Members of the CaCCoAOMT gene family were identified in the genome of the pepper cultivar ‘Zunla 1’ using HMMER-based searches. A total of 11 CCoAOMT genes were identified in the pepper genome and were systematically designated as CaCCoAOMT1 through CaCCoAOMT11, according to their chromosomal positions ( Table 1 ). The lengths of CaCCoAOMT proteins in pepper range from 143 to 380 amino acids. Their isoelectric points (pI) span from 4.90 to 9.47, and their molecular weights range from 16,461.92 Da to 43,000.98 Da. The instability index of these proteins varies between 27.31 and 45.22, while their aliphatic indices are between 94.34 and 113.68. The grand average of hydropathicity (GRAVY) values range from -0.247 to 0.070. Subcellular localization predictions indicate that CaCCoAOMT1 is localized in the nucleus, whereas the other 10 CaCCoAOMT proteins are predominantly localized to both the cytoplasm and nucleus.

To elucidate the evolutionary relationships within the CCoAOMT gene family, an un-rooted neighbor-joining phylogenetic tree was constructed based on 58 CCoAOMT protein sequences from seven angiosperm species: Capsicum annuum (11 members), Arabidopsis thaliana (7), Oryza sativa (6), Camellia sinensis (10), Gossypium raimondii (6), Linum usitatissimum (6), and Solanum tuberosum (12) ( Figure 1 ; Supplementary Table S1 ). The phylogenetic analysis ( Figure 1 ) delineated the CCoAOMT proteins into seven distinct clades, each characterized by distinct species distribution patterns. Notably, members of CaCCoAOMT were absent from clades I, III, and VI. The comparative analysis revealed that CaCCoAOMT1 was grouped within clade V, alongside six rice homologs (OsCCoAOMT1–6), two Arabidopsis members (AtCCoAOMT3 and AtCCoAOMT4), two tea plant homologs (CsCCoAOMT3 and CsCCoAOMT4), and single representatives from flax (LuCCoAOMT1), cotton (GrCCoAOMT4), and potato (StCCoAOMT9). This grouping supports the hypothesis that CaCCoAOMT1 shares a closer evolutionary relationship with rice and other species in clade V. Clade IV comprised four CaCCoAOMT members (CaCCoAOMT2, CaCCoAOMT3, CaCCoAOMT4, and CaCCoAOMT10) that clustered with three potato orthologs (StCCoAOMT2, StCCoAOMT3, and StCCoAOMT4). This observation provides further insight into the evolution of these genes in Capsicum and their relationship with other Solanaceae species. Additionally, CaCCoAOMT9 formed a distinct subclade within clade II, in association with AtCCoAOMT1 and three potato paralogs (StCCoAOMT1, StCCoAOMT11, and StCCoAOMT12). This clustering suggests specific evolutionary trajectories for CaCCoAOMT9 within clade II. Notably, clade VII comprised five CaCCoAOMT proteins (CaCCoAOMT5, CaCCoAOMT6, CaCCoAOMT7, CaCCoAOMT8, and CaCCoAOMT11), which were clustered alongside three isoforms from potato (StCCoAOMT6, StCCoAOMT7, and StCCoAOMT8), a homolog from Arabidopsis (AtCCoAOMT2), two variants from the tea plant (CsCCoAOMT9 and CsCCoAOMT10), and two homologs from cotton (GrCCoAOMT3 and GrCCoAOMT6). These findings suggest a shared evolutionary origin for these CaCCoAOMT members, further supporting their potential functional similarities.

Chromosomal localization analysis revealed that 9 CaCCoAOMT genes were physically mapped to four chromosomes (Chr01, Chr02, Chr04, and Chr08) in Capsicum annuum, displaying distinct distribution patterns ( Supplementary Figure S1 ). Notably, two chromosomal regions exhibited prominent gene clustering: Chr04 harbored the highest proportion of genes (36.36%, 4 out of 11), followed by Chr02 (27.27%, 3 out of 11), while Chr01 and Chr08 each contained a single gene locus. Interestingly, CaCCoAOMT10 and CaCCoAOMT11 were not anchored to any chromosomal scaffold. Intra species collinearity found a collinear relationship between CaCCoAOMT9 and CaCCoAOMT2 ( Figure 2A ), indicating that their positions and arrangement in the genome may have high similarity or identical structural features. Through inter species collinearity analysis, a single orthologous gene pair was identified between Capsicum annuum and Arabidopsis thaliana, indicating that these two species have low conservation in genome structure and may have significant genome rearrangements or evolutionary differentiation ( Figure 2B ), this is consistent with the results of phylogenetic analysis ( Figure 1 ). Whereas five conserved syntenic pairs were found between C. annuum and Nicotiana tabacum ( Figure 2B ). This suggests a greater divergence of C. annuum CCoAOMTs from Brassicaceae lineage genes, possibly due to lineage-specific expansions or differential gene loss.

To further investigate the structural characteristics of CaCCoAOMT proteins, we performed a conserved motif analysis utilizing the MEME suite. Our analysis identified ten conserved motifs, labeled Motif 1 through Motif 10, across the CaCCoAOMT family ( Figure 3 ), with each member exhibiting a single conserved AdoMet-MTase superfamily domain. Interestingly, the number of motifs per protein varied considerably, ranging from two to seven. Despite this variation, motif composition patterns were largely consistent within phylogenetic subgroups, corresponding to their clustering in the phylogenetic tree. Exon-intron structure analysis revealed substantial diversity in gene architecture. The number of introns within CaCCoAOMT genes varied from one (in CaCCoAOMT6 and CaCCoAOMT8) to nine (in CaCCoAOMT1), with intermediate members containing two (CaCCoAOMT5, CaCCoAOMT7, CaCCoAOMT10, and CaCCoAOMT11), three (CaCCoAOMT2, CaCCoAOMT3, and CaCCoAOMT4), or four (CaCCoAOMT9) introns. The observed gradient in structural complexity, particularly the high intron content in CaCCoAOMT1, indicates that functional specialization and regulatory diversification have been influenced by evolutionary pressures within this gene family.

To elucidate the regulatory potential of the CaCCoAOMT genes, we conducted a systematic analysis of the cis-acting elements within their 2000 bp promoter regions. The ‘Plant growth and development’ category was predominantly composed of light-responsive elements, with Box4 being universally present across all CaCCoAOMT promoters. Notably, CaCCoAOMT5 and CaCCoAOMT6 exhibited the highest number of these elements. In addition to being crucial for regulating transcriptional activity under light, Box4 also play a critical role in responding to drought and salt stress, indicating that these genes may be involved in regulating plant light adaptation and stress resistance. In the ‘Phytohormone responsive’ category, ABA-responsive elements (ABRE) were identified in CaCCoAOMT2, CaCCoAOMT7, and CaCCoAOMT11. ABRE elements typically appear in the promoter regions of genes related to environmental stress response. These genes are activated by ABA signaling through the action of ABRE elements, helping plants cope with stress such as drought, salt, and low temperature. The ‘Abiotic and biotic stress’ category was the least represented, with MYB binding sites associated with drought inducibility (MBS) found in CaCCoAOMT3, CaCCoAOMT4, and CaCCoAOMT9 ( Figure 4 ). MYB transcription factors are closely related to the response of plants to environmental stresses such as drought, salinity, pests and diseases. MBS elements play an important role in the regulation of these genes. In addition, the role of MBS components in lignin synthesis, anthocyanin synthesis, and other pathways promotes the accumulation of these important chemicals in plants during growth and development.

To investigate the potential functional roles of the CaCCoAOMT gene family in pepper, we systematically analyzed their expression profiles across different tissues and throughout various stages of fruit development. As illustrated in Figure 5 and Table S2, CaCCoAOMT1 demonstrated elevated expression levels in leaf tissues (ZL1-Leaf) and during the initial stage of fruit development (ZL1-F-Dev1). Regarding tissue-specific expression, CaCCoAOMT2 and CaCCoAOMT3 were predominantly expressed in roots (ZL1-Root) and leaves, whereas CaCCoAOMT4 and CaCCoAOMT5 exhibited strong root-specific expression. Importantly, CaCCoAOMT6 showed no detectable expression in any of the tissues analyzed. In the context of fruit developmental stages, CaCCoAOMT7 was markedly upregulated during the late fruit maturation phase (ZL1-F-Dev8), while CaCCoAOMT8 was specifically activated at the mid-developmental stage (ZL1-F-Dev3). Furthermore, CaCCoAOMT9 and CaCCoAOMT10 displayed coordinated expression patterns in root tissues and at the fourth fruit developmental stage (ZL1-F-Dev4). CaCCoAOMT11 was expressed in both leaf and floral tissues (ZL1-Flower). Collectively, these findings indicate that CaCCoAOMT genes are subject to dynamic and finely tuned regulation across various tissues and during fruit development, suggesting their diverse roles in organogenesis and developmental processes in pepper.

Drought stress elicited significant adaptive morphological and cytological alterations across various organs of pepper plants. Notably, the leaves exhibited pronounced curling, whereas the stems and roots demonstrated deformation ( Figure 6 ). Microscopic analysis of root cross-sections revealed a substantial expansion of lignified regions, identified by red staining, in the drought-treated group compared to the control group, observable under both 20× and 200× magnification. In stem cross-sections, evidence of mechanical tissue damage was apparent, including shrinkage and rupture of epidermal cells, alongside intensified red coloration of xylem vessels in longitudinal sections. Leaf curling was associated with a disorganized arrangement of palisade tissue under 20× magnification, while 200× images revealed compromised cellular membrane integrity and marked plasmolysis.

We employed quantitative real-time PCR (qRT-PCR) to analyze the expression patterns of CaCCoAOMT genes under drought stress ( Figure 7 , Table S3). In the control group, most CaCCoAOMT genes exhibited higher basal expression levels in root tissues compared to stems and leaves. Under drought conditions, the expression of CaCCoAOMT1 and CaCCoAOMT9 in roots was significantly upregulated, while CaCCoAOMT2, CaCCoAOMT3, CaCCoAOMT4, CaCCoAOMT5, CaCCoAOMT6, and CaCCoAOMT7 were markedly downregulated. In stems, drought treatment resulted in increased expression of CaCCoAOMT6 and CaCCoAOMT8, whereas CaCCoAOMT7, CaCCoAOMT9, and CaCCoAOMT11 consistently showed reduced expression. In leaf tissues, compared with the control, there was no significant change in the expression levels of 11 CaCCoAOMT induced by drought stress. The responses of CaCCoAOMT genes were consistent with phenotypic alterations under drought stress. The enhanced lignification observed in roots coincided with the significant upregulation of CaCCoAOMT1 and CaCCoAOMT9 in root tissues, suggesting that these genes actively contribute to drought-induced lignin biosynthesis and reinforcement of cell walls. Such reinforcement likely improves mechanical stability and reduces water loss by limiting apoplastic permeability. Conversely, the downregulation or unchanged expression of other CaCCoAOMT members under drought stress may reflect functionally divergent paralogs, some of which could act as regulatory isoforms with tissue-specific or developmental-stage–dependent roles, rather than as primary drought response factors. Pearson correlation analysis was performed on the RNAseq and qRT-PCR datasets to illustrate their relationship. The results indicate that there may be differences, which could be caused by various factors including batch effects, sample processing, or growth conditions ( Supplementary Table S4 ). Overall, the strong and specific induction of CaCCoAOMT1 and CaCCoAOMT9 in roots after drought stress supports their identification as primary candidates for contributing to drought tolerance in pepper.

To elucidate the subcellular localization of the CaCCoAOMT1 and CaCCoAOMT2 proteins in Capsicum annuum, we constructed the pCAMBIA2300-35S-CaCCoAOMT1-EGFP and pCAMBIA2300-35S-CaCCoAOMT2-EGFP plasmids ( Figure 8A ). These fusion proteins were transiently expressed in tobacco leaves via Agrobacterium-mediated transformation. The localization of the proteins was assessed using GFP fluorescence signals. The results indicated that both CaCCoAOMT1 and CaCCoAOMT2 were localized in the cytoplasm as well as the nucleus ( Figures 8B, C ). While the prediction suggested a different localization pattern ( Table 1 ), the experimental data clearly showed that CaCCoAOMT1 is distributed in both the cytoplasm and nucleus. Computational prediction tools often rely on amino acid sequence motifs and may not fully capture post-translational modifications, protein–protein interactions, or cell type–specific factors that affect protein localization in vivo. A GFP control, which lacked the protein fusion, was employed for comparative analysis.

Caffeoyl-CoA O-methyltransferase (CCoAOMT) is an important enzyme in plants, primarily involved in the methylation of caffeic acid, thereby generating a variety of crucial plant metabolites. S-adenosylmethionine (SAM), as a universal methyl donor, plays a key role in numerous methylation reactions. In this study, molecular docking analysis was performed to investigate the interactions between different CCoAOMT homologs and SAM, aiming to elucidate their potential roles in plant metabolism (Figure 9). The binding energies of CCoAOMT1, CCoAOMT3, CCoAOMT4, CCoAOMT9, and CCoAOMT11 with SAM were calculated as -6.822 kcal/mol, -6.014 kcal/mol, -5.447 kcal/mol, -6.557 kcal/mol, and -7.435 kcal/mol, respectively. These results revealed the diversity of the CCoAOMT family in their interactions with SAM, with the differences in binding energies reflecting their functional divergence in catalytic activity and metabolic processes. Members with stronger binding affinities, such as CCoAOMT1, CCoAOMT9, and CCoAOMT11, are likely to play predominant roles in lignin biosynthesis and phenylpropanoid metabolism, whereas those with weaker affinities, such as CCoAOMT4, may serve more limited regulatory functions or exhibit tissue- or condition-specific activity. This is consistent with the observation that CCoAOMT1 and CCoAOMT9 are significantly upregulated under drought stress, indicating that they not only possess high catalytic potential at the enzyme–substrate interaction level but are also transcriptionally induced under stress conditions, thereby contributing to plant stress responses. As SAM is the methyl donor for a wide range of methyltransferase reactions, the efficiency of its interaction with CCoAOMTs directly influences the biosynthesis and accumulation of metabolic products. The differential affinities among CCoAOMT family members suggest that plants may achieve fine-tuned regulation of metabolic pathways through functional diversification of these enzymes, thereby enabling dynamic modulation of metabolite synthesis during different developmental stages or under varying environmental conditions.

Seedlings of Capsicum annuum cultivar ‘ZunLa 1’, preserved in the Germplasm Resource Bank of the Pepper Research Institute, Guizhou Academy of Agricultural Sciences, were used as experimental materials. The selected seeds were surface-sterilized and then cultivated in pots (20 cm diameter × 20 cm height) filled with nutrient-rich substrate. Plants were grown in a controlled greenhouse under identical environmental conditions for both treatments: a 30/26°C (day/night) temperature regime, 16/8-hour light/dark photoperiod, and ~60–65% relative humidity. After 20 days of growth, seedlings were divided into two groups, with 15 plants per treatment (three biological replicates, each replicate consisting of 5 plants). For the drought-stressed group, irrigation was withheld for 14 days to impose natural drought conditions. Drought severity was verified by measuring soil volumetric water content using a soil moisture probe, which dropped from ~75% to ~30% at the end of the treatment. For the well-watered control group, plants were maintained under the same environmental conditions but irrigated every 2 days to sustain the substrate at ~75–80% of field capacity. At the end of the 14-day treatment, roots, stems, and leaves were collected from both control and drought-stressed plants, immediately flash-frozen in liquid nitrogen, and stored at –80°C for subsequent RNA extraction and expression analysis.

The pepper genome data was downloaded from the Sol Genomics Network (https://www.sgn.cornell.edu/organism/Capsicum_annuum/genome) (Qin et al., 2014), and the HMM model file (PF01596) for the CaCCoAOMT gene family in pepper was obtained from the Pfam (v 37.1) website (https://pfam.xfam.org/) (El-Gebali et al., 2019). The HMMER (v 3.3) software was used to search for CCoAOMT proteins within the pepper protein sequences (Potter et al., 2018). Domain annotation of the sequences was performed using the pfamscan tool and the SMART database, with the sequences containing the “AdoMet_MTases” domain being identified as the final candidate sequences for the CaCCoAOMT gene family.

The online tool ExPASy (https://web.expasy.org/) was used to analyze the physicochemical properties of the proteins, while ProtComp v. 9.0 (http://linux1.softberry.com/berry.phtml) was employed to predict their subcellular localization. Multiple sequence alignment analyses of the CCoAOMT protein sequences from C. annuum and other species were performed (Wu et al., 2024). A phylogenetic tree was constructed for the CCoAOMT genes of D. farinosus using the Clustal W program in MEGA 7.0 software with the neighbor-joining (NJ) method and 1000 bootstrap replications (Li et al., 2025). The CaCCoAOMT protein sequences were uploaded to the NCBI Conserved Domain Database and the MEME Suite to obtain information on conserved domains and motif patterns (Bailey et al., 2015). The promoter regions, consisting of 2000 bp upstream of the start codon of the CaCCoAOMT genes, were extracted using the TBtools (v2.142) GXF Sequences Extract tool (Chen et al., 2023). The extracted sequences were then submitted to the PlantCARE database for analysis of cis-acting regulatory elements (Lescot et al., 2002). Based on the 11 identified CaCCoAOMT sequences and the genome annotation file of pepper (Zunla-1), collinearity analysis was performed using the TBtools (v2.142) One Step McscanX tool with the specified parameters. The homologous relationships between CaCCoAOMT genes and those in Arabidopsis thaliana and Solanum lycopersicum were analyzed using MCScanX (Wang et al., 2012). Segmental and tandem duplication events in the CaCCoAOMT genes were identified and visualized using TBtools-II v2.142. Subcellular localization prediction of CaCCoAOMT was conducted using the online website wolf-psort (https://www.genscript.com/wolf-psort.html).

The transcriptome annotation files of C. annuum (Zunla-1) at different developmental stages were retrieved from the GEO database (https://www.ncbi.nlm.nih.gov/geo/, accession number GSE45037), including root, stem, leaf, bud, flower, and different fruit development stages. The TBtools-II v2.142 software was used to generate expression heatmaps for the CaCCoAOMT gene family in pepper.

Roots, stems, and leaves from representative drought-treated and control pepper plants were immediately placed in FAA fixative (70% ethanol: formaldehyde: glacial acetic acid = 90:5:5) and fixed at 4°C for 24–48 h. After fixation, samples were dehydrated through a graded ethanol series and embedded in Technovit 7100 resin to prepare hard tissue sections. Embedded blocks were sectioned into 5–8 µm slices using a Leica RM2265 microtome. After de-plastification, the sections were stained with phloroglucinol to detect the distribution and degree of lignification. Sections were de-plastified and stained with 1% phloroglucinol in ethanol for 2–5 min, followed by a few drops of concentrated HCl to induce coloration. Lignified cell walls appeared red under light microscopy. Slides were temporarily mounted with neutral balsam and observed under an Olympus BX53 microscope at 20× and 200× magnification (Gritsch et al., 2015). For each treatment, three biological replicates (three independent plants per group) were processed. From each replicate, at least five sections per tissue type (root, stem, leaf) were examined, and representative images were captured.

Total RNA was extracted from the roots, stems, and leaves of pepper plants under control and drought stress conditions using the TRIzol™ Reagent Kit (Invitrogen). cDNA was synthesized from 1 μg of RNA using the HiPro™ (H-) 1st Strand cDNA Synthesis Kit with gDNA Eraser (Beijing PruTone). Primers for the CaCCoAOMT genes were designed based on their CDS sequences ( Supplementary Table S2 ) and synthesized by Genewiz Biotechnology (Shanghai, China), with CaEIF5A2 used as the internal reference gene. qRT-PCR was conducted using the ProQ™ qPCR EvaGreen Master Mix (PruTone), with an ABI ViiA7 Real-Time PCR System (Life Technologies, USA) as the PCR instrument. The reaction volume was 25 μL, consisting of 2 μL cDNA, 0.5 μL of each forward and reverse primer, 10 μL of ProQ™ qPCR EvaGreen Master Mix, and ddH2O to a final volume. The PCR conditions were as follows: initial denaturation at 94°C for 3 minutes, followed by 40 cycles of 94°C for 10 seconds, 60°C for 20 seconds, and 72°C for 30 seconds. The relative expression levels of genes were calculated using the 2^-ΔΔCT method (Livak and Schmittgen, 2001). Gene expression levels were analyzed using Excel 2019, statistical significance was assessed using SPSS 19.0, differences between control and drought-stressed groups were tested using independent-samples t-test (p < 0.05), and graphs were generated using GraphPad Prism 8.0.

To determine the subcellular localization of CaCCoAOMT1 and CaCCoAOMT2, we constructed the pCAMBIA2300-35S-CaCCoAOMT1-EGFP and pCAMBIA2300-35S-CaCCoAOMT2-EGFP plasmids. These fusion proteins were transiently expressed in tobacco leaves via Agrobacterium-mediated transformation. GFP fluorescence signals were used to observe the subcellular localization of the proteins. The localization was examined using a confocal microscope, with GFP signals visualized at an excitation wavelength of 488 nm.

The PDB structure of the CCoAOMT protein was predicted using Phyre2 (https://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index). Molecular docking was performed with AutoDock software. The docking results were visualized using PyMOL.