicum annuum L., commonly known as chili pepper, is a globally significant crop renowned for its distinctive pungency and rich nutritional profile. Their economic importance is underscored by their extensive cultivation and the diverse products derived from them, including fresh produce, spices, and pharmaceuticals. Advancements in genomic research, notably the sequencing of the pepper genome, have paved the way for in-depth studies into gene families that influence key agronomic traits, thereby enhancing breeding programs aimed at improving yield, disease resistance, and stress tolerance [1,2,3].

The AT-hook motif nuclear-localized (AHL) gene family is defined by the presence of an AT-hook motif, a small DNA-binding domain that specifically interacts with AT-rich DNA regions, as well as a conserved plant and prokaryote conserved (PPC) domain. Over the past decade, the role of AHLs in regulating various plant growth and developmental processes has been increasingly recognized. These include the elongation of hypocotyls [4,5,6], the formation of pollen walls and flower development [7,8], root growth [9,10], petiole elongation [11], and the modulation of phytohormone signaling [11]. Furthermore, AHL genes have been implicated in plant responses to pathogen infections, as well as to environmental stresses such as salt and drought [12,13,14,15]. Recent studies have also broadened our understanding of the AHL gene family in important crops like cotton, soybean, maize, and rice [16,17,18,19]. These findings emphasize that diverse roles of AHLs in plant development, offering new strategies for crop improvement.

In recent years, significant progress has been made in understanding the roles of specific AHL genes in plant flower development and male sterility. In Arabidopsis thaliana, AHL22 was identified as a critical regulator of flowering time by modulating FLOWERING LOCUS T (FT) chromatin state [20]. Similarly, AHL16 suppressed transposon activation and ensured timely flowering [21]. TEK (TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK) was found essential for male fertility. TEK regulates AGPs in anthers critical for pollen wall formation. Mutations caused microspore development defects and sterility [22]. AHL15 promoted somatic embryogenesis, highlighting its biotechnological potential [23]. In rice, PERSISTENT TAPETAL CELL2 (PTC2) regulated tapetal programmed cell death and pollen wall patterning, with its loss leading to male sterility [7]. However, despite these advancements, comprehensive investigations into the AHL gene family in chili pepper remain scarce.

Given the economic importance of chili pepper and the potential roles of AHL genes in plant development and stress responses, a genome-wide analysis of the AHL gene family in chili pepper is warranted. This study aims to identify and characterize the CaAHL genes, analyze their expression profiles across different tissues and developmental stages, and explore their potential functions in relation to genic male sterility. The findings from this research will provide valuable insights into the molecular mechanisms governing chili pepper development and facilitate the development of improved cultivars through targeted breeding strategies.

2. Results

2.1. Identification and Basic Information About the CaAHL Gene Family

The Hidden Markov Model (HMM) profile of the PPC domain (PF03479) was used as a query sequence to identify AHL proteins in the Zhangshugang genome database. An initial search yielded 47 AHL candidates from the reference genome. After verification, partial AHL genes with incomplete PPC domains, considered pseudogenes, were excluded. Ultimately, 45 AHL proteins were confirmed for further analysis. To facilitate subsequent studies, these pepper AHL genes were designated as CaAHL1 to CaAHL45 based on their positions across the 12 chromosomes in the Zhangshugang reference genome. Comprehensive details, including gene names, chromosomal locations, protein lengths, molecular weights, theoretical isoelectric points (pI), instability indices, grand average of hydropathicity, and predicted subcellular localizations, are provided in Table 1.

The CaAHLs exhibited significant variation in protein length and physicochemical properties. The protein lengths ranged from 111 amino acids (CaAHL24) to 578 amino acids (CaAHL39). Correspondingly, the molecular weights (MW) varied from 11.71 kDa (CaAHL40) to 60.51 kDa (CaAHL39). The theoretical isoelectric points (pI) spanned from 4.44 (CaAHL34) to 11.25 (CaAHL40), while the instability indices ranged from 30.43 (CaAHL24) to 63.62 (CaAHL31). The grand average of hydropathicity (GRAVY) values ranged from -0.76 (CaAHL4) to 0.18 (CaAHL27). Subcellular localization predictions for the 45 CaAHL proteins revealed diverse distribution patterns: 20 localized to the nucleus, 17 to the chloroplast, five to the cytoplasm, and one each to the plasma membrane, vacuole membrane, and endoplasmic reticulum (ER) (Table 1).

2.2. Chromosome Distribution of the CaAHL Gene Family

The 45 CaAHLs were unevenly distributed across 9 of the 12 chromosomes in the Zhangshugang genome (Figure 1a). Most CaAHLs were concentrated near the distal ends of the chromosomes, with only a few located in central regions. Chromosome 1 had the highest number of CaAHLs, with a total of 21, followed by chromosomes 12 and 3, which contained 7 and 6 genes, respectively. In contrast, no CaAHLs were identified on chromosomes 10 and 11 (Figure 1b).

Interestingly, the CaAHLs were enriched in four specific regions of the genome: the proximal and distal ends of chromosome 1, the distal end of chromosome 3, and the proximal end of chromosome 12. These regions were designated as cluster 1, cluster 2, cluster 3, and cluster 4, respectively. Cluster 1 comprises five genes (CaAHL1 to CaAHL5), cluster 2 contains 12 genes (CaAHL10 to CaAHL21), cluster 3 includes three genes (CaAHL27 to CaAHL29), and cluster 4 consists of four genes (CaAHL39 to CaAHL42) (Figure. 1a). The uneven distribution and clustering of CaAHLs suggest a possible accumulation of genes through tandem duplication events in specific genomic regions.

2.3. Phylogenetic Analysis of the CaAHL Gene Family

We subsequently performed a phylogenetic analysis of the 45 CaAHLs, using their protein sequences to construct an unrooted tree for classification of the CaAHL gene family (Figure 2). The phylogenetic analysis revealed that the CaAHL gene family is divided into four main branches: branch A, containing 15 genes; branch B, containing 9 genes; branch C, containing 6 genes; and branch D, containing 15 genes. This classification provides insight into the evolutionary relationships within the CaAHL gene family. Combined with chromosome localization analysis, gene clusters were mostly distributed in the branch D, suggesting that the differentiation of gene function may gradually occur with gene duplication.

Next, we analyzed the tandem duplications within the CaAHL gene family and highlighted the genes in the four CaAHL gene clusters that may exhibit tandem repeats by using different colors in the phylogenetic tree (Figure 2). Overall, we observed that the five genes in cluster 1 grouped together with the five genes in cluster 2 in the phylogenetic tree, while the remaining three genes in cluster 1 clustered with the three genes in cluster 3 (Figure 2). We speculate that this pattern may result from the duplication of entire gene clusters on chromosomes during evolutionary processes. From the perspective of gene clusters, we found that five genes in cluster 1 (CaAHL13, CaAHL14, CaAHL16, CaAHL17, and CaAHL18) and four genes in cluster 4 (CaAHL39, CaAHL40, CaAHL41, and CaAHL42) were clustered together in the phylogenetic tree, respectively, suggesting the occurrence of tandem duplications within these clusters. In summary, the CaAHLs may undergo both inter-chromosomal replication and intra-cluster duplication.

2.4. Analysis of CaAHLs Conserved Motifs

We conducted an analysis of the phylogenetic relationships and conservative motifs of CaAHL proteins in pepper. The results indicated that most CaAHLs contained motifs 1, 2, 3, and 4. Nearly all CaAHLs included motif 1 and motif 3, except for CaAHL41, which lacked motif 1, and CaAHL17 and CaAHL18, which lacked motif 3. Additionally, 82.22% and 73.33% of CaAHLs contained motif 4 and motif 2, respectively (Figure 3 and Table S1). Some CaAHLs contained multiple identical motifs. For example, CaAHL11, CaAHL12, and CaAHL15 each contained two motif 4 elements, while CaAHL31 and CaAHL4 contained two and three motif 10 elements, respectively. Additionally, we found that CaAHL11 and CaAHL15, CaAHL13 and CaAHL14, as well as CaAHL23 and CaAHL24 exhibited highly similar motif structures, which is consistent with their clustering in the phylogenetic analysis (Figure 3 and Table S1). Overall, CaAHLs in the same subgroup in phylogenetic tree had similar structures and conserved motif distributions (Figure 3 and Table S1), indicating that CaAHLs contain highly conserved amino acid residues and that CaAHLs in the same cluster may have similar roles.

2.5. Cis-Regulatory Element Analysis of the CaAHLs Promoter

Since CaAHLs play important roles in response to plant growth, development, and stress responses, we utilized PlantCARE to analyze the cis-regulatory elements in the pre-2000 bp region of the CaAHLs promoter to explore their possible functions. For the promoter of the CaAHLs, we screened cis-regulatory elements related to growth, development, stress, and hormone responses. Using TBtools, these elements were categorized into 18 distinct classes. (Figure 4 and Figure 5). The analysis revealed that the promoters of CaAHL22 and CaAHL24 are enriched with abscisic acid-responsive cis-regulatory elements, containing 5 and 4 elements, respectively. Promoters of CaAHL7, CaAHL1, and CaAHL34 exhibit a higher abundance of anaerobic-responsive elements, with 5, 4, and 4 elements, respectively. The promoter of CaAHL28 shows a significant presence of gibberellin-responsive elements. Similarly, the promoters of CaAHL23 and CaAHL4 are enriched with jasmonic acid-responsive elements, containing 7 and 5 elements, respectively. CaAHL28 and CaAHL29 exhibit higher numbers of MYB binding site-related elements, with 6 and 5 elements, respectively, while the promoter of CaAHL21 contains 4 salicylic acid-responsive elements (Figure 4 and Figure 5). These distinct distributions of cis-regulatory elements suggest potential functional differentiation among the CaAHLs.

2.6. Tissue-Specific Expression Profiles of the CaAHLs in Peppers

To explore whether the CaAHLs plays a role in the tissue development of pepper, the expression profiles of CaAHLs in the roots, stems, leaves, pericarp, and placenta were observed using RNA-seq data from the pepper line 6421 (Figure 6). The results revealed that several genes, including CaAHL1, CaAHL26, CaAHL5, CaAHL8, CaAHL10, CaAHL44, CaAHL2, and CaAHL20, exhibited root- and stem-specific expression (Figure 6). Certain genes, such as CaAHL9, CaAHL20, CaAHL27, CaAHL28, and CaAHL11 to CaAHL18, showed higher expression levels in seeds at 20 and 25 days after flowering (DAF). Some genes were specifically expressed in certain tissues or developmental stages. For instance, CaAHL29 displayed high expression in seeds at 60 DAF, suggesting a potential role in seed maturation. CaAHL36, CaAHL23, CaAHL12, and CaAHL16 to CaAHL18 exhibited elevated expression during various stages of bud development, indicating their involvement in floral development. CaAHL21 showed specific expression in the placenta at 35 DAF, implying a role in placenta development (Figure 6). These findings suggest that most CaAHL genes are specifically expressed in roots, stems, and seeds at 20 and 25 DAF, highlighting their significant roles in root, stem, and seed development. The tissue-specific expression patterns of certain genes indicate functional differentiation among the CaAHL family members.

2.7. CaAHL23 as a Potential Regulator in Pepper Male Sterility

This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. To investigate the relationship between CaAHL genes and male sterility, we focused on genes specifically expressed during flower development. Through transcriptome data and real-time quantitative data (Figure S1), we found that CaAHL23 is specifically expressed in buds and exhibits a gene expression pattern highly similar to that of the well-characterized Arabidopsis TEK gene [22], suggesting a potential role in genic male sterility. To explore this hypothesis, we first conducted a subcellular localization analysis of CaAHL23, which confirmed its nuclear localization (Figure 7a), consistent with the previously reported localization of TEK. Subsequently, yeast one-hybrid assays were performed to identify downstream genes potentially regulated by CaAHL23. These assays demonstrated that CaAHL23 binds directly to the promoter region of CaCYP703A2 (Figure 7b). Given the high sequence homology between CaCYP703A2 and the tomato sterility-related gene SlCYP703A2 (Figure S2), we hypothesize that CaAHL23 may regulate pepper fertility by modulating the expression of CaCYP703A2. Subsequently, we detected the expression of CaCYP703A2 during flower development in the sterile line material WX2A and its fertile line WX2B, and expression profile data in the wild type (Figure 8). The results showed that compared with fertile lines, the expression of CaCYP703A2 was significantly reduced in sterile lines during flower development. These findings provide new insights into the genetic mechanisms underlying male sterility in pepper and highlights CaAHL23 as a candidate gene for further functional studies.

3. Discussion

The comprehensive analysis of the CaAHL gene family presented in this study offers valuable insights into the structure, evolutionary relationships, and functional roles of these genes in pepper. The identification and characterization of 45 CaAHL genes provide a foundational understanding of their molecular properties and potential regulatory functions in plant development and fertility. The identification of 45 CaAHL genes and detailed analysis of their protein characteristics, including length, molecular weight, theoretical isoelectric point, instability index, and predicted subcellular localization, reflect the molecular diversity of the CaAHL gene family (Table 1). These findings are consistent with previous studies that demonstrated functional diversification within gene families related to plant development [24,25,26]. Chromosomal distribution analysis revealed four gene clusters (Figure 1), suggesting that tandem duplications have contributed to the expansion of the CaAHL gene family. This observation aligns with studies in other species, where tandem duplications play a critical role in the evolution and functional diversification of gene families [27,28,29].

Phylogenetic analysis grouped the 45 CaAHL genes into four distinct subgroups and highlighted both inter-chromosomal replication and intra-cluster duplication as mechanisms driving gene family expansion (Figure 2). The conservation of motif distributions among members of the same subgroup indicates functional similarities and evolutionary conservation (Figure 3), consistent with observations in other plant species [30,31]. The presence of highly conserved amino acid residues underscores the evolutionary importance of these motifs in maintaining gene function.

Cis-regulatory element analysis revealed elements related to stress response, hormone signaling, and developmental processes, suggesting that CaAHL genes may be involved in diverse regulatory networks (Figure 4 and 5). The tissue-specific expression analysis showed that many CaAHL genes are highly expressed in roots, stems, and seeds at 20 and 25 days after flowering (DAF) (Figure 6), highlighting their potential roles in organ development. Notably, the functional differentiation indicated by distinct expression patterns among family members suggests specialization in different developmental or stress response pathways, as similarly reported in studies of AHL family genes in Arabidopsis [6,8,22,32,33].

A critical finding of this study is the potential regulatory role of CaAHL23 in pepper male sterility. Subcellular localization experiments confirmed its nuclear localization (Figure 7a), consistent with the Arabidopsis TEK gene, known for its role in pollen wall development and male sterility [22]. Yeast one-hybrid assays demonstrated that CaAHL23 binds directly to the promoter region of CaCYP703A2 (Figure 7b), a gene whose homologs in Arabidopsis, cotton, and rice are essential for pollen wall formation. Knockout studies of these homologs have resulted in male sterility [34,35]. At the same time, the expression of CaCYP703A2 gene in the sterile line was significantly reduced during flower development compared with fertile lines (Figure 8). Therefore, we hypothesize that CaAHL23 regulates pepper fertility by modulating CaCYP703A2 expression, making it a promising target for further studies on fertility regulation and hybrid breeding.

Our findings provide a comprehensive view of the CaAHL gene family's structure, evolution, and functional roles in pepper. The evidence for the involvement of CaAHL23 in male sterility opens new avenues for exploring its regulatory mechanisms and potential applications in hybrid breeding. Future research should focus on functional validation of CaAHL23 and its regulatory targets through genome-editing technologies and field trials to assess its role in fertility and crop improvement.

4. Materials and Methods

4.1. Retrieval and Identification of AHL Genes in Pepper

In this study, the candidate AHL proteins were retrieved as follows: first, the protein, nucleotide, and genome sequences of Zhangshugang genomes were downloaded from the Pepper Genomics Database (http://ted.bti.cornell.edu/cgi-bin/pepper/search) [36]. Second, the HMM of AHL protein (PF03479) was downloaded from the Pfam database (http://pfam-legacy.xfam.org/) [37]. Finally, the TBtools-II (v2.149) software [38] was used to search the predicted AHL proteins using the cut-off value of the default parameter.

4.2. Sequence Analysis and Structural Characteristics

Protein lengths, molecular weights, theoretical isoelectric points (pI), instability indices, and grand average of hydropathicity of CaAHL proteins were analyzed using the TBtools-II (v2.149) software [38]. The supposed subcellular localizations of CaAHL proteins were predicted using the online tool WoLF PSORT (https://wolfpsort.hgc.jp) [39]. The protein sequences were submitted to the MEME program (https://meme-suite.org/meme/tools/meme) [40] to assess conserved motifs.

4.3. Chromosome Localization, Tandem Duplication, and Synteny Analysis

Chromosome locations and gene position in pepper were obtained by searching the Sol Genomics Network. Chromosome mapping of the CaAHL gene family was visualized using MG2C (http://mg2c.iask.in/mg2c_v2.1) [41]. Tandem duplication events were further confirmed using the following criteria: (1) the alignment length had a coverage rate of more than 70% of the full length of the CaAHL genes; (2) the identity of the aligned region was over 70%, (3) and an array of two or more genes was less than 100 kb distance. TBtools-II (v2.149) software [38] was used to analyze the synteny of CaAHL genes among the three pepper genomes.

4.4. Phylogenetic Analysis

The phylogenetic tree was generated in the following three steps: first, the CaAHL protein sequences were imported into Clustal X to produce a multiple sequence alignment file. Second, the alignment result was used to build an unrooted tree using MEGA11 with a bootstrap of 1000 replicates and neighbor-joining (NJ) methods [42]. Third, the newly produced phylogenetic tree was visualized using the Interactive Tree of Life online website (https://itol.embl.de/) [43].

4.5. RNA-Seq Analysis of CaAHL Genes

Transcriptome sequencing (RNA-seq) data of development was used to explore the distribution of gene expression in pepper (the elite Capsicum line 6421) [44] to gain insight into the expression profiles of the CaAHL gene family in different tissues across periods. The treatment methods of all samples were based on those published by Liu et al.[44]. All data of AHL genes were normalized (log2(FPKM+1)), and a heatmap was drawn using TBtools-II (v2.149) software [38].

4.6. RNA Extraction and RT-qPCR Analysis

Capsicum line 6421 was cultivated under controlled conditions at 27°C during the day and 22°C at night, with a 16-hour light and 8-hour dark photoperiod. Total RNA was isolated using the RNAprep Pure Plant Kit (DP421, TIANGEN, China) following the manufacturer’s protocol and subsequently reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, China). Quantitative real-time PCR (RT-qPCR) was performed on a QuantStudio 3 Real-Time PCR System (ABI Life, USA) using 2× ChamQ Universal SYBR qPCR Master Mix (TransGen, China) in accordance with the provided instructions. The CaActin7 gene was used as the reference gene, with the forward primer sequence 5′-CTCGAGCAGTGTTTCCCAGT-3′ and the reverse primer sequence 5′-AGCTTCATCACCCACATAGGC-3′. Gene-specific primers were designed for CaAHL23 and CaCYP703A2 as follows: CaAHL23 forward primer, 5′-GAGTCTAGCGGTGGACCTAT-3′, and reverse primer, 5′-AGGTTCTCTCATCTCCAGGG-3′; CaCYP703A2 forward primer, 5′-TCAATCGACTCCCTCCTGGT-3′, and reverse primer, 5′-CGGAGATAGACCAATGGCCC-3′. RT-qPCR experiments were conducted with three biological replicates and three technical replicates per sample. Relative gene expression levels were determined using the 2^−ΔΔCt method.

4.7. Subcellular Localization

The full-length ORF sequences of CaAHL23 without the termination codon were cloned into the pCAMBIA1300-GFP vector and transformed into Agrobacterium tumefaciens GV3101. CaAHL23 fusion constructs was transformed into tobacco (Nicotiana benthamiana) leaves. After three days, the fluorescence signals were observed and captured using a confocal laser scanning microscope (Zeiss LSM510 META, Germany).

4.8. Y1H Assays

Pepper floral buds (F1-F9) were collected for cDNA library construction via a Matchmaker GAL4 One-Hybrid System (Clontech) following the manufacturer’s instructions. A 244-bp fragment (2769-3012 upstream of ATG) from the CaCYP703A2 promoter was synthesized by Tsingke Tech (Beijing, China) and subcloned and inserted into a pAbAi vector, and the pCaCYP703A2pro-AbAi bait was transformed into the yeast strain Y1HGold. Y1H library screening assays were performed against the cDNA expression library using the Matchmaker GAL4 One Hybrid System (Clontech). Competent Y1HGold yeast cells containing pCaCYP703A2pro-AbAi as bait were transformed with the contents of the cDNA library, plated onto selective SD/-Leu media supplemented with 100 ng/mL AbA, and incubated at 30℃. The p53-AbAi Control Vector and pGADT7-Rec53 Control Insert from Clontech were used as the positive controls. Yeast plasmids from positive colonies were extracted and transformed into Escherichia coli DH5α competent cells. The prey fragments from the positive colonies were identified by Sanger sequencing. To verify the interaction of CaAHL23 with the bait, the full-length cDNA sequences of CaAHL23 were amplified and fused in frame with the GAL4 activation domain of pGADT7rec (Clontech), forming pGADT7-CaAHL23.