Genome-wide characterization and their roles in abiotic stress responses of HD-Zip transcription factors in pepper (Capsicum annuum)

Genome-wide characterization and their roles in abiotic stress responses of HD-Zip transcription factors in pepper (Capsicum annuum) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide characterization and their roles in abiotic stress responses of HD-Zip transcription factors in pepper (Capsicum annuum) Xiaoqin Wang, Xiaoya Zhou, Kunhao Xie, Xiaojie Feng, Lu Liu, Lihong Gao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6320983/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jun, 2025 Read the published version in BMC Plant Biology → Version 1 posted 13 You are reading this latest preprint version Abstract Background Capsicum annuum is a globally cultivated crop of significant agricultural and economic importance. However, its productivity and fruit quality are frequently challenged by a range of abiotic stresses. The HD-Zip (Homeodomain-Leucine Zipper) gene family, unique to plants, is known to play pivotal regulatory roles in abiotic stress adaptation, yet its functional roles in pepper remain largely unexplored. Results This study systematically analyzed the HD-Zip gene family in pepper through bioinformatics, expression profiling, and responses to abiotic stresses and phytohormones to elucidate their roles in stress tolerance. Results revealed 40 HD-Zip transcription factors unevenly distributed across 12 chromosomes, encoding proteins ranging from 211 to 842 amino acids. Subcellular localization predictions indicated nuclear localization for all members, with a subset also showing cytoplasmic localization. Collinearity analysis demonstrated that CaHD-Zip gene expansion was predominantly driven by segmental duplication, with high conservation across dicotyledons. Promoter regions of CaHD-Zip genes were enriched in cis-regulatory elements associated with light and hormonal responses, as well as stress adaptation. Tissue-specific and developmental stage-dependent expression patterns highlighted functional diversification within the family. Notably, some members were specifically induced by abiotic stresses (cold, heat, drought, and salt) and stress-related phytohormones (ABA, MeJA, ET, and SA), suggesting their involvement in stress signaling. Strikingly, CaHD-Zip18 and CaHD-Zip29 were significantly upregulated under all four stresses, implicating them as core regulators of multi-stress responses. Subsequent stress simulation assays and qRT-PCR validation confirmed the reliability of transcriptomic findings. Conclusion This study delivers the first systematic exploration of HD-Zip transcription factors in Capsicum annuum under abiotic stress, providing foundational knowledge and candidate genes for improving stress resilience in pepper breeding programs. Capsicum annuum HD-Zip gene family Abiotic stress Gene expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction As a globally significant economic crop, pepper serves as both a staple vegetable and a vital spice[ 1 ]. According to 2023 FAO statistics, global fresh pepper production increased significantly from 36.29 to 40.20 million metric tons within a single year, with China notably contributing 40% of total production and retaining its status as the primary producer[ 2 ]. Beyond its role as a fresh vegetable, pepper derives subst antial economic value from its distinctive secondary metabolites, particularly capsaicinoids and carotenoids.These compounds demonstrate unique bioactive properties that enable diverse commercial applications: as natural colorants and flavor enhancers in food processing; analgesic and anti-inflammatory agents in pharmaceuticals; and specialized components in cosmetic formulations and industrial coatings[ 3 – 5 ]. Global climate change has exacerbated abiotic stresses, including extreme temperatures, soil salinization, and drought, which pose serious threats to the yield and quality of Capsicum annuum , leading to substantial economic losses[ 6 – 8 ]. These compounds demonstrate unique bioactive properties that enable diverse commercial applications: as natural colorants and flavor enhancers in food processing; analgesic and anti-inflammatory agents in pharmaceuticals; and specialized components in cosmetic formulations and industrial coatings. Therefore, elucidating the molecular regulatory networks governing pepper’s response to abiotic stress is essential not only for breeding stress-tolerant cultivars but also for ensuring the sustainable development of the global pepper industry. Within stress-response regulatory networks, transcription factors (TFs) act as central signaling hubs that decode environmental and developmental signals through cis-element recognition and target gene regulation[ 9 – 12 ]. The HD-Zip (Homeodomain-Leucine Zipper) family, a plant-exclusive TF class[ 13 , 14 ], contains two signature domains: a DNA-binding homeodomain (HD) and a dimerization-mediating leucine zipper (LZ) [ 15 , 16 ]. Phylogenetic and structural analyses classify the HD-Zip family into four evolutionarily distinct subfamilies (I-IV) [ 17 ]. Subfamily I retains the core HD-LZ architecture, while Subfamily II acquires a conserved N-terminal sequence coupled with a CPSCE motif[ 18 , 19 ]. Subfamilies III and IV demonstrate enhanced structural sophistication, each possessing a START (steroidogenic acute regulatory protein-related lipid transfer) domain and its associated SAD domain downstream of LZ [ 20 ].A defining feature of Subfamily III is its C-terminal MEKHLA motif[ 17 ]. These domain architectures directly determine subfamily-specific functional specialization in developmental regulation and stress adaptation[ 21 ]. The HD-Zip I subfamily orchestrates three core biological functions: developmental programming, abiotic stress adaptation, and hormonal cross-regulation through signal integration. Under osmotic stress conditions, such as drought and salinity, HD-Zip I members activate ABA-dependent acclimation mechanisms, enhancing cellular resilience[ 13 , 22 ]. In Arabidopsis, AtHB7 and AtHB12 establish ABA homeostasis via self-regulatory feedback loops, a conserved mechanism also demonstrated in Gossypium species[ 23 , 24 ]. These regulators fine-tune stomatal patterning (e.g., AtHB7 -mediated density reduction) and osmolyte biosynthesis (e.g., Zmhdz10 driven proline accumulation), synergistically optimizing hydraulic efficiency under combined drought and cold stress[ 25 – 27 ]. The HD-Zip II subfamily is a principal regulator of photomorphogenic responses and organogenetic patterning, particularly influencing leaf morphogenesis [ 14 , 28 , 29 ]. For example, HAT3 and ATHB4 regulate leaf polarity through a bidirectional inhibitory circuit[ 30 ]. Meanwhile, HAT1 modulates organogenesis through interactions with TPL/TPR transcriptional co-repressors[ 31 , 32 ]. HD-Zip III transcription factors coordinate three developmental axes: vascular tissue patterning, meristem maintenance, and lateral organ positioning[ 33 , 34 ]. The HD-Zip IV subfamily is specifically expressed in epidermal and subepidermal cells, where it contributes to trichome formation, anthocyanin accumulation, and cuticle biosynthesis, thereby governing epidermal development and environmental adaptation[ 35 ]. While HD-Zip genes have been extensively characterized in model plants ( Arabidopsis, Oryza sativa ) and major crops ( Solanum lycopersicum, Malus domestica, Prunus persica, Passiflora edulis )[ 36 – 38 ], a systematic genomic investigation of this gene family has not yet been conducted in Capsicum annuum , the most extensively cultivated Solanaceae crop worldwide.To address this gap, we performed a genome-wide analysis to systematically identify and functionally characterize the HD-Zip gene family in Capsicum annuum , focusing on the following objectives: 1) Identification of 40 CaHD-Zip genes and establishment of a cross-species phylogenetic framework; 2) Investigation of their evolutionary trajectories through synteny analysis and gene structure characterization; 3) Comprehensive transcriptional profiling across 13 tissues and organs, as well as under four hormone treatments and four abiotic stress conditions.This study not only sheds light on the phylogenetic characteristics and expression regulatory networks of CaHD-Zip genes but also provides key candidate genes and a theoretical foundation for elucidating the molecular mechanisms underlying HD-Zip mediated development and stress adaptation in pepper. These findings have significant scientific implications for accelerating the molecular breeding of stress-resilient Capsicum annuum cultivars. 2. Materials and Methods 2.1 Identification, Subcellular Localization Prediction, and Physicochemical Characterization of CaHD-Zip Family Genes. The HD-Zip transcription factor family in pepper was systematically identified using the latest high-quality telomere-to-telomere (T2T) genome assembly ( Zunla-1_v3.0 ) obtained from PepperBase ( http://www.bioinformaticslab.cn/PepperBase/ ) [ 39 ]. HMM profiles corresponding to the HD-Zip domain (PF00046 and PF02183) were retrieved from the Pfam database ( http://pfam.xfam.org/ ) and employed in a genome-wide search using HMMER v3.3.2 ( http://hmmer.janelia.org/ ) [ 40 ], resulting in the preliminary identification of candidate HD-Zip genes. These candidates were subsequently validated for conserved domains using both the NCBI Conserved Domain Database (CDD) ( https://www.ncbi.nlm.nih.gov/cdd/ ), [ 41 ] and SMART database ( http://smart.embl-heidelberg.de/ ) [ 42 ], respectively. The validated HD-Zip genes were systematically named based on their chromosomal positions and sequence conservation, then classified into subfamilies through phylogenetic analysis of their protein sequences. Subcellular localization predictions were performed using DeepLoc-2.0 ( https://services.healthtech.dtu.dk/services/DeepLoc-2.0/ ) and WoLF PSORT ( https://www.genscript.com/wolf-psort.html ). Additionally, while key physicochemical properties including molecular weight, isoelectric point, and were calculated using the ExPASy ProtParam tool. ( https://web.expasy.org/protparam/ ) [ 43 ]. 2.2 Phylogenetic Analysis of CaHD-Zip Genes Phylogenetic analysis was performed using HD-Zip protein sequences from pepper and the reference dataset comprising Arabidopsis thaliana sequences obtained from PlantTFDB ( http://planttfdb.gao-lab.org/index.php ). Multiple sequence alignment was conducted with the MUSCLE algorithm implemented in MEGA7 ( https://www.megasoftware.net/ )[ 44 ]followed by phylogenetic tree construction through the maximum likelihood method(ML) with 1,000 bootstrap replicates to evaluate node support. The phylogenetic tree was visualized using TVBOT ( https://www.chiplot.online/tvbot.html ), while Jalview (V2.11.2.4) ( http://www.jalview.org/ ) was utilized for conserved domain annotation and sequence refinement to ensure analytical accuracy[ 45 ]. 2.3 Gene Structure, Conserved Motif, and Cis-Regulatory Element Analysis Gene structure analysis was performed using the General Feature Format (GFF3) annotation files through TBtools' Visualize Gene Structure Basic module. Conserved protein motifs were identified using the MEME Suite ( https://meme-suite.org/meme/tools/meme ) with parameter constraints allowing detection of up to 10 motifs ranging from 5 to 200 amino acids in length. Promoter regions, defined as the 2,000 bp upstream sequences from translation initiation sites, were extracted using TBtools. These promoter sequences were subsequently analyzed in PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ )[ 46 ], for cis-regulatory element annotation, followed by element classification and visualization using TBtools. 2.4 Chromosomal Mapping, Synteny Analysis, and Gene Duplication Events The chromosomal distribution of HD-Zip genes in pepper was determined using TBtools’ Gene Location Visualization module, based on GFF3 genome annotation. Intragenomic synteny patterns were visualized through TBtools’ Advanced Circos module, revealing chromosomal arrangement of paralogous genes. Cross-species collinearity analysis with six representative species ( Arabidopsis thaliana, Cucumis sativus, Solanum lycopersicum, Zea mays, Solanum tuberosum , and Oryza sativa ) was conducted using MCScanX. Gene duplication events were characterized by calculating nonsynonymous (Ka) and synonymous (Ks) substitution rates with TBtools' Simple Ka/Ks Calculator module. Selection pressure was determined based on Ka/Ks ratios: >1 indicated positive selection, = 1 suggested neutral evolution, or < 1 signified purifying selection[ 47 ]. Divergence times were estimated using the formula T = Ks/(2×6.1×10⁻⁹)×10⁻⁶, where 6.1×10⁻⁹ represents the average substitution rate per site per year, and T corresponds to the divergence time in million years (Mya)[ 48 ]. 2.5 RNA-Seq Data Analysis RNA-seq datasets were obtained from the NCBI Sequence Read Archive (SRA) with accession numbers detailed in Table S1 , comprising three experimental groups: 1) 11 organ types of pepper (seeds, embryos, stem, root, leaf, floral buds, flowers, anthers, ovary, placenta, early fruit, breaker fruit and mature fruit ); 2) four abiotic stresses (cold, heat, drought, and salt stress) sampled at 0, 3, 6, 12, 24 h; 3) four phytohormone treatments (ABA, MeJA, ET, and SA) measured at 0, 3, 6, 12, 24, 72 h. TPM-normalized CaHD-Zip expression profiles were hierarchically clustered using Euclidean distance metrics in TBtools, visualized as heatmaps[ 49 ], with heatmaps generated to visualize clustering patterns. Differentially expressed genes (DEGs) were identified using the DESeq2 function in TBtools, with a significance threshold of p 2. 2.6 Plant Materials, Growth Conditions, and Stress Treatments Sterilized seeds of the pepper variety “Bola No. 2”, purchased from Hunan Xingshu Seed Industry, were sown in 50 cell seedling trays. After two weeks of seedling growth, plants were transplanted into nutrient-rich pots (7 cm diameter × 6.5 cm height) and grown under controlled environmental conditions in a growth chamber (24 ± 1°C, 16 h light/8 h dark photoperiod) until six true-leaf stage. Abiotic stress treatments included: Cold stress (10°C), heat stress (40°C), salt stress (400 mM NaCl), and drought stress (20% PEG6000 solution).Hormonal treatments: Leaves were sprayed with 100 µM MeJA or 100 µM ABA, while the control group received distilled water. The third fully expanded leaves were harvested after 12 h (hormones) or 24 h (abiotic stresses) of treatment initiation (n = 3 biological replicates). Samples were immediately snap-frozen in liquid N₂ and stored at -80°C until RNA extraction. 2.7 RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis Total RNA was extracted from pepper samples using the RNA Prep Pure Kit (Accurate Biology, China) according to the manufacturer’s instructions. RNA purity (A260/A280 ratio) and concentration were assessed using a spectrophotometer to ensure that sample quality met the requirements for downstream analyses. First-strand complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the Fast Quant RT Kit (Accurate Biology, China), which contains a genomic DNA elimination step, according to the manufacturer’s instructions. The reaction system contained reverse transcriptase, random primers, and reaction buffer to ensure effective genomic DNA elimination and the synthesis of high-quality cDNA. qRT-PCR reactions were prepared using Super Real Pre Mix Plus with SYBR Green dye, incorporating gene-specific primers synthesized by Sangon Biotech (Shanghai, China). qRT-PCR was performed on a Gentier 96/96E real-time PCR system (Tianlong Technology, Xi’an, China) under the following cycling conditions: initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Melting curve analysis was performed to validate amplicon specificity. All samples were assessed in triplicate, and gene expression was normalized against the reference gene CaActin . The sequences of all primers used are listed in Table S2. Relative gene expression was quantified using the 2⁻ΔΔCt method [ 50 ], with results presented as fold changes in the treatment groups relative to the control. 2.8 Data Analysis All experimental data were compiled using Microsoft Excel 2010, subjected to statistical analysis using SPSS, and visualized with Origin software. 3. Result 3.1 Physicochemical Characteristics and Subcellular Localization of the CaHD-Zip Gene Family Systematic genome-wide screening identified 40 HD-Zip genes (designated CaHD-Zip1 to CaHD-Zip40 ) in Capsicum annuum (Table 1 ). The deduced proteins spanned 211–842 amino acids, with molecular weights ranging from 24.2 to 93.6 kDa. Instability index analysis (37.29–87.67) revealed structural instability in most CaHD-Zip proteins, with CaHD-Zip01, 06, 13 , and 17 exhibiting the highest values (> 70). Subcellular localization predictions indicated nuclear targeting for all CaHD-Zip proteins, while CaHD-Zip03, 09, 14 , and 38 were also predicted to localize to the cytoplasm. These dual-localized proteins exhibited lower instability indices, potentially reflecting enhanced structural stability conducive to functional plasticity. All CaHD-Zip proteins exhibited negative Grand Average of Hydropathicity (GRAVY) values (-1.067 to -0.11), indicating a hydrophilic molecular profile, consistent with the aqueous solubility and nuclear localization typical of transcription factors. Table 1 Properties and Localization of Pepper HD-Zip Proteins Gene Name Sequence ID Number of Amino Acid Molecular Weight Theoretical pI Instability Index Aliphatic Index GRAVY Localizations CaHD-Zip01 ZLC01G0000550.1 211 24442.53 5.12 72.73 67.54 -0.87 Nucleus CaHD-Zip02 ZLC01G0007630.1 282 31939.82 6.97 59.44 62.59 -0.887 Nucleus CaHD-Zip03 ZLC01G0011000.2 842 93610.97 5.2 57.44 77.57 -0.381 Cytoplasm/Nucleus CaHD-Zip04 ZLC01G0015850.1 835 91048.11 5.9 50.24 76.5 -0.352 Nucleus CaHD-Zip05 ZLC01G0025820.1 392 42420.18 7.08 54.4 61.76 -0.675 Nucleus CaHD-Zip06 ZLC01G0036190.1 309 34321.6 6.77 70.6 71.65 -0.764 Nucleus CaHD-Zip07 ZLC02G0005020.1 241 27002.57 9.2 47.37 74.52 -0.687 Nucleus CaHD-Zip08 ZLC02G0008000.1 281 31954.43 5.66 56.71 63.52 -0.914 Nucleus CaHD-Zip09 ZLC02G0011430.1 830 91725.93 5.86 47.44 87.67 -0.113 Cytoplasm/Nucleus CaHD-Zip10 ZLC02G0020890.2 729 80189.76 5.66 42.63 78.49 -0.366 Nucleus CaHD-Zip11 ZLC02G0028560.1 272 30280.13 8.4 49.85 69.96 -0.688 Nucleus CaHD-Zip12 ZLC02G0033860.1 310 35324.22 5.73 59.61 62 -0.897 Nucleus CaHD-Zip13 ZLC02G0035010.1 287 32422.6 4.82 74.16 61.43 -0.885 Nucleus CaHD-Zip14 ZLC03G0002650.1 835 91788.13 6.06 47.75 87.4 -0.127 Cytoplasm/Nucleus CaHD-Zip15 ZLC03G0002950.1 772 86442.01 6.42 51.69 73.87 -0.528 Nucleus CaHD-Zip16 ZLC03G0011730.1 322 37089 5.03 52.26 59.04 -0.899 Nucleus CaHD-Zip17 ZLC03G0016630.1 720 79488.02 6.21 52.86 83.79 -0.312 Nucleus CaHD-Zip18 ZLC03G0019410.1 220 25791.81 5.7 46.16 66.95 -1.067 Nucleus CaHD-Zip19 ZLC03G0023390.1 815 88475.32 6.07 45.67 77.06 -0.221 Nucleus CaHD-Zip20 ZLC04G0008080.1 248 28332.95 8.69 54.5 80.93 -0.83 Nucleus CaHD-Zip21 ZLC04G0010300.2 294 33693.05 5.03 51.06 65 -1.001 Nucleus CaHD-Zip22 ZLC04G0024840.1 728 80148.8 5.5 42.92 84.11 -0.324 Nucleus CaHD-Zip23 ZLC05G0017140.1 323 36807.44 4.64 52.8 68.51 -0.793 Nucleus CaHD-Zip24 ZLC06G0009310.2 734 80878.42 6.08 52.29 80.49 -0.35 Nucleus CaHD-Zip25 ZLC06G0020410.1 313 35570.18 8.13 60.25 63.26 -0.831 Nucleus CaHD-Zip26 ZLC06G0023950.1 222 25688.64 5.17 48.49 65.95 -0.941 Nucleus CaHD-Zip27 ZLC07G0017730.1 211 24259.64 9.42 37.29 72.61 -0.987 Nucleus CaHD-Zip28 ZLC08G0005190.3 840 92409.89 5.77 53.57 88.04 -0.11 Nucleus CaHD-Zip29 ZLC08G0010520.1 243 27633.75 5.27 58.24 70.29 -0.823 Nucleus CaHD-Zip30 ZLC09G0007190.1 777 85895.57 5.48 42.16 77.52 -0.369 Nucleus CaHD-Zip31 ZLC09G0023620.1 245 28426.89 7.77 54.1 70.41 -0.876 Nucleus CaHD-Zip32 ZLC10G0006050.1 731 80565.15 5.6 39.94 84.17 -0.327 Nucleus CaHD-Zip33 ZLC10G0022120.1 333 37177 8.78 55.76 68.92 -0.725 Nucleus CaHD-Zip34 ZLC11G0001840.1 841 92165.03 5.84 51.47 85.28 -0.11 Nucleus CaHD-Zip35 ZLC11G0017670.1 296 34063.72 4.8 55.4 66.52 -0.847 Nucleus CaHD-Zip36 ZLC11G0018300.1 311 35611.57 5.73 62.75 58.68 -0.951 Nucleus CaHD-Zip37 ZLC11G0027120.1 209 24081.97 5.61 76.52 61.53 -1.002 Nucleus CaHD-Zip38 ZLC12G0012540.4 835 92404.88 6.04 47.14 87.14 -0.137 Cytoplasm/Nucleus CaHD-Zip39 ZLC12G0026280.1 354 39565.87 4.47 56 56.75 -0.888 Nucleus CaHD-Zip40 ZLC12G0029280.1 721 81691.4 5.96 45.9 76.63 -0.509 Nucleus 3.2 Multiple Sequence Alignment and Functional Site Analysis of CaHD-Zip Proteins in Pepper Multiple sequence alignment revealed two highly conserved functional domains in CaHD-Zip proteins: a DNA-binding homeodomain (HD, PF00046) and a leucine zipper motif (LZ, PF02183) (Fig. 1 ). The homeodomain contains a conserved core motif, ‘WFQNR’, embedded within the third α-helix (recognition helix), which is highly conserved across family members[ 51 ]. Tryptophan (W) stabilizes the protein–DNA complex by interacting with DNA base stacking via hydrophobic interactions[ 52 ]. Arginine (R), through its positively charged side chain, forms electrostatic interactions with the DNA phosphate backbone, further reinforcing DNA binding. The LZ domain adopts a heptad repeat pattern “L-X-L-X-L”, where "L" denotes conserved leucine residues and "X" represents variable amino acids. This domain architecture is widely conserved across species, underscoring its evolutionary importance in gene regulatory processes. 3.3 Classification and Phylogenetic Relationships of CaHD-Zip Genes To resolve evolutionary relationships between Capsicum annuum and Arabidopsis thaliana HD-Zip genes, we reconstructed a maximum likelihood phylogeny (Fig. 2).The analysis incorporated 40 CaHD-Zip and 58 AtHD-Zip genes. Based on tree topology and prior classifications, all 98 genes were classified into four subfamilies (I-IV). The distribution of CaHD-Zip genes across subfamilies showed significant disparity: subfamily I contained 14 genes, IV had 11, II comprised 10, while III was the smallest group with 5 members. This expansion pattern suggests lineage-specific diversification of subfamilies I and IV in pepper, potentially driven by functional Figure 2. Phylogenetic tree of HD-Zip proteins from Capsicum and Arabidopsis thaliana . The tree was constructed using the maximum likelihood (ML) method in IQ-TREE 2 and evaluated with 1000 bootstrap replicates. Blue circles indicate Capsicum proteins ; red pentagrams denote A. thaliana proteins. Different branch colors represent distinct HD-Zip subfamilies. adaptation. HD-Zip III and IV genes clustered on the same branch, suggesting recent divergence from a shared ancestor and subsequent functional differentiation. Conserved orthologs exhibited one-to-one correspondence in subfamilies I and II: CaHD-Zip31/AtHB51 , CaHD-Zip36/AtHB13 , and CaHD-Zip13/AtHB1 (Subfamily I); CaHD-Zip37/AtHB18, CaHD-Zip02/AtHB2 , and CaHD-Zip06/HAT63 (Subfamily II). In contrast, subfamily IV displayed one-to-many orthologous relationships— CaHD-Zip15 corresponding to GL2.1 and GL2.2 , CaHD-Zip03 to HDG4 and HDG5 , CaHD-Zip04 to AHDP.1 and AHDP.2 —indicating species-specific gene duplications driving functional diversification. Together, these phylogenetic findings demonstrate conserved subfamily architecture with lineage-specific expansions shaping functional innovation. 3.4 Chromosomal Distribution, Collinearity, and Duplication Analysis of CaHD-Zip Genes in Pepper Chromosomal mapping of 40 CaHD-Zip loci in Capsicum annuum showed a non-uniform distribution across 12 chromosomes (Fig. S1 ). Chromosomes 1–3 contained 6–7 CaHD-Zip genes, forming gene-dense regions, whereas chromosomes 5 and 7–10 showed sparse distribution (1–2 genes). Gene duplication analysis using MCScanX identified 15 segmentally duplicated CaHD-Zip gene pairs (Fig. 3 a). All duplicated pairs exhibited Ka/Ks ratios < 0.5 (Table S3), indicative of strong purifying selection. Divergence times ranged from 51.05 to 320.75 Mya, with the most recent duplication event ( CaHD-Zip18/26 ) occurring at ~ 51.05 Mya. Comparative analysis revealed stronger synteny with dicots ( Solanum lycopersicum : 77, S. tuberosum : 75, A. thaliana : 56) than with monocots ( Oryza sativa : 20, Zea mays : 14) (Fig. 3 b; Table S4-1), suggesting greater evolutionary conservation in dicotyledonous lineages. Most CaHD-Zip genes showed asymmetric syntenic relationships, particularly CaHD-Zip02 with four orthologs ( ATHB2/HAT1/HAT2/ATH4 ) across three dicots (Table S4-2). Seven CaHD-Zip genes ( CaHD-Zip02/06/10/19/21/34/39 ) maintained pan-lineage synteny among all analyzed species, demonstrating ancient evolutionary conservation. 3.5 Gene Architecture, Conserved Motifs, and Cis-Element Profiling of CaHD-Zip Family 3.5.1 Gene Structure and Conserved Motif Analysis MEME analysis of 40 CaHD-Zip genes identified 2 ~ 10 conserved motifs per gene (Fig. 4 b; Fig. S2). Motifs 1 and 2 were universally conserved across all CaHD-Zip proteins, suggesting their role as core structural elements essential for gene family stability. In contrast, Motifs 3 ~ 10 displayed subfamily-specific distributions, being largely restricted to HD-Zip III and IV, which may reflect their roles in subfamily-specific functional diversification. Specifically, HD-Zip III members contained Motifs 1, 2, 4, 5, and 7, while HD-Zip IV genes carried additional Motifs 3, 6, 9, and 10, indicating that distinct motif combinations may regulate subfamily-specific functions. Exon-intron architecture analysis revealed 2 to 18 exons per gene. Subfamily III genes (e.g., CaHD-Zip09/14/28/34/38 ) consistently harbored 18 exons, the maximum observed, while subfamily I members (e.g., CaHD-Zip01/18/ 26 ) contained only 2 exons (Fig. 4 d; Table S5). Intron numbers ranged from 1 to 17, and untranslated regions (UTRs) varied from 0 to 5 per gene. Notably, subfamily IV members CaHD-Zip22 and CaHD-Zip32 carried 5 UTRs, whereas ~ 50% of genes lacked annotated UTRs. These structural variations collectively highlight the evolutionary plasticity and functional modularity of the CaHD-Zip gene family in Capsicum annuum . 3.5.2 Conserved Domain Architecture and Subfamily Specific Signatures Domain architecture analysis identified six conserved domains within CaHD-Zip proteins: Homeodomain (HD), START (Steroidogenic Acute Regulatory Protein-related lipid transfer), HALZ (Homeobox-associated leucine zipper), MEKHLA (Membrane occupation and recognition nexus), bZIP (Basic leucine zipper), and HD-Zip_N, exhibiting subfamily-specific combinations. Subfamily I retained a minimal structure composed of HD and HALZ, while Subfamily II incorporated HD-Zip_N in addition to HD and HALZ. Subfamily IV displayed a unique domain configuration including HD, START, HALZ, and MEKHLA. Notably, Subfamily III encompassed all six domains, with the concurrent presence of START, MEKHLA, and bZIP forming a characteristic functional triad that may underpin its regulatory specialization. The HD domain was universally conserved across all CaHD-Zip genes, reaffirming its indispensable role in DNA binding. In contrast, the selective presence of START and MEKHLA among subfamilies suggests their involvement in functional divergence and subfamily-specific regulatory evolution (Fig. 4 c). 3.5.3 Promoter Cis-Elements and Their Functional Categorization in CaHD-Zip Genes Promoter analysis identified 899 cis-regulatory elements (CREs) across CaHD-Zip loci, categorized into 14 functional modules (Fig. 5 a; Table S6-1/2). Photoresponsive elements dominated (49.61%, 446/899), with Box4 (14.68%) and G-Box (8.23%) co-enriched in CaHD-Zip07/11/24 (Table S6-1), suggesting photoadaptive regulatory roles. Among hormone-responsive elements, methyl jasmonate (MeJA)-associated CREs constituted 11.35% (102/899), with CaHD-Zip15 containing the highest count (12 elements). CaHD-Zip38 and CaHD-Zip16 exhibited combinatorial enrichment of ABRE (abscisic acid-responsive), CGTCA-motif (MeJA-responsive), and TCA-element (salicylic acid-responsive) elements, forming a putative cross-hormonal regulatory hub (Fig. 5 b; Table S6-2). Stress-responsive elements comprised 19.20% of CREs, prominently enriched in CaHD-Zip23 and CaHD-Zip16 (Fig. 5 d). Both loci harbored MYB-binding sites (MBS), anaerobic response elements (ARE), and low-temperature-responsive elements (LTR), implicating a MYB-LTR-ARE regulatory axis in stress adaptation. Growth-regulatory CREs accounted for 54.46% of total elements, with CaHD-Zip33 uniquely containing all developmental motifs (e.g., CAT-box, Circadian, O2-site,GATA-motif) (Fig. 5 c). This architectural singularity positions CaHD-Zip33 as a developmental hub gene regulating core growth pathways in Capsicum annuum . Collectively, the CRE landscape reveals light-dominant transcriptional regulation integrated with hormone-stress crosstalk, forming an adaptive framework for environmental responsiveness and developmental control. 3.6 Expression Profiling of the CaHD-Zip Gene Family in Pepper Tissues Using NCBI transcriptome datasets, we analyzed expression profiles of 40 CaHD-Zip genes across 11 Capsicum annuum tissues encompassing floral organs, three stages of fruit development, and vegetative tissues such as root, stem, and leaf (Fig. 6 ). Transcripts per million (TPM)-based hierarchical clustering segregated CaHD-Zip genes into four co-expression clusters (A-D). Cluster A genes ( HD-Zip21 ~ HD-Zip11 ) (Fig. 6 A). were predominantly expressed in meristematic tissues including stem, root, embryos, seeds, and placenta, with especially high levels in the ovary (peak TPM > 25.31) Cluster B genes ( HD-Zip33/18/29 )(Fig. 6 B) showed distinct expression features during fruit development, particularly in the breaker and mature fruit stages. Cluster C genes ( HD-Zip26 ~ HD-Zip30 ) (Fig. 6 C) displayed consistently low expression across all tissues (TPM < 5 in 90% of cases), suggesting that these genes may be selectively expressed under specific developmental or stress-related conditions. Cluster D genes ( HD-Zip25 ~ HD-Zip20 ) (Fig. 6 D) exhibited moderate to high expression levels in seeds and floral buds, implying potential roles in floral organ formation and seed development. Collectively, CaHD-Zip genes exhibit stage- and tissue-partitioned expression patterns: Cluster A dominates meristem activity, Cluster B drives late fruit metabolism, Cluster C enables stress adaptation, and Cluster D maintains developmental homeostasis. 3.7 Hormonal Response of the CaHD-Zip Gene Family Under Abiotic Stress Conditions Using NCBI transcriptome datasets (Table S1 ), we analyzed CaHD-Zip expression under four stress hormones: abscisic acid (ABA), methyl jasmonate (MeJA), ethylene (ET), and salicylic acid (SA) at 0,3,6,12,24 h post-treatment (Fig. 7 a). Transcripts per million (TPM) analysis revealed hormone-specific temporal regulation patterns.ABA and MeJA treatments primarily induced gene expression at later stages (12–24 h), indicating involvement in stress adaptation. For instance, under 24 h ABA exposure, CaHD-Zip29 and CaHD-Zip16 were upregulated to 105.8 and 40.6 TPM, respectively (control: 3.2 and 8.1 TPM); MeJA upregulated CaHD-Zip11 to 62.3 TPM. In contrast, ET and SA triggered rapid responses at 3–6 h. ET rapidly induced CaHD-Zip11 expression to 57.6 TPM (control: 22.6 TPM) at 3 h, while SA downregulated CaHD-Zip21 to 13.0 TPM at 6 h (control: 43.0 TPM). Venn diagram analysis (Fig. S3) identified maximal DEGs at ABA 12 h (13 genes), MeJA 24 h (11), SA 6 h (12), and ET 6 h (10). Notably, CaHD-Zip16 and CaHD-Zip18 exhibited significant responsiveness to all four hormones across at least two timepoints (Fig. 7 b), positioning them as central integrators of hormone signal crosstalk. These findings indicate a division of hormonal response phases: ABA/MeJA mediate delayed transcriptional activation, while ET/SA orchestrate early signaling events. CaHD-Zip16 and CaHD-Zip18 likely function as regulatory hubs bridging diverse hormonal pathways. 3.8 Expression Profiling and Differential Expression Analysis of CaHD-Zip Genes Under Abiotic Stresses Utilizing transcriptome datasets from NCBI, we analyzed TPM-based expression profiles of 40 CaHD-Zip genes in Capsicum annuum under four major abiotic stress conditions—cold, heat, drought, and salinity across six timepoints (0, 3, 6, 12, 24, and 72 hours). The spatiotemporal dynamics revealed stress-specific regulatory patterns. 3.8.1 Cold Stress Responsive Expression Patterns of CaHD-Zip Genes Hierarchical clustering of TPM values identified three co-expression clusters (A-C) under cold stress (10°C) (Fig. 8 a). Cluster A ( CaHD-Zip31 ~ CaHD-Zip15; ) showed progressive suppression, exemplified by CaHD-Zip25 (20.58 to 0.74 TPM, 96.4% reduction) and CaHD-Zip36 (23.63 to 9.52 TPM, 59.7% decrease) from 0–72 h. Cluster B ( CaHD-Zip26 ~ CaHD-Zip19 ) peaked at 12 h, with CaHD-Zip29 showing 80.3-fold induction (0 to 281.22 TPM) followed by 35-fold retention at 72 h. Cluster C ( CaHD-Zip02 ~ CaHD-Zip09 ) exhibited sustained activation, with CaHD-Zip07 (20.73-fold, 10.78 TPM) and CaHD-Zip18 (14.93-fold, 34.54 TPM) reaching maxima at 72 h. Venn analysis (Fig. 8 e) identified five DEGs ( CaHD-Zip36/29/07/18/25 ) showing consistent differential expression (FDR < 0.01) across all timepoints. The regulatory dynamics featured sustained activation ( CaHD-Zip07/18 ), progressive suppression ( CaHD-Zip25/36 ), and transient induction ( CaHD-Zip29 ), indicating phase-specific cold adaptation strategies. 3.8.2 Heat Stress Responsive Expression Patterns of CaHD-Zip Genes Hierarchical clustering of TPM-based transcriptomic profiles under heat stress (40°C) revealed four distinct co-expression clusters (A–D) among the CaHD-Zip gene family (Fig. 8 b). Cluster A ( CaHD-Zip34 ~ CaHD-Zip06 ) exhibited transient induction at 12 h, with CaHD-Zip29 peaking at 36.28 TPM (10.4-fold vs. 0 h), followed by a decline to near-baseline levels at 72 h (8.76 TPM). Cluster B ( CaHD-Zip39 ~ CaHD-Zip09 ) showed rapid early activation, exemplified by CaHD-Zip18 , which rose from 2.31 to 21.8 TPM at 3 h (8.43-fold) and peaked at 31.82 TPM by 6 h. CaHD-Zip23 and CaHD-Zip16 also displayed similar early induction patterns, indicating involvement in thermotolerance priming. Cluster C ( CaHD-Zip33 ~ CaHD-Zip14 ) demonstrated significant early repression, as CaHD-Zip25 and CaHD-Zip36 were downregulated by 92.6% (20.5 to 1.51 TPM) and 56.2% (23.63 to 10.34 TPM) respectively, at 6 h, suggesting potential roles as negative regulators of heat response. Cluster D ( CaHD-Zip12 ~ CaHD-Zip04 ) maintained stable expression (fold-change < 2), indicating minimal involvement in heat-induced transcriptional reprogramming. Venn diagram analysis (Fig. 8 f) identified six core heat-responsive DEGs ( CaHD-Zip16/18/23/25/29/35 ) with significant differential expression across all treatment stages (FDR < 0.01). Among these, CaHD-Zip16/18/23/29/35 were persistently upregulated, while CaHD-Zip25 was consistently downregulated, delineating two opposing regulatory trajectories involved in orchestrating pepper’s transcriptional adaptation to thermal stress. 3.8.3 Expression Profiling of CaHD-Zip Genes Under Drought Stress Hierarchical clustering of TPM based transcriptomic profiles under drought stress segregated CaHD-Zip genes into four distinct co-expression modules (A–D) (Fig. 8 c). Cluster A ( CaHD-Zip17 ~ CaHD-Zip25 ; 6 members) showed sustained transcriptional suppression, exemplified by CaHD-Zip25 (94.7% reduction: 20.58 to 1.09 TPM) from 12–72 h. Cluster B ( CaHD-Zip08 ~ CaHD-Zip31 ; 7 members) exhibited transient early activation (3–6 h), followed by a gradual decline to approximately 50% of peak levels by 72 h, as observed for CaHD-Zip23 (6 h peak: 32.16 TPM, 6.0-fold induction). Cluster C ( CaHD-Zip40 ~ CaHD-Zip37 ; 12 members) demonstrated progressive transcriptional upregulation, with CaHD-Zip29 achieving a 31.5-fold induction (3.50 to 113.67 TPM) at 72 h. Cluster D ( CaHD-Zip30 ~ CaHD-Zip04 ; 13 members) exhibited temporally dynamic expression patterns, with certain members activated and others repressed in a stage-specific manner (e.g., CaHD-Zip06 induced at 12 h; CaHD-Zip03 repressed at 72 h). Venn diagram analysis (Fig. 8 g) identified seven core drought-responsive DEGs ( CaHD-Zip35/16/29/01/18/23/25 ) with significant expression changes across all timepoints (FDR < 0.05). Most of these DEGs belonged to Cluster C and exhibited persistent upregulation, highlighting their potential roles as central regulatory hubs in the long-term drought stress adaptation network of Capsicum annuum . 3.8.4 Expression Profiling of the CaHD-Zip Gene Family Under Salt Stress Hierarchical clustering of TPM-based transcriptomes under salt stress (200 mM NaCl) grouped CaHD-Zip genes into five regulatory modules (Clusters A–E) (Fig. 8 d). Cluster A ( CaHD-Zip12 ~ CaHD-Zip21 ) showed late-phase induction, exemplified by CaHD-Zip21 increasing from 19.62 to 30.15 TPM by 72 h. Cluster B ( CaHD-Zip16 ~ CaHD-Zip11 ) exhibited persistent upregulation throughout the stress period, peaking at 24 h. Cluster C ( CaHD-Zip30 ~ CaHD-Zip35 ) comprised early-inducible genes such as CaHD-Zip23 , which was upregulated 6.9-fold by 6 h (5.36 to 37.11 TPM), suggesting involvement in initial salt signaling. Cluster D ( CaHD-Zip25 ~ CaHD-Zip04 ) showed strong and sustained repression, with CaHD-Zip25 decreasing 45.77-fold by 24 h (20.58 to 0.44 TPM), suggesting negative regulatory functions in salt tolerance. Cluster E ( CaHD-Zip36 ~ CaHD-Zip22 ) exhibited stage-specific responses, as exemplified by CaHD-Zip06 upregulated to 17.57 TPM at 12 h (65.85% increase), possibly mediating short-term salt adaptation. Venn analysis (Fig. 8 h) revealed five key DEGs (CaHD-Zip16/29/01/18/25) differentially expressed at all timepoints. CaHD-Zip16/29/01/18 were consistently induced, whereas CaHD-Zip25 was persistently downregulated, delineating contrasting regulatory modes essential for sustained salt stress adaptation. 3.8.5 Key Regulators Shared Across Abiotic Stress Conditions Further Venn analysis (Fig. 8 i) revealed that CaHD-Zip29 , CaHD-Zip18 , and CaHD-Zip25 were consistently differentially expressed across all timepoints under cold, heat, drought, and salt stresses, implicating them as core regulators in broad-spectrum stress responses. Among them, CaHD-Zip29 and CaHD-Zip18 were persistently upregulated, suggesting positive regulatory roles in promoting abiotic stress adaptation. In contrast, CaHD-Zip25 was consistently downregulated, indicating its potential function as a negative regulator. Collectively, these genes appear to represent key regulatory hubs orchestrating stress signaling, transcriptional modulation, and long-term environmental resilience in Capsicum annuum . 3.9 Validation of Transcriptome-Based Expression Patterns via qRT-PCR Based on transcriptome profiling, eight representative CaHD-Zip genes ( HD-Zip16, HD-Zip29, HD-Zip01, HD-Zip18, HD-Zip25, HD-Zip23, HD-Zip35, and HD-Zip36 ) were selected for qRT-PCR validation under four abiotic stress conditions (cold, heat, drought, salinity) and two hormone treatments (ABA and MeJA) (Fig. 9 ). Under cold stress, qRT-PCR analysis revealed significant upregulation of all tested genes except HD-Zip25 and HD-Zip36 , suggesting a positive regulatory role of the remaining genes in cold acclimation. Under heat, drought, and salinity stress conditions, HD-Zip25 was consistently downregulated, while the other seven genes exhibited pronounced induction, indicating a potential negative regulatory role of HD-Zip25 in abiotic stress responses. Under ABA and MeJA treatment, HD-Zip36 remained transcriptionally unchanged, HD-Zip25 was downregulated, and the other six genes were significantly upregulated. Overall, the qRT-PCR results showed strong concordance with transcriptomic sequencing data, confirming the reliability of transcriptomic analysis and supporting the differential expression patterns of CaHD-Zip genes under hormone and abiotic stress regulation. These findings provide robust experimental evidence for future studies on the functional roles and regulatory mechanisms of HD-Zip genes in stress responses, and identify potential gene targets for enhancing abiotic stress tolerance in plants. 4. Discussion The HD-ZIP (Homeodomain-Leucine Zipper) gene family comprises plant-specific transcription factors that orchestrate diverse developmental processes and mediate stress responses in plants [ 13 , 14 ]. Despite extensive characterization in numerous economically important crops, knowledge regarding their expression dynamics in pepper under abiotic stresses remains limited. In this study, we identified 40 CaHD-ZIP genes and performed a genome-wide analysis of their structural features, chromosomal localization, tissue-specific expression patterns, and responses to abiotic stresses and hormone signaling pathways. Three candidate HD-ZIP transcription factors responsive to multiple abiotic stress conditions were identified, offering a molecular foundation for advancing functional studies of the HD-ZIP family in pepper stress adaptation. 4.1 Phylogenetic relationships and functional conservation of the HD-Zip gene family The CaHD-Zip genes in pepper are classified into four subfamilies (HD-Zip I to IV), displaying evolutionary divergence patterns that are conserved across diverse plant lineages [ 37 , 38 ]. We found that HD-Zip I subfamily members are the most abundant, whereas the HD-Zip III subfamily comprises comparatively fewer genes. This trend mirrors observations in Arabidopsis thaliana, Solanum tuberosum , and Cucumis sativus [ 53 – 55 ]. The reduced representation of HD-Zip III genes may reflect stronger purifying selection during evolution—a pattern corroborated in several dicot species [ 55 ]. Additionally, a one-to-one orthologous relationship exists between CaHD-Zip genes in pepper and AtHD-Zip genes in Arabidopsis , such as CaHD-Zip31 with ATHB51 , and CaHD-Zip37 with ATHB18 . These results underscore the evolutionary conservation of the HD-Zip gene family and its potentially conserved roles in regulating growth and development across species [ 36 ]. 4.2 Gene expansion mechanisms and collinearity Gene duplication represents a fundamental driver of plant genome evolution, enabling gene family expansion through segmental duplication and tandem duplication, thereby enhancing adaptability to environmental stimuli [ 56 ]. Our analysis revealed that the expansion of the HD-Zip gene family in pepper was primarily driven by segmental duplication, with 15 segmentally duplicated gene pairs identified, whereas no tandem duplication events were detected. This pattern aligns with observations in sesame and cassava, where HD-Zip gene expansion similarly relies on segmental duplication [ 57 ]. Moreover, the divergence times of these segmental duplicates span a broad evolutionary timeframe, ranging from 320.75 million years ago ( HD-Zip11 and HD-Zip20 ) to 51.05 million years ago ( HD-Zip18 and HD-Zip26 ). These findings suggest that the HD-Zip gene family in pepper underwent multiple independent duplication events, potentially linked to species-specific genome duplication or chromosomal rearrangement events [ 58 ]. Synteny analysis revealed strong collinearity between pepper HD-Zip genes and those of dicot species (tomato, potato, Arabidopsis), while collinearity with monocots such as rice and maize was comparatively weak[ 59 – 61 ]. This disparity likely reflects deep evolutionary divergence and genome structural differences, further suggesting that HD-Zip genes in dicots have been subject to intensified selective constraints. Additionally, seven CaHD-Zip genes displayed conserved synteny across all five species analyzed, underscoring their functionally conserved roles in plant development and environmental adaptation—a finding consistent with previous studies in watermelon [ 62 ]. 4.3 Core regulatory roles of the HD-Zip gene family in abiotic stress responses The HD-Zip gene family regulates abiotic stress responses through multiple coordinated mechanisms, including ABA-dependent signaling pathways, lignin biosynthesis, membrane stability modulation, and miRNA-guided gene regulation, thereby enhancing plant resilience. Numerous HD-Zip genes have been implicated in salt tolerance across various species, such as Gossypium hirsutum GhHB4 -like and GhHB12 [ 63 , 64 ], Miscanthus sinensis MsHDZ23 [ 65 ], and Brassica napus BnaHDZ149 [ 66 ], which exert their functions primarily via ABA-related or stress-inducible pathways. Cold stress responses typically involve ABA signaling and membrane protein stabilization, exemplified by Malus domestica MdHDZ14 , which is significantly upregulated under low-temperature conditions [ 38 ], as well as Helianthus annuus HaHB1 and Arabidopsis thaliana AtHB13 , which enhance cold tolerance by maintaining membrane integrity[ 67 ]. In heat stress adaptation, Raphanus sativus RsHDZ17 enhances thermotolerance when overexpressed in both radish and Arabidopsis thaliana [ 11 ], while Capsicum annuum HD-Zip15 has been shown to directly target HSFA6a and activate CaHSFA2 , playing a central role in thermotolerance regulation[ 7 ]. Moreover, under drought stress, HD-Zip genes such as Passiflora edulis PeHB31 [ 36 ]and Zea may s ZmHDZ9 [ 68 ] promote lignin biosynthesis, thereby contributing to enhanced drought resistance. In our study, CaHD-Zip29 and CaHD-Zip18 were significantly upregulated in Capsicum annuum under cold, heat, salt, and drought conditions, indicating robust multi-stress responsiveness. Phylogenetic analysis revealed that both genes are closely related to Arabidopsis thaliana AtHB7 and AtHB12 , and their orthologs in other species—such as Oryza sativa HOX22 [ 69 ], Salix suchowensis SsHox36/SsHox51 [ 70 ], and Sesamum indicum SiHDZ31/16 [ 71 ]have been associated with drought and salt stress responses. Additionally, these genes exhibit strong conservation under thermal stress. For instance, Passiflora edulis PeHB17/07 and Raphanus sativus RsHDZ17 are highly induced under heat stress[ 11 , 36 ], while Hordeum vulgare HvHD-Zip19 [ 72 ] and Malus domestica MdHDZ21 [ 38 ] show specific responses to cold stress. Collectively, these findings not only underscore the evolutionary conservation of HD-Zip genes in abiotic stress adaptation but also suggest that CaHD-Zip29 and CaHD-Zip18 , as orthologs of AtHB7/AtHB12 , may serve as integrative regulators coordinating multiple abiotic stress signaling pathways in Capsicum annuum . 4.4 Regulatory roles of CaHD-Zip genes in pepper development We systematically profiled the expression patterns of 40 CaHD-Zip genes, categorizing them into four subgroups, and uncovered a multi-layered regulatory framework coordinating meristem maintenance, fruit ripening, floral organogenesis, and environmental adaptation. Class A genes exhibited strong expression in meristematic and reproductive tissues (e.g., seeds, embryos, placentae), a pattern conserved with Arabidopsis thaliana AtHB23 , which modulates shoot apical meristem activity [ 36 ], and Passiflora edulis PeHB31 , which promotes ovule development [ 73 ]. Arabidopsis thaliana AtHB2 mediates red/far-red light-induced shade avoidance and lateral root development [ 74 ], while Zea mays ZmHOX32 improves photosynthetic efficiency by modulating leaf meristem activity [ 75 ], highlighting the HD-Zip family's extensive role in plant morphogenesis. Class B genes, including CaHD-Zip29 , were markedly upregulated during fruit maturation, mirroring Passiflora edulis PeHB7/PeHB17/PeHB18 expression [ 36 ], thereby suggesting a conserved role in ripening. Additional support stems from Musa acuminata HD-Zip I genes regulating ACS/ACO [ 76 ], LeHB-1 promoting ACO1 Mrna [ 77 ], and Cucumis sativus CsHDZ11/37 being highly expressed in fruits [ 53 ]. Class C genes exhibited low baseline expression across tissues but were robustly induced by drought, salinity, cold stress, and heat stress (e.g., CaHD-Zip01 ), similar to Eucalyptus grandis HD-Zip38 [ 78 ] and Hordeum vulgare HvHD-ZipI16/II1 under drought [ 72 ], implicating their role as central stress-responsive factors in Capsicum annuum . Class D genes showed strong, specific expression in floral buds and seeds, suggesting functions in floral primordium differentiation or pollination. Such roles are exemplified by Citrus sinensis PtHB13 [ 79 ], and Cucumis sativus CsHDZ08/Z22 [ 53 ]. Collectively, these results unveil distinct spatiotemporal expression profiles of the CaHD-Zip gene family across Capsicum annuum tissues and developmental stages, laying a molecular foundation for elucidating their roles in pepper development. 5. Conclusions In summary, we identified 40 HD-Zip transcription factors in Capsicum annuum , with gene family expansion predominantly driven by segmental duplication and exhibited strong evolutionary conservation among dicot species. Spatiotemporal expression profiling revealed distinct regulatory patterns across meristematic tissues, fruit development, and floral differentiation. Notably, CaHD-Zip18 and CaHD-Zip29 were consistently upregulated under multiple abiotic stresses, whereas CaHD-Zip25 was significantly downregulated, suggesting their respective roles as core positive and negative regulators in stress adaptation. The expression dynamics of these genes were further validated by quantitative reverse transcription PCR (qRT-PCR). As the first comprehensive investigation of the HD-Zip family in Capsicum annuum under abiotic stress, this study offers valuable insights into their regulatory functions and provides key candidate genes for the development of stress-tolerant cultivars. Future research integrating CRISPR-based gene editing and molecular interaction analyses will be critical to unravelling the functional mechanisms of these transcription factors and advancing molecular breeding strategies in Capsicum annuum . Abbreviations ABA abscisic acid bZIP Basic leucine zipper Ca Capsicum annuum CDS Coding sequences ET ethylene GRAVY Grand Average of Hydropathicity HALZ Homeobox-associated leucine zipper HD homeodomain HMM Hidden markov model Ka Nonsynonymous Substitution Rate Ks Synonymous Substitution Rate LZ leucine zipper MeJA methyl jasmonate MEKHLA Membrane occupation and recognition nexus ML maximum likelihood PEG-6000 Polyethylene Glycol 6000 pI Isoelectric point PPI Protein–protein interaction qRT-PCR Quantitative Real Time Polymerase Chain Reaction R Arginine SA salicylic acid SPSS Statistical Package for the Social Sciences START Steroidogenic Acute Regulatory Protein-related lipid transfer TPM Transcripts per million W Tryptophan Declarations Supplementary information The online version contains supplementary material available at…. Supplementary Material: Supplementary Table S1: RNA-seq datasets from NCBI SRA for Capsicum annuum across organ types, abiotic stresses, and phytohormone treatments. Supplementary Table S2: The primer sequences used for qRT-PCR. Supplementary Table S3: Ka/Ks Ratios of 15 Segmentally Duplicated CaHD-Zip Genes. Supplementary Table S4: Collinearity analysis results of Capsicum (intraspecific and interspecific). Supplementary Table S5: Structural Characteristics of the Capsicum HD-Zip Gene Family. Supplementary Table S6: Statistics on the functional classification of CaHD-zip genes. Supplementary Figure S1: Chromosomal Localization of 40 Capsicum CaHD-Zip Genes . Supplementary Figure S2: SeqLogo Representation of Motif1-10. Supplementary Figure S3: Spatiotemporal Distribution Analysis of Differentially Expressed Genes (DEGs) Under Different Hormone Treatments. Acknowledgements We are sincerely grateful to Prof. Jimin Sun (Chinese Academy of Sciences) and Dr. Yuchao Tang, Postdoctoral Fellow (Beijing Forestry University) for their important revisions and insightful suggestions that significantly improved the quality of this manuscript. Author contributions X.W. designed the experiments, conducted data analysis and visualization, and was responsible for drafting and revising the manuscript. X.Z., K.X., and X.F. contributed to data analysis and visualization. L.L., L.G. and M.S. participated in the experimental design and assisted with data analysis. Y.L. and C.H. provided funding and contributed to the final revision of the manuscript. All authors reviewed and approved the final manuscript. Funding The earmarked fund for CARS, Grant/Award Number: CARS-24-B-04 and CARS-23-B05; The National Key Research and Development Program of China (2022YFD1602403 and 2023YFD2300704 ); The Major Science and Technology Special Project of Ordos (ZD20232318); Talent Project in the Field of Science and Technology Innovation of Hohhot. This research acknowledged the support of the Key Laboratory of Horticultural Crop Biology and Germplasm Innovation, the Ministry of Agriculture, China. Data availability Data is provided within the manuscript or supplementary information files. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Zhou C-X, Zhang W, Yu B-G, Yang H-F, Zhao Q-Y, Wang Y, et al. Globalanalysis ofspatio-temporal variation inmineral nutritional quality ofpepper( Capsicum spp.)fruitanditsregulatory variables: A meta-analysis. Food Research International. 2024;193:114855. Tripodi P, Kumar S. The Capsicum Crop: An Introduction. In: Ramchiary N, Kole C, editors. The Capsicum Genome. Cham: Springer International Publishing; 2019. p. 1–8. Moseley A, Laipply K, Rubinstein J. Capsaicin Mediates Remote Ischemic Pre-Conditioning to Explain Improved Cardiovascular Mortality With Chili Pepper Intake. Journal of the American College of Cardiology. 2020;75:1865. Parvez GMM. CURRENT ADVANCES IN PHARMACOLOGICAL ACTIVITY AND TOXIC EFFETCS OF VARIOUS CAPSICUM SPECIES. International Journal of Pharmaceutical Sciences and Research. 8. Baenas N, Belović M, Ilic N, Moreno DA, García-Viguera C. Industrial use of pepper (Capsicum annum L.) derived products: technological benefits and biological advantages. Food Chemistry. 2019;274:872–85. Zhang X, Ma X, Wang S, Liu S, Shi S. Physiological and Genetic Aspects of Resistance to Abiotic Stresses in Capsicum Species. Plants. 2024;13:3013. Mou S, He W, Jiang H, Meng Q, Zhang T, Liu Z, et al. Transcription factor CaHDZ15 promotes pepper basal thermotolerance by activating HEAT SHOCK FACTORA6a. Plant Physiology. 2024;195:812–31. Chhapekar SS, Jaiswal V, Ahmad I, Gaur R, Ramchiary N. Progress and Prospects in Capsicum Breeding for Biotic and Abiotic Stresses. In: Vats S, editor. Biotic and Abiotic Stress Tolerance in Plants. Singapore: Springer; 2018. p. 279–322. Nakashima K, Ito Y, Yamaguchi-Shinozaki K. Transcriptional Regulatory Networks in Response to Abiotic Stresses in Arabidopsis and Grasses. Plant Physiology. 2009;149:88–95. Capella M, Ribone PA, Arce AL, Chan RL. Arabidopsis thaliana HomeoBox 1 (AtHB1), a Homedomain-Leucine Zipper I (HD-Zip I) transcription factor, is regulated by PHYTOCHROME-INTERACTING FACTOR 1 to promote hypocotyl elongation. New Phytologist. 2015;207:669–82. Wang K, Xu L, Wang Y, Ying J, Li J, Dong J, et al. Genome-wide characterization of homeodomain-leucine zipper genes reveals enhances the heat tolerance in radish (Raphanus sativus L.). Physiologia Plantarum. 2022;174:e13789. Navarro MA, Navarro C, Hernández LE, Garnica M, Franco-Zorrilla JM, Burko Y, et al. GLABRA2 transcription factor integrates arsenic tolerance with epidermal cell fate determination. New Phytologist. 2024;244:1882–900. Leung J, Merlot S, Giraudat J. The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. Plant Cell. 1997;9:759–71. Sessa G, Carabelli M, Sassi M, Ciolfi A, Possenti M, Mittempergher F, et al. A dynamic balance between gene activation and repression regulates the shade avoidance response in Arabidopsis. Genes Dev. 2005;19:2811–5. Henriksson E, Olsson ASB, Johannesson H, Johansson H, Hanson J, Engström P, et al. Homeodomain Leucine Zipper Class I Genes in Arabidopsis. Expression Patterns and Phylogenetic Relationships. Plant Physiology. 2005;139:509–18. A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif. Ariel FD, Manavella PA, Dezar CA, Chan RL. The true story of the HD-Zip family. Trends Plant Sci. 2007;12:419–26. Harris JC, Hrmova M, Lopato S, Langridge P. Modulation of plant growth by HD-Zip class I and II transcription factors in response to environmental stimuli. New Phytologist. 2011;190:823–37. Meijer AH, de Kam RJ, d’Erfurth I, Shen W, Hoge JHC. HD-Zip proteins of families I and II from rice: interactions and functional properties. Mol Gen Genet. 2000;263:12–21. Elhiti M, Stasolla C. Structure and function of homodomain-leucine zipper (HD-Zip) proteins. Plant Signal Behav. 2009;4:86–8. Roodbarkelari F, Groot EP. Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis. New Phytologist. 2017;213:95–104. Perotti MF, Ribone PA, Chan RL. Plant transcription factors from the homeodomain-leucine zipper family I. Role in development and stress responses. IUBMB Life. 2017;69:280–9. Romani F, Ribone PA, Capella M, Miguel VN, Chan RL. A matter of quantity: Common features in the drought response of transgenic plants overexpressing HD-Zip I transcription factors. Plant Sci. 2016;251:139–54. Söderman E, Mattsson J, Engström P. The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid. Plant J. 1996;10:375–81. Mishra KB, Iannacone R, Petrozza A, Mishra A, Armentano N, La Vecchia G, et al. Engineered drought tolerance in tomato plants is reflected in chlorophyll fluorescence emission. Plant Sci. 2012;182:79–86. Zhao S, Wang H, Jia X, Gao H, Mao K, Ma F. The HD-Zip I transcription factor -like confers tolerance to salinity in transgenic apple (). Physiologia Plantarum. 2021;172:1452–64. Turchi L, Baima S, Morelli G, Ruberti I. Interplay of HD-Zip II and III transcription factors in auxin-regulated plant development. J Exp Bot. 2015;66:5043–53. Turchi L, Carabelli M, Ruzza V, Possenti M, Sassi M, Peñalosa A, et al. Arabidopsis HD-Zip II transcription factors control apical embryo development and meristem function. Development. 2013;140:2118–29. Carabelli M, Turchi L, Ruzza V, Morelli G, Ruberti I. Homeodomain-Leucine Zipper II family of transcription factors to the limelight: central regulators of plant development. Plant Signal Behav. 2013;8:e25447. Merelo P, Ram H, Pia Caggiano M, Ohno C, Ott F, Straub D, et al. Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity. Proceedings of the National Academy of Sciences. 2016;113:11973–8. Causier B, Ashworth M, Guo W, Davies B. The TOPLESS interactome: a framework for gene repression in Arabidopsis. Plant Physiol. 2012;158:423–38. Liu T, Longhurst AD, Talavera-Rauh F, Hokin SA, Barton MK. The Arabidopsis transcription factor ABIG1 relays ABA signaled growth inhibition and drought induced senescence. Elife. 2016;5:e13768. Prigge MJ, Clark SE. Evolution of the class III HD-Zip gene family in land plants. Evolution & Development. 2006;8:350–61. Zhu Y, Song D, Xu P, Sun J, Li L. A HD-ZIP III gene, PtrHB4, is required for interfascicular cambium development in Populus. Plant Biotechnol J. 2018;16:808–17. Gao Y, Gao S, Xiong C, Yu G, Chang J, Ye Z, et al. Comprehensive analysis and expression profile of the homeodomain leucine zipper IV transcription factor family in tomato. Plant Physiology and Biochemistry. 2015;96:141–53. Ma F, Song S, Li C, Huang D, Wu B, Xing W, et al. Passion fruit HD-ZIP genes: Characterization, expression variance, and overexpression PeHB31 enhanced drought tolerance via lignin pathway. International Journal of Biological Macromolecules. 2024;276:133603. Wang Z, Wu X, Zhang B, Xiao Y, Guo J, Liu J, et al. Genome-wide identification, bioinformatics and expression analysis of HD-Zip gene family in peach. BMC Plant Biology. 2023;23:122. Zhang Q, Chen T, Wang X, Wang J, Gu K, Yu J, et al. Genome-wide identification and expression analyses of homeodomain-leucine zipper family genes reveal their involvement in stress response in apple (Malus × domestica). Horticultural Plant Journal. 2022;8:261–78. Zhang K, Wang X, Chen S, Liu Y, Zhang L, Yang X, et al. The gap-free assembly of pepper genome reveals transposable-element-driven expansion and rapid evolution of pericentromeres. Plant Communications. 2025;6:101177. Potter SC, Luciani A, Eddy SR, Park Y, Lopez R, Finn RD. HMMER web server: 2018 update. Nucleic Acids Res. 2018;46:W200–4. Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020;48:D265–8. Letunic I, Bork P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Research. 2018;46:D493–6. Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003;31:3784–8. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33:1870–4. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:1189–91. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30:325–7. Wang D, Zhang S, He F, Zhu J, Hu S, Yu J. How do variable substitution rates influence Ka and Ks calculations? Genomics Proteomics Bioinformatics. 2009;7:116–27. Lynch M, Conery JS. The Evolutionary Fate and Consequences of Duplicate Genes. Science. 2000;290:1151–5. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant. 2020;13:1194–202. Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 2013;3:71–85. Kock KH, Kimes PK, Gisselbrecht SS, Inukai S, Phanor SK, Anderson JT, et al. DNA binding analysis of rare variants in homeodomains reveals homeodomain specificity-determining residues. Nat Commun. 2024;15:3110. González FG, Capella M, Ribichich KF, Curín F, Giacomelli JI, Ayala F, et al. Field-grown transgenic wheat expressing the sunflower gene HaHB4 significantly outyields the wild type. J Exp Bot. 2019;70:1669–81. Sharif R, Xie C, Wang J, Cao Z, Zhang H, Chen P, et al. Genome wide identification, characterization and expression analysis of HD-ZIP gene family in Cucumis sativus L. under biotic and various abiotic stresses. International Journal of Biological Macromolecules. 2020;158:502–20. Li W, Dong J, Cao M, Gao X, Wang D, Liu B, et al. Genome-wide identification and characterization of HD-ZIP genes in potato. Gene. 2019;697:103–17. Li Z, Zhang C, Guo Y, Niu W, Wang Y, Xu Y. Evolution and expression analysis reveal the potential role of the HD-Zip gene family in regulation of embryo abortion in grapes (Vitis vinifera L.). BMC Genomics. 2017;18:744. Cannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biology. 2004;4:10. Ding Z, Fu L, Yan Y, Tie W, Xia Z, Wang W, et al. Genome-wide characterization and expression profiling of HD-Zip gene family related to abiotic stress in cassava. PLoS One. 2017;12:e0173043. Soltis PS, Soltis DE. The role of hybridization in plant speciation. Annu Rev Plant Biol. 2009;60:561–88. Lopez-Ortiz C, Dutta SK, Natarajan P, Peña-Garcia Y, Abburi V, Saminathan T, et al. Genome-wide identification and gene expression pattern of ABC transporter gene family in Capsicum spp. PLoS One. 2019;14:e0215901. Islam MAU, Nupur JA, Shafiq M, Ali Q, Sami A, Shahid MA. In silico and computational analysis of zinc finger motif-associated homeodomain (ZF-HD) family genes in chilli (Capsicum annuum L). BMC Genomics. 2023;24:603. Pan X, Hu M, Wang Z, Guan L, Jiang X, Bai W, et al. Identification, systematic evolution and expression analyses of the AAAP gene family in Capsicum annuum. BMC Genomics. 2021;22:463. Yan X, Yue Z, Pan X, Si F, Li J, Chen X, et al. The HD-ZIP Gene Family in Watermelon: Genome-Wide Identification and Expression Analysis under Abiotic Stresses. Genes. 2022;13:2242. Ren A, Wen T, Xu X, Wu J, Zhao G. Cotton HD-Zip I transcription factor GhHB4-like regulates the plant response to salt stress. International Journal of Biological Macromolecules. 2024;278:134857. He X, Luo X, Wang T, Liu S, Zhang X, Zhu L. GhHB12 negatively regulates abiotic stress tolerance in Arabidopsis and cottonGhHB12. Environmental and Experimental Botany. 2020;176:104087. Liu N, Yu R, Deng W, Hu R, He G, He K, et al. MsHDZ23, a Novel Miscanthus HD-ZIP Transcription Factor, Participates in Tolerance to Multiple Abiotic Stresses. International Journal of Molecular Sciences. 2024;25:3253. Fang Y, Wang L, Liu K, Wu H, Zheng Y, Duan Y, et al. Genome-wide investigation of HD-ZIP gene family and functional characterization of BnaHDZ149 and BnaHDZ22 in salt and drought response in Brassica napus L. Plant Science. 2024;346:112130. Cabello JV, Arce AL, Chan RL. The homologous HD-Zip I transcription factors HaHB1 and AtHB13 confer cold tolerance via the induction of pathogenesis-related and glucanase proteins. Plant J. 2012;69:141–53. Jiao P, Jiang Z, Miao M, Wei X, Wang C, Liu S, et al. Zmhdz9, an HD-Zip transcription factor, promotes drought stress resistance in maize by modulating ABA and lignin accumulationHD-Zip. International Journal of Biological Macromolecules. 2024;258:128849. Zhang S, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer AH, et al. Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Mol Biol. 2012;80:571–85. Wang Y, Wang H, Yu C, Yan X, Chu J, Jiang B, et al. Comprehensive bioinformation analysis of homeodomain-leucine zipper gene family and expression pattern of HD-Zip I under abiotic stress in Salix suchowensis. BMC Genomics. 2024;25:182. Wei M, Liu A, Zhang Y, Zhou Y, Li D, Dossa K, et al. Genome-wide characterization and expression analysis of the HD-Zip gene family in response to drought and salinity stresses in sesame. BMC Genomics. 2019;20:748. Li Y, Xiong H, Cuo D, Wu X, Duan R. Genome-wide characterization and expression profiling of the relation of the HD-Zip gene family to abiotic stress in barley (Hordeum vulgare L.). Plant Physiology and Biochemistry. 2019;141:250–8. Perotti MF, Ribone PA, Cabello JV, Ariel FD, Chan RL. AtHB23 participates in the gene regulatory network controlling root branching, and reveals differences between secondary and tertiary roots. The Plant Journal. 2019;100:1224–36. C S, A M, G S, T W, M O, T A, et al. Shade avoidance responses are mediated by the ATHB-2 HD-zip protein, a negative regulator of gene expression. Development (Cambridge, England). 1999;126. Miao X, Zhu W, Jin Q, Song Z, Li L. ZmHOX32 is related to photosynthesis and likely functions in plant architecture of maize. Front Plant Sci. 2023;14. Yang Y-Y, Shan W, Kuang J-F, Chen J-Y, Lu W-J. Four HD-ZIPs are involved in banana fruit ripening by activating the transcription of ethylene biosynthetic and cell wall-modifying genes. Plant Cell Rep. 2020;39:351–62. Lin Z, Hong Y, Yin M, Li C, Zhang K, Grierson D. A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening. The Plant Journal. 2008;55:301–10. Zhang J, Wu J, Guo M, Aslam M, Wang Q, Ma H, et al. Genome-wide characterization and expression profiling of Eucalyptus grandis HD-Zip gene family in response to salt and temperature stress. BMC Plant Biology. 2020;20:451. Ma Y-J, Li P-T, Sun L-M, Zhou H, Zeng R-F, Ai X-Y, et al. HD-ZIP I Transcription Factor (PtHB13) Negatively Regulates Citrus Flowering through Binding to FLOWERING LOCUS C Promoter. Plants (Basel). 2020;9:114. Additional Declarations No competing interests reported. Supplementary Files Supplement.zip Cite Share Download PDF Status: Published Journal Publication published 05 Jun, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 07 May, 2025 Reviews received at journal 27 Apr, 2025 Reviewers agreed at journal 19 Apr, 2025 Reviewers agreed at journal 19 Apr, 2025 Reviewers agreed at journal 18 Apr, 2025 Reviews received at journal 16 Apr, 2025 Reviewers agreed at journal 07 Apr, 2025 Reviewers agreed at journal 06 Apr, 2025 Reviewers invited by journal 04 Apr, 2025 Editor assigned by journal 01 Apr, 2025 Editor invited by journal 31 Mar, 2025 Submission checks completed at journal 28 Mar, 2025 First submitted to journal 28 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6320983","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445266079,"identity":"30ad8478-50b5-43ba-8f93-04031e3f98db","order_by":0,"name":"Xiaoqin Wang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqin","middleName":"","lastName":"Wang","suffix":""},{"id":445266080,"identity":"a4065921-87bb-47dd-ab0d-7fc02c340eb9","order_by":1,"name":"Xiaoya Zhou","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoya","middleName":"","lastName":"Zhou","suffix":""},{"id":445266081,"identity":"32c7af37-f99c-420e-bd3a-6013435be2ff","order_by":2,"name":"Kunhao Xie","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kunhao","middleName":"","lastName":"Xie","suffix":""},{"id":445266082,"identity":"e7f58c01-2d26-4409-9644-be6b5e40ecf4","order_by":3,"name":"Xiaojie Feng","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaojie","middleName":"","lastName":"Feng","suffix":""},{"id":445266083,"identity":"a2b74ff8-90ab-4e99-8f02-69c0b7393832","order_by":4,"name":"Lu Liu","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Liu","suffix":""},{"id":445266084,"identity":"16a93434-b6a5-4f0f-b50d-1b3b6cee5190","order_by":5,"name":"Lihong Gao","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lihong","middleName":"","lastName":"Gao","suffix":""},{"id":445266085,"identity":"9a2a8893-d75a-471d-9cbd-78e65ca99057","order_by":6,"name":"Mintao Sun","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mintao","middleName":"","lastName":"Sun","suffix":""},{"id":445266086,"identity":"9933f78b-44f6-41ea-98fd-5abb9bdf49cd","order_by":7,"name":"Yansu Li","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yansu","middleName":"","lastName":"Li","suffix":""},{"id":445266087,"identity":"24996979-34c2-4b3e-8d27-dbf7ccf7b50b","order_by":8,"name":"Chaoxing He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACgwMMbCCah4GB+QDJWtgSSNMC0mVApJbjzc8e/Kg4LGPOv+bjhx81dQz8sxvwa7E/c8zcsOdMGo/ljLebJXuOHWaQuHOAgC03ctgkeNtseAxunN3GwMN2gMFAIoGAlvtv2CT/tkkAtZx5xvjnXx0RWm7wsEmDbTnfw8bM28ZMhJYzaWbSMkC/GNxgM5aW7TvMI3GDkJbjh59Jvqk4bG9w/vDDj2++1cnxzyCgBQGg7uEhVj0Q8B8gQfEoGAWjYBSMKAAAtM1D4i0jXt0AAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Chaoxing","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2025-03-27 13:23:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6320983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6320983/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-06798-y","type":"published","date":"2025-06-05T15:57:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81379746,"identity":"a48354a7-b216-4c9a-b788-a1134652150f","added_by":"auto","created_at":"2025-04-25 12:26:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1635814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiple Sequence Alignment and Conserved Motif Analysis of CaHD-Zip Proteins.\u003c/strong\u003e Multiple sequence alignment of CaHD-Zip proteins highlights conserved motifs. Key functional regions, including the HD (homeodomain) and LZ (leucine zipper) domains, are boxed to emphasize their evolutionary conservation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/d738be461bf97b282eb8c295.png"},{"id":81379749,"identity":"65c89097-6c34-4428-b983-a12968b69d40","added_by":"auto","created_at":"2025-04-25 12:26:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6135628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of HD-Zip proteins from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCapsicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe tree was constructed using the maximum likelihood(ML) method in IQ-TREE 2 and evaluated with 1000 bootstrap replicates. Blue circles indicate \u003cem\u003eCapsicum proteins\u003c/em\u003e; red pentagrams denote \u003cem\u003eA. thaliana\u003c/em\u003eproteins. Different branch colors represent distinct HD-Zip subfamilies.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/72f2295673cfee6f4d9c7d0a.png"},{"id":81379750,"identity":"8d36b28b-0db8-481d-b739-a2897c43b2ab","added_by":"auto","created_at":"2025-04-25 12:26:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5158373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic synteny analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaHD-Zip\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes\u003c/strong\u003e. (a) Intraspecific synteny analysis within the \u003cem\u003eCapsicum annuum\u003c/em\u003e genome. Red lines indicate syntenic relationships among \u003cem\u003eCaHD-Zip\u003c/em\u003e genes, while blue trapezoids and red curves represent gene density distribution along the chromosomes. (b) Comparative genomic synteny analysis between \u003cem\u003eCapsicum annuum\u003c/em\u003e and five other plant species. \u003cem\u003eC. annuum\u003c/em\u003e represents pepper, \u003cem\u003eA. thaliana\u003c/em\u003e represents Arabidopsis, while \u003cem\u003eS. lycopersicum\u003c/em\u003e (tomato), \u003cem\u003eO. sativa\u003c/em\u003e (rice), \u003cem\u003eS. tuberosum\u003c/em\u003e(potato), and \u003cem\u003eZ. mays\u003c/em\u003e (maize) serve as reference species in the synteny analysis. Gray lines connect all syntenic gene pairs, while red lines specifically highlight syntenic relationships involving \u003cem\u003eHD-Zip\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/90f769cf4b522527cb037626.png"},{"id":81380094,"identity":"ec40354f-70e2-44eb-8ac7-1ded9b342046","added_by":"auto","created_at":"2025-04-25 12:34:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2200441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic Relationships, Conserved Motifs, Gene Structures, and Domain Architectures of Capsicum \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHD-Zip\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Genes.\u003c/strong\u003e (a) Phylogenetic tree of \u003cem\u003eHD-Zip\u003c/em\u003e genes constructed using the maximum likelihood (ML) method. (b) Motif composition of HD-Zip proteins, with differently colored blocks representing distinct conserved motifs. (c) Domain distribution of HD-Zip proteins, where different colors denote individual domain types. (d) Gene structure analysis, with yellow bars representing coding sequences (CDS) and green bars indicating untranslated regions (UTRs).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/1e1786bacae7f647ad924a1e.png"},{"id":81379753,"identity":"da127692-7741-4147-ae23-6721b8198f22","added_by":"auto","created_at":"2025-04-25 12:26:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2622097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrediction and Functional Categorization of Cis-Regulatory Elements in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaHD-Zip\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eGene Family.\u003c/strong\u003e (a) Distribution of cis-regulatory elements identified within the 2000 bp upstream promoter regions of \u003cem\u003eCaHD-Zip\u003c/em\u003egenes. (b) Venn diagram showing the overlap of hormone-responsive elements. (c) Venn diagram of growth- and development-related elements. (d) Venn diagram of stress-responsive elements.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/d10e7446a19d4ca269e8a6cd.png"},{"id":81379757,"identity":"63373d73-80c3-473d-add8-d50c4358ffa8","added_by":"auto","created_at":"2025-04-25 12:26:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1879245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative Expression Analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaHD-Zip\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Genes in Different \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCapsicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eTissues.\u003c/strong\u003e This figure illustrates the transcripts per million (TPM) based expression profiles of \u003cem\u003eCaHD-Zip\u003c/em\u003e genes across 11 distinct tissues in \u003cem\u003eCapsicum annuum\u003c/em\u003e. The analyzed tissues include seeds, embryos, stem, root, leaf, floral buds, flowers, anthers, ovary, placenta, and three developmental stages of early fruit (fruit1), breaker fruit (fruit2), and mature fruit (fruit3). A heatmap is used to visualize expression variation, where red indicates high expression, blue indicates low expression, and the color gradient reflects relative abundance across tissues.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/eebef50e5cf8854a98eb2c90.png"},{"id":81379758,"identity":"27c8dee4-0598-4bb4-b372-2d6a4fb046d7","added_by":"auto","created_at":"2025-04-25 12:26:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2431281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatiotemporal Expression Analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaHD-Zip\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eGenes in Response to Hormonal Treatments. \u003c/strong\u003e(a) Heatmap showing TPM-based expression profiles of \u003cem\u003eCaHD-Zip\u003c/em\u003egenes in response to ABA, MeJA, ET, and SA treatments across five timepoints (0, 3, 6, 12, and 24 h). The color scale represents relative expression levels, with red indicating high expression and blue indicating low expression. (b) Venn diagram analysis of differentially expressed \u003cem\u003eCaHD-Zip\u003c/em\u003e genes (DEGs; |log2FC| ≥1, adjusted p \u0026lt;0.05) under ABA, MeJA, ET, and SA treatments.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/a5425c79963cbc1ce1f98aa7.png"},{"id":81379751,"identity":"eb1e5c9f-d517-43d2-846b-ce086bd3417e","added_by":"auto","created_at":"2025-04-25 12:26:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3408180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression Profiles and Differentially Expressed Genes (DEGs) Analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaHD-Zip\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Genes Under Different Abiotic Stress.\u003c/strong\u003eConditions (a–d) TPM-based expression profiles of \u003cem\u003eCaHD-Zip\u003c/em\u003e genes under cold (a), heat (b), drought (c), and salt (d) stress at 0, 3, 6, 12, 24, and 72 hours. The color gradient represents relative expression levels, with red indicating high expression and blue indicating low expression. (e -h) Venn diagrams of DEGs under different abiotic stress conditions, illustrating the overlap of differentially expressed genes at various time points under cold (e), heat (f), drought (g), and salt (h) stress. (i) A Venn diagram depicting shared DEGs across all four abiotic stress conditions, highlighting the core \u003cem\u003eCaHD-Zip\u003c/em\u003egenes responsive to cold, heat, drought, and salt stress.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/71a093806712d57b11708217.png"},{"id":81379772,"identity":"b5129e16-db55-4e30-b0da-1c9ed3084054","added_by":"auto","created_at":"2025-04-25 12:26:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1181132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStress-responsive expression validation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCaHD-Zip\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes.\u003c/strong\u003e qRT-PCR analysis of eight \u003cem\u003eCaHD-Zip\u003c/em\u003egenes under abiotic stresses (cold, heat, drought, salinity) and hormonal treatments (ABA, MeJA). Relative expression was calculated using the 2⁻ΔΔCt method and expressed as fold-change (2⁻ΔΔCt values) relative to untreated controls. Error bars: ±SD (n=3). *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/6cc356116ad08016b8544de3.png"},{"id":84242842,"identity":"a5fd2cc6-0e9e-447a-971e-fedb98814df6","added_by":"auto","created_at":"2025-06-09 16:12:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26619005,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/10b598b8-4e79-4748-8342-4f563f5e49c5.pdf"},{"id":81380896,"identity":"b769aed2-279e-489c-a72d-93647de4eb32","added_by":"auto","created_at":"2025-04-25 12:42:12","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9071774,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement.zip","url":"https://assets-eu.researchsquare.com/files/rs-6320983/v1/5ffe4975808f61c7813f38a3.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide characterization and their roles in abiotic stress responses of HD-Zip transcription factors in pepper (Capsicum annuum)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs a globally significant economic crop, pepper serves as both a staple vegetable and a vital spice[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to 2023 FAO statistics, global fresh pepper production increased significantly from 36.29 to 40.20\u0026nbsp;million metric tons within a single year, with China notably contributing 40% of total production and retaining its status as the primary producer[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Beyond its role as a fresh vegetable, pepper derives subst antial economic value from its distinctive secondary metabolites, particularly capsaicinoids and carotenoids.These compounds demonstrate unique bioactive properties that enable diverse commercial applications: as natural colorants and flavor enhancers in food processing; analgesic and anti-inflammatory agents in pharmaceuticals; and specialized components in cosmetic formulations and industrial coatings[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Global climate change has exacerbated abiotic stresses, including extreme temperatures, soil salinization, and drought, which pose serious threats to the yield and quality of \u003cem\u003eCapsicum annuum\u003c/em\u003e, leading to substantial economic losses[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These compounds demonstrate unique bioactive properties that enable diverse commercial applications: as natural colorants and flavor enhancers in food processing; analgesic and anti-inflammatory agents in pharmaceuticals; and specialized components in cosmetic formulations and industrial coatings. Therefore, elucidating the molecular regulatory networks governing pepper\u0026rsquo;s response to abiotic stress is essential not only for breeding stress-tolerant cultivars but also for ensuring the sustainable development of the global pepper industry.\u003c/p\u003e \u003cp\u003eWithin stress-response regulatory networks, transcription factors (TFs) act as central signaling hubs that decode environmental and developmental signals through cis-element recognition and target gene regulation[\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The HD-Zip (Homeodomain-Leucine Zipper) family, a plant-exclusive TF class[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], contains two signature domains: a DNA-binding homeodomain (HD) and a dimerization-mediating leucine zipper (LZ) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Phylogenetic and structural analyses classify the HD-Zip family into four evolutionarily distinct subfamilies (I-IV) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Subfamily I retains the core HD-LZ architecture, while Subfamily II acquires a conserved N-terminal sequence coupled with a CPSCE motif[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Subfamilies III and IV demonstrate enhanced structural sophistication, each possessing a START (steroidogenic acute regulatory protein-related lipid transfer) domain and its associated SAD domain downstream of LZ [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].A defining feature of Subfamily III is its C-terminal MEKHLA motif[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These domain architectures directly determine subfamily-specific functional specialization in developmental regulation and stress adaptation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The HD-Zip I subfamily orchestrates three core biological functions: developmental programming, abiotic stress adaptation, and hormonal cross-regulation through signal integration. Under osmotic stress conditions, such as drought and salinity, HD-Zip I members activate ABA-dependent acclimation mechanisms, enhancing cellular resilience[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In Arabidopsis, \u003cem\u003eAtHB7\u003c/em\u003e and \u003cem\u003eAtHB12\u003c/em\u003e establish ABA homeostasis via self-regulatory feedback loops, a conserved mechanism also demonstrated in Gossypium species[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These regulators fine-tune stomatal patterning (e.g., \u003cem\u003eAtHB7\u003c/em\u003e-mediated density reduction) and osmolyte biosynthesis (e.g., \u003cem\u003eZmhdz10\u003c/em\u003e driven proline accumulation), synergistically optimizing hydraulic efficiency under combined drought and cold stress[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The HD-Zip II subfamily is a principal regulator of photomorphogenic responses and organogenetic patterning, particularly influencing leaf morphogenesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For example, \u003cem\u003eHAT3\u003c/em\u003e and \u003cem\u003eATHB4\u003c/em\u003e regulate leaf polarity through a bidirectional inhibitory circuit[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Meanwhile, \u003cem\u003eHAT1\u003c/em\u003e modulates organogenesis through interactions with TPL/TPR transcriptional co-repressors[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. HD-Zip III transcription factors coordinate three developmental axes: vascular tissue patterning, meristem maintenance, and lateral organ positioning[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The HD-Zip IV subfamily is specifically expressed in epidermal and subepidermal cells, where it contributes to trichome formation, anthocyanin accumulation, and cuticle biosynthesis, thereby governing epidermal development and environmental adaptation[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile \u003cem\u003eHD-Zip\u003c/em\u003e genes have been extensively characterized in model plants (\u003cem\u003eArabidopsis, Oryza sativa\u003c/em\u003e) and major crops (\u003cem\u003eSolanum lycopersicum, Malus domestica, Prunus persica, Passiflora edulis\u003c/em\u003e)[\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], a systematic genomic investigation of this gene family has not yet been conducted in \u003cem\u003eCapsicum annuum\u003c/em\u003e, the most extensively cultivated Solanaceae crop worldwide.To address this gap, we performed a genome-wide analysis to systematically identify and functionally characterize the \u003cem\u003eHD-Zip\u003c/em\u003e gene family in \u003cem\u003eCapsicum annuum\u003c/em\u003e, focusing on the following objectives: 1) Identification of 40 \u003cem\u003eCaHD-Zip\u003c/em\u003e genes and establishment of a cross-species phylogenetic framework; 2) Investigation of their evolutionary trajectories through synteny analysis and gene structure characterization; 3) Comprehensive transcriptional profiling across 13 tissues and organs, as well as under four hormone treatments and four abiotic stress conditions.This study not only sheds light on the phylogenetic characteristics and expression regulatory networks of \u003cem\u003eCaHD-Zip\u003c/em\u003e genes but also provides key candidate genes and a theoretical foundation for elucidating the molecular mechanisms underlying HD-Zip mediated development and stress adaptation in pepper. These findings have significant scientific implications for accelerating the molecular breeding of stress-resilient \u003cem\u003eCapsicum annuum\u003c/em\u003e cultivars.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Identification, Subcellular Localization Prediction, and Physicochemical Characterization of \u003cem\u003eCaHD-Zip\u003c/em\u003e Family Genes.\u003c/h2\u003e \u003cp\u003eThe HD-Zip transcription factor family in pepper was systematically identified using the latest high-quality telomere-to-telomere (T2T) genome assembly (\u003cem\u003eZunla-1_v3.0\u003c/em\u003e) obtained from PepperBase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformaticslab.cn/PepperBase/\u003c/span\u003e\u003cspan address=\"http://www.bioinformaticslab.cn/PepperBase/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. HMM profiles corresponding to the HD-Zip domain (PF00046 and PF02183) were retrieved from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and employed in a genome-wide search using HMMER v3.3.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hmmer.janelia.org/\u003c/span\u003e\u003cspan address=\"http://hmmer.janelia.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], resulting in the preliminary identification of candidate \u003cem\u003eHD-Zip\u003c/em\u003e genes. These candidates were subsequently validated for conserved domains using both the NCBI Conserved Domain Database (CDD) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cdd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and SMART database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], respectively. The validated \u003cem\u003eHD-Zip\u003c/em\u003e genes were systematically named based on their chromosomal positions and sequence conservation, then classified into subfamilies through phylogenetic analysis of their protein sequences. Subcellular localization predictions were performed using DeepLoc-2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/DeepLoc-2.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/DeepLoc-2.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and WoLF PSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genscript.com/wolf-psort.html\u003c/span\u003e\u003cspan address=\"https://www.genscript.com/wolf-psort.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Additionally, while key physicochemical properties including molecular weight, isoelectric point, and were calculated using the ExPASy ProtParam tool. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Phylogenetic Analysis of \u003cem\u003eCaHD-Zip\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis was performed using HD-Zip protein sequences from pepper and the reference dataset comprising Arabidopsis thaliana sequences obtained from PlantTFDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://planttfdb.gao-lab.org/index.php\u003c/span\u003e\u003cspan address=\"http://planttfdb.gao-lab.org/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple sequence alignment was conducted with the MUSCLE algorithm implemented in MEGA7 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megasoftware.net/\u003c/span\u003e\u003cspan address=\"https://www.megasoftware.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]followed by phylogenetic tree construction through the maximum likelihood method(ML) with 1,000 bootstrap replicates to evaluate node support. The phylogenetic tree was visualized using TVBOT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/tvbot.html\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/tvbot.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), while Jalview (V2.11.2.4) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.jalview.org/\u003c/span\u003e\u003cspan address=\"http://www.jalview.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized for conserved domain annotation and sequence refinement to ensure analytical accuracy[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Gene Structure, Conserved Motif, and Cis-Regulatory Element Analysis\u003c/h2\u003e \u003cp\u003eGene structure analysis was performed using the General Feature Format (GFF3) annotation files through TBtools' Visualize Gene Structure Basic module. Conserved protein motifs were identified using the MEME Suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with parameter constraints allowing detection of up to 10 motifs ranging from 5 to 200 amino acids in length. Promoter regions, defined as the 2,000 bp upstream sequences from translation initiation sites, were extracted using TBtools. These promoter sequences were subsequently analyzed in PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], for cis-regulatory element annotation, followed by element classification and visualization using TBtools.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Chromosomal Mapping, Synteny Analysis, and Gene Duplication Events\u003c/h2\u003e \u003cp\u003eThe chromosomal distribution of \u003cem\u003eHD-Zip\u003c/em\u003e genes in \u003cem\u003epepper\u003c/em\u003e was determined using TBtools\u0026rsquo; Gene Location Visualization module, based on GFF3 genome annotation. Intragenomic synteny patterns were visualized through TBtools\u0026rsquo; Advanced Circos module, revealing chromosomal arrangement of paralogous genes. Cross-species collinearity analysis with six representative species (\u003cem\u003eArabidopsis thaliana, Cucumis sativus, Solanum lycopersicum, Zea mays, Solanum tuberosum\u003c/em\u003e, and \u003cem\u003eOryza sativa\u003c/em\u003e) was conducted using MCScanX. Gene duplication events were characterized by calculating nonsynonymous (Ka) and synonymous (Ks) substitution rates with TBtools' Simple Ka/Ks Calculator module. Selection pressure was determined based on Ka/Ks ratios: \u0026gt;1 indicated positive selection, =\u0026thinsp;1 suggested neutral evolution, or \u0026lt;\u0026thinsp;1 signified purifying selection[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Divergence times were estimated using the formula T\u0026thinsp;=\u0026thinsp;Ks/(2\u0026times;6.1\u0026times;10⁻⁹)\u0026times;10⁻⁶, where 6.1\u0026times;10⁻⁹ represents the average substitution rate per site per year, and T corresponds to the divergence time in million years (Mya)[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RNA-Seq Data Analysis\u003c/h2\u003e \u003cp\u003eRNA-seq datasets were obtained from the NCBI Sequence Read Archive (SRA) with accession numbers detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, comprising three experimental groups: 1) 11 organ types of pepper (seeds, embryos, stem, root, leaf, floral buds, flowers, anthers, ovary, placenta, early fruit, breaker fruit and mature fruit ); 2) four abiotic stresses (cold, heat, drought, and salt stress) sampled at 0, 3, 6, 12, 24 h; 3) four phytohormone treatments (ABA, MeJA, ET, and SA) measured at 0, 3, 6, 12, 24, 72 h. TPM-normalized \u003cem\u003eCaHD-Zip\u003c/em\u003e expression profiles were hierarchically clustered using Euclidean distance metrics in TBtools, visualized as heatmaps[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], with heatmaps generated to visualize clustering patterns. Differentially expressed genes (DEGs) were identified using the DESeq2 function in TBtools, with a significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Plant Materials, Growth Conditions, and Stress Treatments\u003c/h2\u003e \u003cp\u003eSterilized seeds of the pepper variety \u0026ldquo;Bola No. 2\u0026rdquo;, purchased from Hunan Xingshu Seed Industry, were sown in 50 cell seedling trays. After two weeks of seedling growth, plants were transplanted into nutrient-rich pots (7 cm diameter \u0026times; 6.5 cm height) and grown under controlled environmental conditions in a growth chamber (24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 16 h light/8 h dark photoperiod) until six true-leaf stage. Abiotic stress treatments included: Cold stress (10\u0026deg;C), heat stress (40\u0026deg;C), salt stress (400 mM NaCl), and drought stress (20% PEG6000 solution).Hormonal treatments: Leaves were sprayed with 100 \u0026micro;M MeJA or 100 \u0026micro;M ABA, while the control group received distilled water. The third fully expanded leaves were harvested after 12 h (hormones) or 24 h (abiotic stresses) of treatment initiation (n\u0026thinsp;=\u0026thinsp;3 biological replicates). Samples were immediately snap-frozen in liquid N₂ and stored at -80\u0026deg;C until RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from pepper samples using the RNA Prep Pure Kit (Accurate Biology, China) according to the manufacturer\u0026rsquo;s instructions. RNA purity (A260/A280 ratio) and concentration were assessed using a spectrophotometer to ensure that sample quality met the requirements for downstream analyses. First-strand complementary DNA (cDNA) was synthesized from 1 \u0026micro;g of total RNA using the Fast Quant RT Kit (Accurate Biology, China), which contains a genomic DNA elimination step, according to the manufacturer\u0026rsquo;s instructions. The reaction system contained reverse transcriptase, random primers, and reaction buffer to ensure effective genomic DNA elimination and the synthesis of high-quality cDNA. qRT-PCR reactions were prepared using Super Real Pre Mix Plus with SYBR Green dye, incorporating gene-specific primers synthesized by Sangon Biotech (Shanghai, China). qRT-PCR was performed on a Gentier 96/96E real-time PCR system (Tianlong Technology, Xi\u0026rsquo;an, China) under the following cycling conditions: initial denaturation at 95\u0026deg;C for 2 min, followed by 40 cycles of 95\u0026deg;C for 15 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s. Melting curve analysis was performed to validate amplicon specificity. All samples were assessed in triplicate, and gene expression was normalized against the reference gene \u003cem\u003eCaActin\u003c/em\u003e. The sequences of all primers used are listed in Table S2. Relative gene expression was quantified using the 2⁻ΔΔCt method [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], with results presented as fold changes in the treatment groups relative to the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Data Analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were compiled using Microsoft Excel 2010, subjected to statistical analysis using SPSS, and visualized with Origin software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Physicochemical Characteristics and Subcellular Localization of the \u003cem\u003eCaHD-Zip\u003c/em\u003e Gene Family\u003c/h2\u003e \u003cp\u003eSystematic genome-wide screening identified 40 \u003cem\u003eHD-Zip\u003c/em\u003e genes (designated \u003cem\u003eCaHD-Zip1\u003c/em\u003e to \u003cem\u003eCaHD-Zip40\u003c/em\u003e) in \u003cem\u003eCapsicum annuum\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The deduced proteins spanned 211\u0026ndash;842 amino acids, with molecular weights ranging from 24.2 to 93.6 kDa. Instability index analysis (37.29\u0026ndash;87.67) revealed structural instability in most CaHD-Zip proteins, with \u003cem\u003eCaHD-Zip01, 06, 13\u003c/em\u003e, and \u003cem\u003e17\u003c/em\u003e exhibiting the highest values (\u0026gt;\u0026thinsp;70). Subcellular localization predictions indicated nuclear targeting for all CaHD-Zip proteins, while \u003cem\u003eCaHD-Zip03, 09, 14\u003c/em\u003e, and \u003cem\u003e38\u003c/em\u003e were also predicted to localize to the cytoplasm. These dual-localized proteins exhibited lower instability indices, potentially reflecting enhanced structural stability conducive to functional plasticity. All CaHD-Zip proteins exhibited negative Grand Average of Hydropathicity (GRAVY) values (-1.067 to -0.11), indicating a hydrophilic molecular profile, consistent with the aqueous solubility and nuclear localization typical of transcription factors.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProperties and Localization of Pepper HD-Zip Proteins\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of Amino Acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecular Weight\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTheoretical pI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInstability Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAliphatic Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGRAVY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLocalizations\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC01G0000550.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24442.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e72.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e67.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip02\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC01G0007630.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31939.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e59.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e62.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.887\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip03\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC01G0011000.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93610.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e57.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e77.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCytoplasm/Nucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip04\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC01G0015850.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91048.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC01G0025820.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42420.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.675\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip06\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC01G0036190.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34321.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e71.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip07\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC02G0005020.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27002.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e74.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.687\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip08\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC02G0008000.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31954.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip09\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC02G0011430.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91725.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCytoplasm/Nucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC02G0020890.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e729\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80189.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e78.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC02G0028560.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30280.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e69.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.688\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip12\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC02G0033860.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35324.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e59.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC02G0035010.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32422.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e74.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.885\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip14\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC03G0002650.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91788.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCytoplasm/Nucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC03G0002950.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e772\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86442.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e73.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC03G0011730.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e52.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e59.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.899\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip17\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC03G0016630.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79488.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e52.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e83.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip18\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC03G0019410.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25791.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e66.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-1.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC03G0023390.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88475.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e77.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC04G0008080.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28332.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e80.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip21\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC04G0010300.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33693.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-1.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip22\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC04G0024840.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e728\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80148.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e84.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.324\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC05G0017140.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e323\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36807.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e52.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e68.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip24\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC06G0009310.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e734\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80878.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e52.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e80.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip25\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC06G0020410.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e313\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35570.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip26\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC06G0023950.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25688.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e65.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.941\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip27\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC07G0017730.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24259.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e72.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip28\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC08G0005190.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92409.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e53.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e88.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip29\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC08G0010520.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27633.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e70.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip30\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC09G0007190.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85895.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e77.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.369\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip31\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC09G0023620.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28426.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e70.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC10G0006050.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e731\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80565.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e84.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip33\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC10G0022120.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e68.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.725\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip34\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC11G0001840.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92165.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e85.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip35\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC11G0017670.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34063.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e66.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC11G0018300.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35611.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e58.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.951\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip37\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC11G0027120.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24081.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e76.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-1.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip38\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC12G0012540.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92404.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCytoplasm/Nucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip39\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC12G0026280.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39565.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e56.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.888\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCaHD-Zip40\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZLC12G0029280.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e721\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81691.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Multiple Sequence Alignment and Functional Site Analysis of CaHD-Zip Proteins in Pepper\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMultiple sequence alignment revealed two highly conserved functional domains in CaHD-Zip proteins: a DNA-binding homeodomain (HD, PF00046) and a leucine zipper motif (LZ, PF02183) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The homeodomain contains a conserved core motif, \u0026lsquo;WFQNR\u0026rsquo;, embedded within the third α-helix (recognition helix), which is highly conserved across family members[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Tryptophan (W) stabilizes the protein\u0026ndash;DNA complex by interacting with DNA base stacking via hydrophobic interactions[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Arginine (R), through its positively charged side chain, forms electrostatic interactions with the DNA phosphate backbone, further reinforcing DNA binding. The LZ domain adopts a heptad repeat pattern \u0026ldquo;L-X-L-X-L\u0026rdquo;, where \"L\" denotes conserved leucine residues and \"X\" represents variable amino acids. This domain architecture is widely conserved across species, underscoring its evolutionary importance in gene regulatory processes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Classification and Phylogenetic Relationships of \u003cem\u003eCaHD-Zip\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003eTo resolve evolutionary relationships between \u003cem\u003eCapsicum annuum\u003c/em\u003e and Arabidopsis thaliana \u003cem\u003eHD-Zip\u003c/em\u003e genes, we reconstructed a maximum likelihood phylogeny (Fig.\u0026nbsp;2).The analysis incorporated 40 \u003cem\u003eCaHD-Zip\u003c/em\u003e and 58 \u003cem\u003eAtHD-Zip\u003c/em\u003e genes. Based on tree topology and prior classifications, all 98 genes were classified into four subfamilies (I-IV). The distribution of \u003cem\u003eCaHD-Zip\u003c/em\u003e genes across subfamilies showed significant disparity: subfamily I contained 14 genes, IV had 11, II comprised 10, while III was the smallest group with 5 members. This expansion pattern suggests lineage-specific diversification of subfamilies I and IV in pepper, potentially driven by functional \u003cb\u003eFigure 2. Phylogenetic tree of HD-Zip proteins from\u003c/b\u003e \u003cb\u003eCapsicum\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eArabidopsis thaliana\u003c/b\u003e. The tree was constructed using the maximum likelihood (ML) method in IQ-TREE 2 and evaluated with 1000 bootstrap replicates. Blue circles indicate \u003cem\u003eCapsicum proteins\u003c/em\u003e; red pentagrams denote \u003cem\u003eA. thaliana\u003c/em\u003e proteins. Different branch colors represent distinct HD-Zip subfamilies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eadaptation. HD-Zip III and IV genes clustered on the same branch, suggesting recent divergence from a shared ancestor and subsequent functional differentiation. Conserved orthologs exhibited one-to-one correspondence in subfamilies I and II: \u003cem\u003eCaHD-Zip31/AtHB51\u003c/em\u003e, \u003cem\u003eCaHD-Zip36/AtHB13\u003c/em\u003e, and \u003cem\u003eCaHD-Zip13/AtHB1\u003c/em\u003e (Subfamily I); \u003cem\u003eCaHD-Zip37/AtHB18, CaHD-Zip02/AtHB2\u003c/em\u003e, and \u003cem\u003eCaHD-Zip06/HAT63\u003c/em\u003e (Subfamily II). In contrast, subfamily IV displayed one-to-many orthologous relationships\u0026mdash;\u003cem\u003eCaHD-Zip15\u003c/em\u003e corresponding to \u003cem\u003eGL2.1\u003c/em\u003e and \u003cem\u003eGL2.2\u003c/em\u003e, \u003cem\u003eCaHD-Zip03\u003c/em\u003e to \u003cem\u003eHDG4\u003c/em\u003e and \u003cem\u003eHDG5\u003c/em\u003e, \u003cem\u003eCaHD-Zip04\u003c/em\u003e to \u003cem\u003eAHDP.1\u003c/em\u003e and \u003cem\u003eAHDP.2\u003c/em\u003e\u0026mdash;indicating species-specific gene duplications driving functional diversification. Together, these phylogenetic findings demonstrate conserved subfamily architecture with lineage-specific expansions shaping functional innovation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Chromosomal Distribution, Collinearity, and Duplication Analysis of \u003cem\u003eCaHD-Zip\u003c/em\u003e Genes in Pepper\u003c/h2\u003e \u003cp\u003eChromosomal mapping of 40 \u003cem\u003eCaHD-Zip\u003c/em\u003e loci in \u003cem\u003eCapsicum annuum\u003c/em\u003e showed a non-uniform distribution across 12 chromosomes (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Chromosomes 1\u0026ndash;3 contained 6\u0026ndash;7 \u003cem\u003eCaHD-Zip\u003c/em\u003e genes, forming gene-dense regions, whereas chromosomes 5 and 7\u0026ndash;10 showed sparse distribution (1\u0026ndash;2 genes). Gene duplication analysis using MCScanX identified 15 segmentally duplicated \u003cem\u003eCaHD-Zip\u003c/em\u003e gene pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). All duplicated pairs exhibited Ka/Ks ratios\u0026thinsp;\u0026lt;\u0026thinsp;0.5 (Table S3), indicative of strong purifying selection. Divergence times ranged from 51.05 to 320.75 Mya, with the most recent duplication event (\u003cem\u003eCaHD-Zip18/26\u003c/em\u003e) occurring at ~\u0026thinsp;51.05 Mya.\u003c/p\u003e \u003cp\u003eComparative analysis revealed stronger synteny with dicots (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e: 77, \u003cem\u003eS. tuberosum\u003c/em\u003e: 75, \u003cem\u003eA. thaliana\u003c/em\u003e: 56) than with monocots (\u003cem\u003eOryza sativa\u003c/em\u003e: 20, \u003cem\u003eZea mays\u003c/em\u003e: 14) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb; Table S4-1), suggesting greater evolutionary conservation in dicotyledonous lineages. Most \u003cem\u003eCaHD-Zip\u003c/em\u003e genes showed asymmetric syntenic relationships, particularly \u003cem\u003eCaHD-Zip02\u003c/em\u003e with four orthologs (\u003cem\u003eATHB2/HAT1/HAT2/ATH4\u003c/em\u003e) across three dicots (Table S4-2). Seven \u003cem\u003eCaHD-Zip\u003c/em\u003e genes (\u003cem\u003eCaHD-Zip02/06/10/19/21/34/39\u003c/em\u003e) maintained pan-lineage synteny among all analyzed species, demonstrating ancient evolutionary conservation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Gene Architecture, Conserved Motifs, and Cis-Element Profiling of CaHD-Zip Family\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Gene Structure and Conserved Motif Analysis\u003c/h2\u003e \u003cp\u003eMEME analysis of 40 \u003cem\u003eCaHD-Zip\u003c/em\u003e genes identified 2\u0026thinsp;~\u0026thinsp;10 conserved motifs per gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; Fig. S2). Motifs 1 and 2 were universally conserved across all CaHD-Zip proteins, suggesting their role as core structural elements essential for gene family stability. In contrast, Motifs 3\u0026thinsp;~\u0026thinsp;10 displayed subfamily-specific distributions, being largely restricted to HD-Zip III and IV, which may reflect their roles in subfamily-specific functional diversification. Specifically, HD-Zip III members contained Motifs 1, 2, 4, 5, and 7, while HD-Zip IV genes carried additional Motifs 3, 6, 9, and 10, indicating that distinct motif combinations may regulate subfamily-specific functions.\u003c/p\u003e \u003cp\u003eExon-intron architecture analysis revealed 2 to 18 exons per gene. Subfamily III genes (e.g., \u003cem\u003eCaHD-Zip09/14/28/34/38\u003c/em\u003e) consistently harbored 18 exons, the maximum observed, while subfamily I members (e.g., \u003cem\u003eCaHD-Zip01/18/ 26\u003c/em\u003e) contained only 2 exons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed; Table S5). Intron numbers ranged from 1 to 17, and untranslated regions (UTRs) varied from 0 to 5 per gene. Notably, subfamily IV members \u003cem\u003eCaHD-Zip22\u003c/em\u003e and \u003cem\u003eCaHD-Zip32\u003c/em\u003e carried 5 UTRs, whereas ~\u0026thinsp;50% of genes lacked annotated UTRs. These structural variations collectively highlight the evolutionary plasticity and functional modularity of the \u003cem\u003eCaHD-Zip\u003c/em\u003e gene family in \u003cem\u003eCapsicum annuum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 Conserved Domain Architecture and Subfamily Specific Signatures\u003c/h2\u003e \u003cp\u003eDomain architecture analysis identified six conserved domains within CaHD-Zip proteins: Homeodomain (HD), START (Steroidogenic Acute Regulatory Protein-related lipid transfer), HALZ (Homeobox-associated leucine zipper), MEKHLA (Membrane occupation and recognition nexus), bZIP (Basic leucine zipper), and HD-Zip_N, exhibiting subfamily-specific combinations. Subfamily I retained a minimal structure composed of HD and HALZ, while Subfamily II incorporated HD-Zip_N in addition to HD and HALZ. Subfamily IV displayed a unique domain configuration including HD, START, HALZ, and MEKHLA. Notably, Subfamily III encompassed all six domains, with the concurrent presence of START, MEKHLA, and bZIP forming a characteristic functional triad that may underpin its regulatory specialization. The HD domain was universally conserved across all \u003cem\u003eCaHD-Zip\u003c/em\u003e genes, reaffirming its indispensable role in DNA binding. In contrast, the selective presence of START and MEKHLA among subfamilies suggests their involvement in functional divergence and subfamily-specific regulatory evolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.5.3 Promoter Cis-Elements and Their Functional Categorization in \u003cem\u003eCaHD-Zip\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003ePromoter analysis identified 899 cis-regulatory elements (CREs) across \u003cem\u003eCaHD-Zip\u003c/em\u003e loci, categorized into 14 functional modules (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea; Table S6-1/2). Photoresponsive elements dominated (49.61%, 446/899), with Box4 (14.68%) and G-Box (8.23%) co-enriched in \u003cem\u003eCaHD-Zip07/11/24\u003c/em\u003e (Table S6-1), suggesting photoadaptive regulatory roles. Among hormone-responsive elements, methyl jasmonate (MeJA)-associated CREs constituted 11.35% (102/899), with \u003cem\u003eCaHD-Zip15\u003c/em\u003e containing the highest count (12 elements). \u003cem\u003eCaHD-Zip38\u003c/em\u003e and \u003cem\u003eCaHD-Zip16\u003c/em\u003e exhibited combinatorial enrichment of ABRE (abscisic acid-responsive), CGTCA-motif (MeJA-responsive), and TCA-element (salicylic acid-responsive) elements, forming a putative cross-hormonal regulatory hub (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; Table S6-2). Stress-responsive elements comprised 19.20% of CREs, prominently enriched in \u003cem\u003eCaHD-Zip23\u003c/em\u003e and \u003cem\u003eCaHD-Zip16\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Both loci harbored MYB-binding sites (MBS), anaerobic response elements (ARE), and low-temperature-responsive elements (LTR), implicating a MYB-LTR-ARE regulatory axis in stress adaptation. Growth-regulatory CREs accounted for 54.46% of total elements, with \u003cem\u003eCaHD-Zip33\u003c/em\u003e uniquely containing all developmental motifs (e.g., CAT-box, Circadian, O2-site,GATA-motif) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). This architectural singularity positions \u003cem\u003eCaHD-Zip33\u003c/em\u003e as a developmental hub gene regulating core growth pathways in \u003cem\u003eCapsicum annuum\u003c/em\u003e. Collectively, the CRE landscape reveals light-dominant transcriptional regulation integrated with hormone-stress crosstalk, forming an adaptive framework for environmental responsiveness and developmental control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Expression Profiling of the \u003cem\u003eCaHD-Zip\u003c/em\u003e Gene Family in Pepper Tissues\u003c/h2\u003e \u003cp\u003eUsing NCBI transcriptome datasets, we analyzed expression profiles of 40 \u003cem\u003eCaHD-Zip\u003c/em\u003e genes across 11 \u003cem\u003eCapsicum annuum\u003c/em\u003e tissues encompassing floral organs, three stages of fruit development, and vegetative tissues such as root, stem, and leaf (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Transcripts per million (TPM)-based hierarchical clustering segregated \u003cem\u003eCaHD-Zip\u003c/em\u003e genes into four co-expression clusters (A-D). Cluster A genes (\u003cem\u003eHD-Zip21\u0026thinsp;~\u0026thinsp;HD-Zip11\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). were predominantly expressed in meristematic tissues including stem, root, embryos, seeds, and placenta, with especially high levels in the ovary (peak TPM\u0026thinsp;\u0026gt;\u0026thinsp;25.31) Cluster B genes (\u003cem\u003eHD-Zip33/18/29\u003c/em\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) showed distinct expression features during fruit development, particularly in the breaker and mature fruit stages. Cluster C genes (\u003cem\u003eHD-Zip26\u0026thinsp;~\u0026thinsp;HD-Zip30\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) displayed consistently low expression across all tissues (TPM\u0026thinsp;\u0026lt;\u0026thinsp;5 in 90% of cases), suggesting that these genes may be selectively expressed under specific developmental or stress-related conditions. Cluster D genes (\u003cem\u003eHD-Zip25\u0026thinsp;~\u0026thinsp;HD-Zip20\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) exhibited moderate to high expression levels in seeds and floral buds, implying potential roles in floral organ formation and seed development. Collectively, \u003cem\u003eCaHD-Zip\u003c/em\u003e genes exhibit stage- and tissue-partitioned expression patterns: Cluster A dominates meristem activity, Cluster B drives late fruit metabolism, Cluster C enables stress adaptation, and Cluster D maintains developmental homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Hormonal Response of the \u003cem\u003eCaHD-Zip\u003c/em\u003e Gene Family Under Abiotic Stress Conditions\u003c/h2\u003e \u003cp\u003eUsing NCBI transcriptome datasets (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), we analyzed \u003cem\u003eCaHD-Zip\u003c/em\u003e expression under four stress hormones: abscisic acid (ABA), methyl jasmonate (MeJA), ethylene (ET), and salicylic acid (SA) at 0,3,6,12,24 h post-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Transcripts per million (TPM) analysis revealed hormone-specific temporal regulation patterns.ABA and MeJA treatments primarily induced gene expression at later stages (12\u0026ndash;24 h), indicating involvement in stress adaptation. For instance, under 24 h ABA exposure, \u003cem\u003eCaHD-Zip29\u003c/em\u003e and \u003cem\u003eCaHD-Zip16\u003c/em\u003e were upregulated to 105.8 and 40.6 TPM, respectively (control: 3.2 and 8.1 TPM); MeJA upregulated \u003cem\u003eCaHD-Zip11\u003c/em\u003e to 62.3 TPM. In contrast, ET and SA triggered rapid responses at 3\u0026ndash;6 h. ET rapidly induced \u003cem\u003eCaHD-Zip11\u003c/em\u003e expression to 57.6 TPM (control: 22.6 TPM) at 3 h, while SA downregulated \u003cem\u003eCaHD-Zip21\u003c/em\u003e to 13.0 TPM at 6 h (control: 43.0 TPM).\u003c/p\u003e \u003cp\u003eVenn diagram analysis (Fig. S3) identified maximal DEGs at ABA 12 h (13 genes), MeJA 24 h (11), SA 6 h (12), and ET 6 h (10). Notably, \u003cem\u003eCaHD-Zip16\u003c/em\u003e and \u003cem\u003eCaHD-Zip18\u003c/em\u003e exhibited significant responsiveness to all four hormones across at least two timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), positioning them as central integrators of hormone signal crosstalk. These findings indicate a division of hormonal response phases: ABA/MeJA mediate delayed transcriptional activation, while ET/SA orchestrate early signaling events. \u003cem\u003eCaHD-Zip16\u003c/em\u003e and \u003cem\u003eCaHD-Zip18\u003c/em\u003e likely function as regulatory hubs bridging diverse hormonal pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Expression Profiling and Differential Expression Analysis of \u003cem\u003eCaHD-Zip\u003c/em\u003e Genes Under Abiotic Stresses\u003c/h2\u003e \u003cp\u003eUtilizing transcriptome datasets from NCBI, we analyzed TPM-based expression profiles of 40 \u003cem\u003eCaHD-Zip\u003c/em\u003e genes in \u003cem\u003eCapsicum annuum\u003c/em\u003e under four major abiotic stress conditions\u0026mdash;cold, heat, drought, and salinity across six timepoints (0, 3, 6, 12, 24, and 72 hours). The spatiotemporal dynamics revealed stress-specific regulatory patterns.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.8.1 Cold Stress Responsive Expression Patterns of \u003cem\u003eCaHD-Zip\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003eHierarchical clustering of TPM values identified three co-expression clusters (A-C) under cold stress (10\u0026deg;C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Cluster A (\u003cem\u003eCaHD-Zip31\u0026thinsp;~\u0026thinsp;CaHD-Zip15;\u003c/em\u003e) showed progressive suppression, exemplified by \u003cem\u003eCaHD-Zip25\u003c/em\u003e (20.58 to 0.74 TPM, 96.4% reduction) and \u003cem\u003eCaHD-Zip36\u003c/em\u003e (23.63 to 9.52 TPM, 59.7% decrease) from 0\u0026ndash;72 h. Cluster B (\u003cem\u003eCaHD-Zip26\u0026thinsp;~\u0026thinsp;CaHD-Zip19\u003c/em\u003e) peaked at 12 h, with \u003cem\u003eCaHD-Zip29\u003c/em\u003e showing 80.3-fold induction (0 to 281.22 TPM) followed by 35-fold retention at 72 h. Cluster C (\u003cem\u003eCaHD-Zip02\u0026thinsp;~\u0026thinsp;CaHD-Zip09\u003c/em\u003e) exhibited sustained activation, with \u003cem\u003eCaHD-Zip07\u003c/em\u003e (20.73-fold, 10.78 TPM) and \u003cem\u003eCaHD-Zip18\u003c/em\u003e (14.93-fold, 34.54 TPM) reaching maxima at 72 h. Venn analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ee) identified five DEGs (\u003cem\u003eCaHD-Zip36/29/07/18/25\u003c/em\u003e) showing consistent differential expression (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01) across all timepoints. The regulatory dynamics featured sustained activation (\u003cem\u003eCaHD-Zip07/18\u003c/em\u003e), progressive suppression (\u003cem\u003eCaHD-Zip25/36\u003c/em\u003e), and transient induction (\u003cem\u003eCaHD-Zip29\u003c/em\u003e), indicating phase-specific cold adaptation strategies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.8.2 Heat Stress Responsive Expression Patterns of \u003cem\u003eCaHD-Zip\u003c/em\u003e Genes\u003c/h2\u003e \u003cp\u003eHierarchical clustering of TPM-based transcriptomic profiles under heat stress (40\u0026deg;C) revealed four distinct co-expression clusters (A\u0026ndash;D) among the \u003cem\u003eCaHD-Zip\u003c/em\u003e gene family (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Cluster A (\u003cem\u003eCaHD-Zip34\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eCaHD-Zip06\u003c/em\u003e) exhibited transient induction at 12 h, with \u003cem\u003eCaHD-Zip29\u003c/em\u003e peaking at 36.28 TPM (10.4-fold vs. 0 h), followed by a decline to near-baseline levels at 72 h (8.76 TPM). Cluster B (\u003cem\u003eCaHD-Zip39\u0026thinsp;~\u0026thinsp;CaHD-Zip09\u003c/em\u003e) showed rapid early activation, exemplified by \u003cem\u003eCaHD-Zip18\u003c/em\u003e, which rose from 2.31 to 21.8 TPM at 3 h (8.43-fold) and peaked at 31.82 TPM by 6 h. \u003cem\u003eCaHD-Zip23\u003c/em\u003e and \u003cem\u003eCaHD-Zip16\u003c/em\u003e also displayed similar early induction patterns, indicating involvement in thermotolerance priming. Cluster C (\u003cem\u003eCaHD-Zip33\u0026thinsp;~\u0026thinsp;CaHD-Zip14\u003c/em\u003e) demonstrated significant early repression, as \u003cem\u003eCaHD-Zip25\u003c/em\u003e and \u003cem\u003eCaHD-Zip36\u003c/em\u003e were downregulated by 92.6% (20.5 to 1.51 TPM) and 56.2% (23.63 to 10.34 TPM) respectively, at 6 h, suggesting potential roles as negative regulators of heat response. Cluster D (\u003cem\u003eCaHD-Zip12\u0026thinsp;~\u0026thinsp;CaHD-Zip04\u003c/em\u003e) maintained stable expression (fold-change\u0026thinsp;\u0026lt;\u0026thinsp;2), indicating minimal involvement in heat-induced transcriptional reprogramming. Venn diagram analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ef) identified six core heat-responsive DEGs (\u003cem\u003eCaHD-Zip16/18/23/25/29/35\u003c/em\u003e) with significant differential expression across all treatment stages (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Among these, \u003cem\u003eCaHD-Zip16/18/23/29/35\u003c/em\u003e were persistently upregulated, while \u003cem\u003eCaHD-Zip25\u003c/em\u003e was consistently downregulated, delineating two opposing regulatory trajectories involved in orchestrating pepper\u0026rsquo;s transcriptional adaptation to thermal stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.8.3 Expression Profiling of CaHD-Zip Genes Under Drought Stress\u003c/h2\u003e \u003cp\u003eHierarchical clustering of TPM based transcriptomic profiles under drought stress segregated CaHD-Zip genes into four distinct co-expression modules (A\u0026ndash;D) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Cluster A (\u003cem\u003eCaHD-Zip17\u0026thinsp;~\u0026thinsp;CaHD-Zip25\u003c/em\u003e; 6 members) showed sustained transcriptional suppression, exemplified by \u003cem\u003eCaHD-Zip25\u003c/em\u003e (94.7% reduction: 20.58 to 1.09 TPM) from 12\u0026ndash;72 h. Cluster B (\u003cem\u003eCaHD-Zip08\u0026thinsp;~\u0026thinsp;CaHD-Zip31\u003c/em\u003e; 7 members) exhibited transient early activation (3\u0026ndash;6 h), followed by a gradual decline to approximately 50% of peak levels by 72 h, as observed for \u003cem\u003eCaHD-Zip23\u003c/em\u003e (6 h peak: 32.16 TPM, 6.0-fold induction). Cluster C (\u003cem\u003eCaHD-Zip40\u0026thinsp;~\u0026thinsp;CaHD-Zip37\u003c/em\u003e; 12 members) demonstrated progressive transcriptional upregulation, with \u003cem\u003eCaHD-Zip29\u003c/em\u003e achieving a 31.5-fold induction (3.50 to 113.67 TPM) at 72 h. Cluster D (\u003cem\u003eCaHD-Zip30\u0026thinsp;~\u0026thinsp;CaHD-Zip04\u003c/em\u003e; 13 members) exhibited temporally dynamic expression patterns, with certain members activated and others repressed in a stage-specific manner (e.g., \u003cem\u003eCaHD-Zip06\u003c/em\u003e induced at 12 h; \u003cem\u003eCaHD-Zip03\u003c/em\u003e repressed at 72 h). Venn diagram analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eg) identified seven core drought-responsive DEGs (\u003cem\u003eCaHD-Zip35/16/29/01/18/23/25\u003c/em\u003e) with significant expression changes across all timepoints (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Most of these DEGs belonged to Cluster C and exhibited persistent upregulation, highlighting their potential roles as central regulatory hubs in the long-term drought stress adaptation network of \u003cem\u003eCapsicum annuum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.8.4 Expression Profiling of the \u003cem\u003eCaHD-Zip\u003c/em\u003e Gene Family Under Salt Stress\u003c/h2\u003e \u003cp\u003eHierarchical clustering of TPM-based transcriptomes under salt stress (200 mM NaCl) grouped \u003cem\u003eCaHD-Zip\u003c/em\u003e genes into five regulatory modules (Clusters A\u0026ndash;E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Cluster A (\u003cem\u003eCaHD-Zip12\u0026thinsp;~\u0026thinsp;CaHD-Zip21\u003c/em\u003e) showed late-phase induction, exemplified by \u003cem\u003eCaHD-Zip21\u003c/em\u003e increasing from 19.62 to 30.15 TPM by 72 h. Cluster B (\u003cem\u003eCaHD-Zip16\u0026thinsp;~\u0026thinsp;CaHD-Zip11\u003c/em\u003e) exhibited persistent upregulation throughout the stress period, peaking at 24 h. Cluster C (\u003cem\u003eCaHD-Zip30\u0026thinsp;~\u0026thinsp;CaHD-Zip35\u003c/em\u003e) comprised early-inducible genes such as \u003cem\u003eCaHD-Zip23\u003c/em\u003e, which was upregulated 6.9-fold by 6 h (5.36 to 37.11 TPM), suggesting involvement in initial salt signaling. Cluster D (\u003cem\u003eCaHD-Zip25\u0026thinsp;~\u0026thinsp;CaHD-Zip04\u003c/em\u003e) showed strong and sustained repression, with \u003cem\u003eCaHD-Zip25\u003c/em\u003e decreasing 45.77-fold by 24 h (20.58 to 0.44 TPM), suggesting negative regulatory functions in salt tolerance. Cluster E (\u003cem\u003eCaHD-Zip36\u0026thinsp;~\u0026thinsp;CaHD-Zip22\u003c/em\u003e) exhibited stage-specific responses, as exemplified by \u003cem\u003eCaHD-Zip06\u003c/em\u003e upregulated to 17.57 TPM at 12 h (65.85% increase), possibly mediating short-term salt adaptation. Venn analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eh) revealed five key DEGs (CaHD-Zip16/29/01/18/25) differentially expressed at all timepoints. CaHD-Zip16/29/01/18 were consistently induced, whereas \u003cem\u003eCaHD-Zip25\u003c/em\u003e was persistently downregulated, delineating contrasting regulatory modes essential for sustained salt stress adaptation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.8.5 Key Regulators Shared Across Abiotic Stress Conditions\u003c/h2\u003e \u003cp\u003eFurther Venn analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ei) revealed that \u003cem\u003eCaHD-Zip29\u003c/em\u003e, \u003cem\u003eCaHD-Zip18\u003c/em\u003e, and \u003cem\u003eCaHD-Zip25\u003c/em\u003e were consistently differentially expressed across all timepoints under cold, heat, drought, and salt stresses, implicating them as core regulators in broad-spectrum stress responses. Among them, \u003cem\u003eCaHD-Zip29\u003c/em\u003e and \u003cem\u003eCaHD-Zip18\u003c/em\u003e were persistently upregulated, suggesting positive regulatory roles in promoting abiotic stress adaptation. In contrast, \u003cem\u003eCaHD-Zip25\u003c/em\u003e was consistently downregulated, indicating its potential function as a negative regulator. Collectively, these genes appear to represent key regulatory hubs orchestrating stress signaling, transcriptional modulation, and long-term environmental resilience in \u003cem\u003eCapsicum annuum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Validation of Transcriptome-Based Expression Patterns via qRT-PCR\u003c/h2\u003e \u003cp\u003eBased on transcriptome profiling, eight representative \u003cem\u003eCaHD-Zip\u003c/em\u003e genes (\u003cem\u003eHD-Zip16, HD-Zip29, HD-Zip01, HD-Zip18, HD-Zip25, HD-Zip23, HD-Zip35, and HD-Zip36\u003c/em\u003e) were selected for qRT-PCR validation under four abiotic stress conditions (cold, heat, drought, salinity) and two hormone treatments (ABA and MeJA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Under cold stress, qRT-PCR analysis revealed significant upregulation of all tested genes except \u003cem\u003eHD-Zip25\u003c/em\u003e and \u003cem\u003eHD-Zip36\u003c/em\u003e, suggesting a positive regulatory role of the remaining genes in cold acclimation. Under heat, drought, and salinity stress conditions, \u003cem\u003eHD-Zip25\u003c/em\u003e was consistently downregulated, while the other seven genes exhibited pronounced induction, indicating a potential negative regulatory role of \u003cem\u003eHD-Zip25\u003c/em\u003e in abiotic stress responses. Under ABA and MeJA treatment, \u003cem\u003eHD-Zip36\u003c/em\u003e remained transcriptionally unchanged,\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHD-Zip25 was downregulated, and the other six genes were significantly upregulated. Overall, the qRT-PCR results showed strong concordance with transcriptomic sequencing data, confirming the reliability of transcriptomic analysis and supporting the differential expression patterns of CaHD-Zip genes under hormone and abiotic stress regulation. These findings provide robust experimental evidence for future studies on the functional roles and regulatory mechanisms of HD-Zip genes in stress responses, and identify potential gene targets for enhancing abiotic stress tolerance in plants.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe \u003cem\u003eHD-ZIP\u003c/em\u003e (Homeodomain-Leucine Zipper) gene family comprises plant-specific transcription factors that orchestrate diverse developmental processes and mediate stress responses in plants [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Despite extensive characterization in numerous economically important crops, knowledge regarding their expression dynamics in pepper under abiotic stresses remains limited. In this study, we identified 40 \u003cem\u003eCaHD-ZIP\u003c/em\u003e genes and performed a genome-wide analysis of their structural features, chromosomal localization, tissue-specific expression patterns, and responses to abiotic stresses and hormone signaling pathways. Three candidate \u003cem\u003eHD-ZIP\u003c/em\u003e transcription factors responsive to multiple abiotic stress conditions were identified, offering a molecular foundation for advancing functional studies of the HD-ZIP family in pepper stress adaptation.\u003c/p\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Phylogenetic relationships and functional conservation of the \u003cem\u003eHD-Zip\u003c/em\u003e gene family\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eCaHD-Zip\u003c/em\u003e genes in pepper are classified into four subfamilies (HD-Zip I to IV), displaying evolutionary divergence patterns that are conserved across diverse plant lineages [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We found that HD-Zip I subfamily members are the most abundant, whereas the HD-Zip III subfamily comprises comparatively fewer genes. This trend mirrors observations in \u003cem\u003eArabidopsis thaliana, Solanum tuberosum\u003c/em\u003e, and \u003cem\u003eCucumis sativus\u003c/em\u003e [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The reduced representation of HD-Zip III genes may reflect stronger purifying selection during evolution\u0026mdash;a pattern corroborated in several dicot species [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Additionally, a one-to-one orthologous relationship exists between \u003cem\u003eCaHD-Zip\u003c/em\u003e genes in pepper and \u003cem\u003eAtHD-Zip\u003c/em\u003e genes in \u003cem\u003eArabidopsis\u003c/em\u003e, such as \u003cem\u003eCaHD-Zip31\u003c/em\u003e with \u003cem\u003eATHB51\u003c/em\u003e, and \u003cem\u003eCaHD-Zip37\u003c/em\u003e with \u003cem\u003eATHB18\u003c/em\u003e. These results underscore the evolutionary conservation of the \u003cem\u003eHD-Zip\u003c/em\u003e gene family and its potentially conserved roles in regulating growth and development across species [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Gene expansion mechanisms and collinearity\u003c/h2\u003e \u003cp\u003eGene duplication represents a fundamental driver of plant genome evolution, enabling gene family expansion through segmental duplication and tandem duplication, thereby enhancing adaptability to environmental stimuli [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Our analysis revealed that the expansion of the \u003cem\u003eHD-Zip\u003c/em\u003e gene family in pepper was primarily driven by segmental duplication, with 15 segmentally duplicated gene pairs identified, whereas no tandem duplication events were detected. This pattern aligns with observations in sesame and cassava, where \u003cem\u003eHD-Zip\u003c/em\u003e gene expansion similarly relies on segmental duplication [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Moreover, the divergence times of these segmental duplicates span a broad evolutionary timeframe, ranging from 320.75\u0026nbsp;million years ago (\u003cem\u003eHD-Zip11\u003c/em\u003e and \u003cem\u003eHD-Zip20\u003c/em\u003e) to 51.05\u0026nbsp;million years ago (\u003cem\u003eHD-Zip18\u003c/em\u003e and \u003cem\u003eHD-Zip26\u003c/em\u003e). These findings suggest that the \u003cem\u003eHD-Zip\u003c/em\u003e gene family in pepper underwent multiple independent duplication events, potentially linked to species-specific genome duplication or chromosomal rearrangement events [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSynteny analysis revealed strong collinearity between pepper \u003cem\u003eHD-Zip\u003c/em\u003e genes and those of dicot species (tomato, potato, Arabidopsis), while collinearity with monocots such as rice and maize was comparatively weak[\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. This disparity likely reflects deep evolutionary divergence and genome structural differences, further suggesting that \u003cem\u003eHD-Zip\u003c/em\u003e genes in dicots have been subject to intensified selective constraints. Additionally, seven \u003cem\u003eCaHD-Zip\u003c/em\u003e genes displayed conserved synteny across all five species analyzed, underscoring their functionally conserved roles in plant development and environmental adaptation\u0026mdash;a finding consistent with previous studies in watermelon [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Core regulatory roles of the \u003cem\u003eHD-Zip\u003c/em\u003e gene family in abiotic stress responses\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eHD-Zip\u003c/em\u003e gene family regulates abiotic stress responses through multiple coordinated mechanisms, including ABA-dependent signaling pathways, lignin biosynthesis, membrane stability modulation, and miRNA-guided gene regulation, thereby enhancing plant resilience. Numerous \u003cem\u003eHD-Zip\u003c/em\u003e genes have been implicated in salt tolerance across various species, such as \u003cem\u003eGossypium hirsutum GhHB4\u003c/em\u003e-like and \u003cem\u003eGhHB12\u003c/em\u003e [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], \u003cem\u003eMiscanthus sinensis MsHDZ23\u003c/em\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], and \u003cem\u003eBrassica napus BnaHDZ149\u003c/em\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], which exert their functions primarily via ABA-related or stress-inducible pathways. Cold stress responses typically involve ABA signaling and membrane protein stabilization, exemplified by \u003cem\u003eMalus domestica MdHDZ14\u003c/em\u003e, which is significantly upregulated under low-temperature conditions [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], as well as \u003cem\u003eHelianthus annuus HaHB1\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana AtHB13\u003c/em\u003e, which enhance cold tolerance by maintaining membrane integrity[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In heat stress adaptation, \u003cem\u003eRaphanus sativus RsHDZ17\u003c/em\u003e enhances thermotolerance when overexpressed in both radish and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], while \u003cem\u003eCapsicum annuum HD-Zip15\u003c/em\u003e has been shown to directly target \u003cem\u003eHSFA6a\u003c/em\u003e and activate \u003cem\u003eCaHSFA2\u003c/em\u003e, playing a central role in thermotolerance regulation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, under drought stress, \u003cem\u003eHD-Zip\u003c/em\u003e genes such as \u003cem\u003ePassiflora edulis PeHB31\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]and \u003cem\u003eZea may\u003c/em\u003es \u003cem\u003eZmHDZ9\u003c/em\u003e [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] promote lignin biosynthesis, thereby contributing to enhanced drought resistance.\u003c/p\u003e \u003cp\u003eIn our study, \u003cem\u003eCaHD-Zip29\u003c/em\u003e and \u003cem\u003eCaHD-Zip18\u003c/em\u003e were significantly upregulated in \u003cem\u003eCapsicum annuum\u003c/em\u003e under cold, heat, salt, and drought conditions, indicating robust multi-stress responsiveness. Phylogenetic analysis revealed that both genes are closely related to \u003cem\u003eArabidopsis thaliana AtHB7\u003c/em\u003e and \u003cem\u003eAtHB12\u003c/em\u003e, and their orthologs in other species\u0026mdash;such as \u003cem\u003eOryza sativa HOX22\u003c/em\u003e [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], \u003cem\u003eSalix suchowensis SsHox36/SsHox51\u003c/em\u003e [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], and \u003cem\u003eSesamum indicum SiHDZ31/16\u003c/em\u003e [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]have been associated with drought and salt stress responses. Additionally, these genes exhibit strong conservation under thermal stress. For instance, \u003cem\u003ePassiflora edulis PeHB17/07\u003c/em\u003e and \u003cem\u003eRaphanus sativus RsHDZ17\u003c/em\u003e are highly induced under heat stress[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], while \u003cem\u003eHordeum vulgare HvHD-Zip19\u003c/em\u003e [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] and \u003cem\u003eMalus domestica MdHDZ21\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] show specific responses to cold stress. Collectively, these findings not only underscore the evolutionary conservation of \u003cem\u003eHD-Zip\u003c/em\u003e genes in abiotic stress adaptation but also suggest that \u003cem\u003eCaHD-Zip29\u003c/em\u003e and \u003cem\u003eCaHD-Zip18\u003c/em\u003e, as orthologs of \u003cem\u003eAtHB7/AtHB12\u003c/em\u003e, may serve as integrative regulators coordinating multiple abiotic stress signaling pathways in \u003cem\u003eCapsicum annuum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Regulatory roles of CaHD-Zip genes in pepper development\u003c/h2\u003e \u003cp\u003eWe systematically profiled the expression patterns of 40 \u003cem\u003eCaHD-Zip\u003c/em\u003e genes, categorizing them into four subgroups, and uncovered a multi-layered regulatory framework coordinating meristem maintenance, fruit ripening, floral organogenesis, and environmental adaptation. Class A genes exhibited strong expression in meristematic and reproductive tissues (e.g., seeds, embryos, placentae), a pattern conserved with \u003cem\u003eArabidopsis thaliana AtHB23\u003c/em\u003e, which modulates shoot apical meristem activity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and \u003cem\u003ePassiflora edulis PeHB31\u003c/em\u003e, which promotes ovule development [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. \u003cem\u003eArabidopsis thaliana AtHB2\u003c/em\u003e mediates red/far-red light-induced shade avoidance and lateral root development [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], while \u003cem\u003eZea mays ZmHOX32\u003c/em\u003e improves photosynthetic efficiency by modulating leaf meristem activity [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], highlighting the \u003cem\u003eHD-Zip\u003c/em\u003e family's extensive role in plant morphogenesis. Class B genes, including \u003cem\u003eCaHD-Zip29\u003c/em\u003e, were markedly upregulated during fruit maturation, mirroring \u003cem\u003ePassiflora edulis PeHB7/PeHB17/PeHB18\u003c/em\u003e expression [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], thereby suggesting a conserved role in ripening. Additional support stems from Musa acuminata \u003cem\u003eHD-Zip\u003c/em\u003e I genes regulating ACS/ACO [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], \u003cem\u003eLeHB-1\u003c/em\u003e promoting \u003cem\u003eACO1\u003c/em\u003e Mrna [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], and \u003cem\u003eCucumis sativus CsHDZ11/37\u003c/em\u003e being highly expressed in fruits [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Class C genes exhibited low baseline expression across tissues but were robustly induced by drought, salinity, cold stress, and heat stress (e.g., \u003cem\u003eCaHD-Zip01\u003c/em\u003e), similar to Eucalyptus grandis \u003cem\u003eHD-Zip38\u003c/em\u003e [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] and \u003cem\u003eHordeum vulgare HvHD-ZipI16/II1\u003c/em\u003e under drought [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], implicating their role as central stress-responsive factors in \u003cem\u003eCapsicum annuum\u003c/em\u003e. Class D genes showed strong, specific expression in floral buds and seeds, suggesting functions in floral primordium differentiation or pollination. Such roles are exemplified by \u003cem\u003eCitrus sinensis PtHB13\u003c/em\u003e [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e], and \u003cem\u003eCucumis sativus CsHDZ08/Z22\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Collectively, these results unveil distinct spatiotemporal expression profiles of the \u003cem\u003eCaHD-Zip\u003c/em\u003e gene family across \u003cem\u003eCapsicum annuum\u003c/em\u003e tissues and developmental stages, laying a molecular foundation for elucidating their roles in pepper development.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, we identified 40 HD-Zip transcription factors in \u003cem\u003eCapsicum annuum\u003c/em\u003e, with gene family expansion predominantly driven by segmental duplication and exhibited strong evolutionary conservation among dicot species. Spatiotemporal expression profiling revealed distinct regulatory patterns across meristematic tissues, fruit development, and floral differentiation. Notably, \u003cem\u003eCaHD-Zip18\u003c/em\u003e and \u003cem\u003eCaHD-Zip29\u003c/em\u003e were consistently upregulated under multiple abiotic stresses, whereas \u003cem\u003eCaHD-Zip25\u003c/em\u003e was significantly downregulated, suggesting their respective roles as core positive and negative regulators in stress adaptation. The expression dynamics of these genes were further validated by quantitative reverse transcription PCR (qRT-PCR). As the first comprehensive investigation of the \u003cem\u003eHD-Zip\u003c/em\u003e family in \u003cem\u003eCapsicum annuum\u003c/em\u003e under abiotic stress, this study offers valuable insights into their regulatory functions and provides key candidate genes for the development of stress-tolerant cultivars. Future research integrating CRISPR-based gene editing and molecular interaction analyses will be critical to unravelling the functional mechanisms of these transcription factors and advancing molecular breeding strategies in \u003cem\u003eCapsicum annuum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"552\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eABA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eabscisic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003ebZIP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eBasic leucine zipper\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eCa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003e\u003cem\u003eCapsicum annuum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003eCDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 385px;\"\u003e\n \u003cp\u003eCoding sequences\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eethylene\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eGRAVY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eGrand Average of Hydropathicity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eHALZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eHomeobox-associated leucine zipper\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eHD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003ehomeodomain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003eHMM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 385px;\"\u003e\n \u003cp\u003eHidden markov model\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003eKa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 385px;\"\u003e\n \u003cp\u003eNonsynonymous Substitution Rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003eKs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 385px;\"\u003e\n \u003cp\u003eSynonymous Substitution Rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eLZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eleucine zipper\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eMeJA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003emethyl jasmonate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eMEKHLA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eMembrane occupation and recognition nexus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eML\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003emaximum likelihood\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003ePEG-6000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 385px;\"\u003e\n \u003cp\u003ePolyethylene Glycol 6000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003epI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 385px;\"\u003e\n \u003cp\u003eIsoelectric point\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003ePPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 385px;\"\u003e\n \u003cp\u003eProtein\u0026ndash;protein interaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003eqRT-PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 385px;\"\u003e\n \u003cp\u003eQuantitative Real Time Polymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eArginine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003esalicylic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eSPSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eStatistical Package for the Social Sciences\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eSTART\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eSteroidogenic Acute Regulatory Protein-related lipid transfer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eTPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eTranscripts per million\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003eW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 385px;\"\u003e\n \u003cp\u003eTryptophan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u0026hellip;.\u003c/p\u003e\n\u003cp\u003eSupplementary Material: Supplementary Table S1: RNA-seq datasets from NCBI SRA for \u003cem\u003eCapsicum annuum\u003c/em\u003e across organ types, abiotic stresses, and phytohormone treatments. Supplementary Table S2: The primer sequences used for qRT-PCR. Supplementary Table S3: Ka/Ks Ratios of 15 Segmentally Duplicated \u003cem\u003eCaHD-Zip\u003c/em\u003e Genes. Supplementary Table S4: Collinearity analysis results of \u003cem\u003eCapsicum\u0026nbsp;\u003c/em\u003e(intraspecific and interspecific). Supplementary Table S5: Structural Characteristics of the Capsicum \u003cem\u003eHD-Zip\u003c/em\u003e Gene Family. Supplementary Table S6: Statistics on the functional classification of \u003cem\u003eCaHD-zip\u003c/em\u003e genes. Supplementary Figure S1: Chromosomal Localization of 40 Capsicum \u003cem\u003eCaHD-Zip Genes\u003c/em\u003e. Supplementary Figure S2: SeqLogo Representation of Motif1-10. Supplementary Figure S3: Spatiotemporal Distribution Analysis of Differentially Expressed Genes (DEGs) Under Different Hormone Treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are sincerely grateful to Prof. Jimin Sun (Chinese Academy of Sciences) and Dr. Yuchao Tang, Postdoctoral Fellow (Beijing Forestry University) for their important revisions and insightful suggestions that significantly improved the quality of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.W. designed the experiments, conducted data analysis and visualization, and was responsible for drafting and revising the manuscript. \u0026nbsp;X.Z., K.X., and X.F. contributed to data analysis and visualization. L.L., L.G. and M.S. participated in the experimental design and assisted with data analysis. Y.L. and C.H. provided funding and contributed to the final revision of the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe earmarked fund for CARS, Grant/Award Number: CARS-24-B-04 and CARS-23-B05; The National Key Research and Development Program of China (2022YFD1602403 and 2023YFD2300704 ); The Major Science and Technology Special Project of Ordos (ZD20232318); Talent Project in the Field of Science and Technology Innovation of Hohhot. This research acknowledged the support of the Key Laboratory of Horticultural Crop Biology and Germplasm Innovation, the Ministry of Agriculture, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZhou C-X, Zhang W, Yu B-G, Yang H-F, Zhao Q-Y, Wang Y, et al. Globalanalysis ofspatio-temporal variation inmineral nutritional quality ofpepper( Capsicum spp.)fruitanditsregulatory variables: A meta-analysis. Food Research International. 2024;193:114855.\u003c/li\u003e\n \u003cli\u003eTripodi P, Kumar S. The Capsicum Crop: An Introduction. In: Ramchiary N, Kole C, editors. The Capsicum Genome. Cham: Springer International Publishing; 2019. p. 1\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eMoseley A, Laipply K, Rubinstein J. Capsaicin Mediates Remote Ischemic Pre-Conditioning to Explain Improved Cardiovascular Mortality With Chili Pepper Intake. Journal of the American College of Cardiology. 2020;75:1865.\u003c/li\u003e\n \u003cli\u003eParvez GMM. CURRENT ADVANCES IN PHARMACOLOGICAL ACTIVITY AND TOXIC EFFETCS OF VARIOUS CAPSICUM SPECIES. International Journal of Pharmaceutical Sciences and Research. 8.\u003c/li\u003e\n \u003cli\u003eBaenas N, Belović M, Ilic N, Moreno DA, Garc\u0026iacute;a-Viguera C. Industrial use of pepper (Capsicum annum L.) derived products: technological benefits and biological advantages. Food Chemistry. 2019;274:872\u0026ndash;85.\u003c/li\u003e\n \u003cli\u003eZhang X, Ma X, Wang S, Liu S, Shi S. Physiological and Genetic Aspects of Resistance to Abiotic Stresses in Capsicum Species. Plants. 2024;13:3013.\u003c/li\u003e\n \u003cli\u003eMou S, He W, Jiang H, Meng Q, Zhang T, Liu Z, et al. Transcription factor CaHDZ15 promotes pepper basal thermotolerance by activating HEAT SHOCK FACTORA6a. Plant Physiology. 2024;195:812\u0026ndash;31.\u003c/li\u003e\n \u003cli\u003eChhapekar SS, Jaiswal V, Ahmad I, Gaur R, Ramchiary N. Progress and Prospects in Capsicum Breeding for Biotic and Abiotic Stresses. In: Vats S, editor. Biotic and Abiotic Stress Tolerance in Plants. Singapore: Springer; 2018. p. 279\u0026ndash;322.\u003c/li\u003e\n \u003cli\u003eNakashima K, Ito Y, Yamaguchi-Shinozaki K. Transcriptional Regulatory Networks in Response to Abiotic Stresses in Arabidopsis and Grasses. Plant Physiology. 2009;149:88\u0026ndash;95.\u003c/li\u003e\n \u003cli\u003eCapella M, Ribone PA, Arce AL, Chan RL. Arabidopsis thaliana HomeoBox 1 (AtHB1), a Homedomain-Leucine Zipper I (HD-Zip I) transcription factor, is regulated by PHYTOCHROME-INTERACTING FACTOR 1 to promote hypocotyl elongation. New Phytologist. 2015;207:669\u0026ndash;82.\u003c/li\u003e\n \u003cli\u003eWang K, Xu L, Wang Y, Ying J, Li J, Dong J, et al. Genome-wide characterization of homeodomain-leucine zipper genes reveals enhances the heat tolerance in radish (Raphanus sativus L.). Physiologia Plantarum. 2022;174:e13789.\u003c/li\u003e\n \u003cli\u003eNavarro MA, Navarro C, Hern\u0026aacute;ndez LE, Garnica M, Franco-Zorrilla JM, Burko Y, et al. GLABRA2 transcription factor integrates arsenic tolerance with epidermal cell fate determination. New Phytologist. 2024;244:1882\u0026ndash;900.\u003c/li\u003e\n \u003cli\u003eLeung J, Merlot S, Giraudat J. The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. Plant Cell. 1997;9:759\u0026ndash;71.\u003c/li\u003e\n \u003cli\u003eSessa G, Carabelli M, Sassi M, Ciolfi A, Possenti M, Mittempergher F, et al. A dynamic balance between gene activation and repression regulates the shade avoidance response in Arabidopsis. Genes Dev. 2005;19:2811\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eHenriksson E, Olsson ASB, Johannesson H, Johansson H, Hanson J, Engstr\u0026ouml;m P, et al. Homeodomain Leucine Zipper Class I Genes in Arabidopsis. Expression Patterns and Phylogenetic Relationships. Plant Physiology. 2005;139:509\u0026ndash;18.\u003c/li\u003e\n \u003cli\u003eA novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif.\u003c/li\u003e\n \u003cli\u003eAriel FD, Manavella PA, Dezar CA, Chan RL. The true story of the HD-Zip family. Trends Plant Sci. 2007;12:419\u0026ndash;26.\u003c/li\u003e\n \u003cli\u003eHarris JC, Hrmova M, Lopato S, Langridge P. Modulation of plant growth by HD-Zip class I and II transcription factors in response to environmental stimuli. New Phytologist. 2011;190:823\u0026ndash;37.\u003c/li\u003e\n \u003cli\u003eMeijer AH, de Kam RJ, d\u0026rsquo;Erfurth I, Shen W, Hoge JHC. HD-Zip proteins of families I and II from rice: interactions and functional properties. Mol Gen Genet. 2000;263:12\u0026ndash;21.\u003c/li\u003e\n \u003cli\u003eElhiti M, Stasolla C. Structure and function of homodomain-leucine zipper (HD-Zip) proteins. Plant Signal Behav. 2009;4:86\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eRoodbarkelari F, Groot EP. Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis. New Phytologist. 2017;213:95\u0026ndash;104.\u003c/li\u003e\n \u003cli\u003ePerotti MF, Ribone PA, Chan RL. Plant transcription factors from the homeodomain-leucine zipper family I. Role in development and stress responses. IUBMB Life. 2017;69:280\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eRomani F, Ribone PA, Capella M, Miguel VN, Chan RL. A matter of quantity: Common features in the drought response of transgenic plants overexpressing HD-Zip I transcription factors. Plant Sci. 2016;251:139\u0026ndash;54.\u003c/li\u003e\n \u003cli\u003eS\u0026ouml;derman E, Mattsson J, Engstr\u0026ouml;m P. The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid. Plant J. 1996;10:375\u0026ndash;81.\u003c/li\u003e\n \u003cli\u003eMishra KB, Iannacone R, Petrozza A, Mishra A, Armentano N, La Vecchia G, et al. Engineered drought tolerance in tomato plants is reflected in chlorophyll fluorescence emission. Plant Sci. 2012;182:79\u0026ndash;86.\u003c/li\u003e\n \u003cli\u003eZhao S, Wang H, Jia X, Gao H, Mao K, Ma F. The HD-Zip I transcription factor -like confers tolerance to salinity in transgenic apple (). Physiologia Plantarum. 2021;172:1452\u0026ndash;64.\u003c/li\u003e\n \u003cli\u003eTurchi L, Baima S, Morelli G, Ruberti I. Interplay of HD-Zip II and III transcription factors in auxin-regulated plant development. J Exp Bot. 2015;66:5043\u0026ndash;53.\u003c/li\u003e\n \u003cli\u003eTurchi L, Carabelli M, Ruzza V, Possenti M, Sassi M, Pe\u0026ntilde;alosa A, et al. Arabidopsis HD-Zip II transcription factors control apical embryo development and meristem function. Development. 2013;140:2118\u0026ndash;29.\u003c/li\u003e\n \u003cli\u003eCarabelli M, Turchi L, Ruzza V, Morelli G, Ruberti I. Homeodomain-Leucine Zipper II family of transcription factors to the limelight: central regulators of plant development. Plant Signal Behav. 2013;8:e25447.\u003c/li\u003e\n \u003cli\u003eMerelo P, Ram H, Pia Caggiano M, Ohno C, Ott F, Straub D, et al. Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity. Proceedings of the National Academy of Sciences. 2016;113:11973\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eCausier B, Ashworth M, Guo W, Davies B. The TOPLESS interactome: a framework for gene repression in Arabidopsis. Plant Physiol. 2012;158:423\u0026ndash;38.\u003c/li\u003e\n \u003cli\u003eLiu T, Longhurst AD, Talavera-Rauh F, Hokin SA, Barton MK. The Arabidopsis transcription factor ABIG1 relays ABA signaled growth inhibition and drought induced senescence. Elife. 2016;5:e13768.\u003c/li\u003e\n \u003cli\u003ePrigge MJ, Clark SE. Evolution of the class III HD-Zip gene family in land plants. Evolution \u0026amp; Development. 2006;8:350\u0026ndash;61.\u003c/li\u003e\n \u003cli\u003eZhu Y, Song D, Xu P, Sun J, Li L. A HD-ZIP III gene, PtrHB4, is required for interfascicular cambium development in Populus. Plant Biotechnol J. 2018;16:808\u0026ndash;17.\u003c/li\u003e\n \u003cli\u003eGao Y, Gao S, Xiong C, Yu G, Chang J, Ye Z, et al. Comprehensive analysis and expression profile of the homeodomain leucine zipper IV transcription factor family in tomato. Plant Physiology and Biochemistry. 2015;96:141\u0026ndash;53.\u003c/li\u003e\n \u003cli\u003eMa F, Song S, Li C, Huang D, Wu B, Xing W, et al. Passion fruit HD-ZIP genes: Characterization, expression variance, and overexpression PeHB31 enhanced drought tolerance via lignin pathway. International Journal of Biological Macromolecules. 2024;276:133603.\u003c/li\u003e\n \u003cli\u003eWang Z, Wu X, Zhang B, Xiao Y, Guo J, Liu J, et al. Genome-wide identification, bioinformatics and expression analysis of HD-Zip gene family in peach. BMC Plant Biology. 2023;23:122.\u003c/li\u003e\n \u003cli\u003eZhang Q, Chen T, Wang X, Wang J, Gu K, Yu J, et al. Genome-wide identification and expression analyses of homeodomain-leucine zipper family genes reveal their involvement in stress response in apple (Malus \u0026times; domestica). Horticultural Plant Journal. 2022;8:261\u0026ndash;78.\u003c/li\u003e\n \u003cli\u003eZhang K, Wang X, Chen S, Liu Y, Zhang L, Yang X, et al. The gap-free assembly of pepper genome reveals transposable-element-driven expansion and rapid evolution of pericentromeres. Plant Communications. 2025;6:101177.\u003c/li\u003e\n \u003cli\u003ePotter SC, Luciani A, Eddy SR, Park Y, Lopez R, Finn RD. HMMER web server: 2018 update. Nucleic Acids Res. 2018;46:W200\u0026ndash;4.\u003c/li\u003e\n \u003cli\u003eLu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020;48:D265\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eLetunic I, Bork P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Research. 2018;46:D493\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003eGasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003;31:3784\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eKumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33:1870\u0026ndash;4.\u003c/li\u003e\n \u003cli\u003eWaterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:1189\u0026ndash;91.\u003c/li\u003e\n \u003cli\u003eLescot M, D\u0026eacute;hais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30:325\u0026ndash;7.\u003c/li\u003e\n \u003cli\u003eWang D, Zhang S, He F, Zhu J, Hu S, Yu J. How do variable substitution rates influence Ka and Ks calculations? Genomics Proteomics Bioinformatics. 2009;7:116\u0026ndash;27.\u003c/li\u003e\n \u003cli\u003eLynch M, Conery JS. The Evolutionary Fate and Consequences of Duplicate Genes. Science. 2000;290:1151\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eChen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant. 2020;13:1194\u0026ndash;202.\u003c/li\u003e\n \u003cli\u003eRao X, Huang X, Zhou Z, Lin X. An improvement of the 2\u0026circ;(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 2013;3:71\u0026ndash;85.\u003c/li\u003e\n \u003cli\u003eKock KH, Kimes PK, Gisselbrecht SS, Inukai S, Phanor SK, Anderson JT, et al. DNA binding analysis of rare variants in homeodomains reveals homeodomain specificity-determining residues. Nat Commun. 2024;15:3110.\u003c/li\u003e\n \u003cli\u003eGonz\u0026aacute;lez FG, Capella M, Ribichich KF, Cur\u0026iacute;n F, Giacomelli JI, Ayala F, et al. Field-grown transgenic wheat expressing the sunflower gene HaHB4 significantly outyields the wild type. J Exp Bot. 2019;70:1669\u0026ndash;81.\u003c/li\u003e\n \u003cli\u003eSharif R, Xie C, Wang J, Cao Z, Zhang H, Chen P, et al. Genome wide identification, characterization and expression analysis of HD-ZIP gene family in Cucumis sativus L. under biotic and various abiotic stresses. International Journal of Biological Macromolecules. 2020;158:502\u0026ndash;20.\u003c/li\u003e\n \u003cli\u003eLi W, Dong J, Cao M, Gao X, Wang D, Liu B, et al. Genome-wide identification and characterization of HD-ZIP genes in potato. Gene. 2019;697:103\u0026ndash;17.\u003c/li\u003e\n \u003cli\u003eLi Z, Zhang C, Guo Y, Niu W, Wang Y, Xu Y. Evolution and expression analysis reveal the potential role of the HD-Zip gene family in regulation of embryo abortion in grapes (Vitis vinifera L.). BMC Genomics. 2017;18:744.\u003c/li\u003e\n \u003cli\u003eCannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biology. 2004;4:10.\u003c/li\u003e\n \u003cli\u003eDing Z, Fu L, Yan Y, Tie W, Xia Z, Wang W, et al. Genome-wide characterization and expression profiling of HD-Zip gene family related to abiotic stress in cassava. PLoS One. 2017;12:e0173043.\u003c/li\u003e\n \u003cli\u003eSoltis PS, Soltis DE. The role of hybridization in plant speciation. Annu Rev Plant Biol. 2009;60:561\u0026ndash;88.\u003c/li\u003e\n \u003cli\u003eLopez-Ortiz C, Dutta SK, Natarajan P, Pe\u0026ntilde;a-Garcia Y, Abburi V, Saminathan T, et al. Genome-wide identification and gene expression pattern of ABC transporter gene family in Capsicum spp. PLoS One. 2019;14:e0215901.\u003c/li\u003e\n \u003cli\u003eIslam MAU, Nupur JA, Shafiq M, Ali Q, Sami A, Shahid MA. In silico and computational analysis of zinc finger motif-associated homeodomain (ZF-HD) family genes in chilli (Capsicum annuum L). BMC Genomics. 2023;24:603.\u003c/li\u003e\n \u003cli\u003ePan X, Hu M, Wang Z, Guan L, Jiang X, Bai W, et al. Identification, systematic evolution and expression analyses of the AAAP gene family in Capsicum annuum. BMC Genomics. 2021;22:463.\u003c/li\u003e\n \u003cli\u003eYan X, Yue Z, Pan X, Si F, Li J, Chen X, et al. The HD-ZIP Gene Family in Watermelon: Genome-Wide Identification and Expression Analysis under Abiotic Stresses. Genes. 2022;13:2242.\u003c/li\u003e\n \u003cli\u003eRen A, Wen T, Xu X, Wu J, Zhao G. Cotton HD-Zip I transcription factor GhHB4-like regulates the plant response to salt stress. International Journal of Biological Macromolecules. 2024;278:134857.\u003c/li\u003e\n \u003cli\u003eHe X, Luo X, Wang T, Liu S, Zhang X, Zhu L. GhHB12 negatively regulates abiotic stress tolerance in Arabidopsis and cottonGhHB12. Environmental and Experimental Botany. 2020;176:104087.\u003c/li\u003e\n \u003cli\u003eLiu N, Yu R, Deng W, Hu R, He G, He K, et al. MsHDZ23, a Novel Miscanthus HD-ZIP Transcription Factor, Participates in Tolerance to Multiple Abiotic Stresses. International Journal of Molecular Sciences. 2024;25:3253.\u003c/li\u003e\n \u003cli\u003eFang Y, Wang L, Liu K, Wu H, Zheng Y, Duan Y, et al. Genome-wide investigation of HD-ZIP gene family and functional characterization of BnaHDZ149 and BnaHDZ22 in salt and drought response in Brassica napus L. Plant Science. 2024;346:112130.\u003c/li\u003e\n \u003cli\u003eCabello JV, Arce AL, Chan RL. The homologous HD-Zip I transcription factors HaHB1 and AtHB13 confer cold tolerance via the induction of pathogenesis-related and glucanase proteins. Plant J. 2012;69:141\u0026ndash;53.\u003c/li\u003e\n \u003cli\u003eJiao P, Jiang Z, Miao M, Wei X, Wang C, Liu S, et al. Zmhdz9, an HD-Zip transcription factor, promotes drought stress resistance in maize by modulating ABA and lignin accumulationHD-Zip. International Journal of Biological Macromolecules. 2024;258:128849.\u003c/li\u003e\n \u003cli\u003eZhang S, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer AH, et al. Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Mol Biol. 2012;80:571\u0026ndash;85.\u003c/li\u003e\n \u003cli\u003eWang Y, Wang H, Yu C, Yan X, Chu J, Jiang B, et al. Comprehensive bioinformation analysis of homeodomain-leucine zipper gene family and expression pattern of HD-Zip I under abiotic stress in Salix suchowensis. BMC Genomics. 2024;25:182.\u003c/li\u003e\n \u003cli\u003eWei M, Liu A, Zhang Y, Zhou Y, Li D, Dossa K, et al. Genome-wide characterization and expression analysis of the HD-Zip gene family in response to drought and salinity stresses in sesame. BMC Genomics. 2019;20:748.\u003c/li\u003e\n \u003cli\u003eLi Y, Xiong H, Cuo D, Wu X, Duan R. Genome-wide characterization and expression profiling of the relation of the HD-Zip gene family to abiotic stress in barley (Hordeum vulgare L.). Plant Physiology and Biochemistry. 2019;141:250\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003ePerotti MF, Ribone PA, Cabello JV, Ariel FD, Chan RL. AtHB23 participates in the gene regulatory network controlling root branching, and reveals differences between secondary and tertiary roots. The Plant Journal. 2019;100:1224\u0026ndash;36.\u003c/li\u003e\n \u003cli\u003eC S, A M, G S, T W, M O, T A, et al. Shade avoidance responses are mediated by the ATHB-2 HD-zip protein, a negative regulator of gene expression. Development (Cambridge, England). 1999;126.\u003c/li\u003e\n \u003cli\u003eMiao X, Zhu W, Jin Q, Song Z, Li L. ZmHOX32 is related to photosynthesis and likely functions in plant architecture of maize. Front Plant Sci. 2023;14.\u003c/li\u003e\n \u003cli\u003eYang Y-Y, Shan W, Kuang J-F, Chen J-Y, Lu W-J. Four HD-ZIPs are involved in banana fruit ripening by activating the transcription of ethylene biosynthetic and cell wall-modifying genes. Plant Cell Rep. 2020;39:351\u0026ndash;62.\u003c/li\u003e\n \u003cli\u003eLin Z, Hong Y, Yin M, Li C, Zhang K, Grierson D. A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening. The Plant Journal. 2008;55:301\u0026ndash;10.\u003c/li\u003e\n \u003cli\u003eZhang J, Wu J, Guo M, Aslam M, Wang Q, Ma H, et al. Genome-wide characterization and expression profiling of Eucalyptus grandis HD-Zip gene family in response to salt and temperature stress. BMC Plant Biology. 2020;20:451.\u003c/li\u003e\n \u003cli\u003eMa Y-J, Li P-T, Sun L-M, Zhou H, Zeng R-F, Ai X-Y, et al. HD-ZIP I Transcription Factor (PtHB13) Negatively Regulates Citrus Flowering through Binding to FLOWERING LOCUS C Promoter. Plants (Basel). 2020;9:114.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Capsicum annuum, HD-Zip gene family, Abiotic stress, Gene expression","lastPublishedDoi":"10.21203/rs.3.rs-6320983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6320983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u0026nbsp;\u003c/strong\u003e\u003cem\u003eCapsicum annuum\u003c/em\u003e is a globally cultivated crop of significant agricultural and economic importance. However, its productivity and fruit quality are frequently challenged by a range of abiotic stresses. The \u003cem\u003eHD-Zip\u003c/em\u003e(Homeodomain-Leucine Zipper) gene family, unique to plants, is known to play pivotal regulatory roles in abiotic stress adaptation, yet its functional roles in pepper remain largely unexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eThis study systematically analyzed the \u003cem\u003eHD-Zip\u003c/em\u003egene family in pepper through bioinformatics, expression profiling, and responses to abiotic stresses and phytohormones to elucidate their roles in stress tolerance. Results revealed 40 HD-Zip transcription factors unevenly distributed across 12 chromosomes, encoding proteins ranging from 211 to 842 amino acids. Subcellular localization predictions indicated nuclear localization for all members, with a subset also showing cytoplasmic localization. Collinearity analysis demonstrated that \u003cem\u003eCaHD-Zip\u003c/em\u003e gene expansion was predominantly driven by segmental duplication, with high conservation across dicotyledons. Promoter regions of \u003cem\u003eCaHD-Zip\u003c/em\u003e genes were enriched in cis-regulatory elements associated with light and hormonal responses, as well as stress adaptation. Tissue-specific and developmental stage-dependent expression patterns highlighted functional diversification within the family. Notably, some members were specifically induced by abiotic stresses (cold, heat, drought, and salt) and stress-related phytohormones (ABA, MeJA, ET, and SA), suggesting their involvement in stress signaling. Strikingly, \u003cem\u003eCaHD-Zip18\u003c/em\u003e and \u003cem\u003eCaHD-Zip29\u003c/em\u003e were significantly upregulated under all four stresses, implicating them as core regulators of multi-stress responses. Subsequent stress simulation assays and qRT-PCR validation confirmed the reliability of transcriptomic findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eThis study delivers the first systematic exploration of HD-Zip transcription factors in \u003cem\u003eCapsicum annuum\u003c/em\u003e under abiotic stress, providing foundational knowledge and candidate genes for improving stress resilience in pepper breeding programs.\u003c/p\u003e","manuscriptTitle":"Genome-wide characterization and their roles in abiotic stress responses of HD-Zip transcription factors in pepper (Capsicum annuum)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 12:26:06","doi":"10.21203/rs.3.rs-6320983/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-07T09:31:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-27T19:45:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274195530778253351480249377576255807914","date":"2025-04-20T03:45:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313327421580686674079026752602968553877","date":"2025-04-19T10:18:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131159985399318888736767557821537080050","date":"2025-04-18T16:51:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-16T12:07:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161327873070638588324334311716758662585","date":"2025-04-07T21:44:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280779661296331513102116044746125335636","date":"2025-04-06T12:52:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-04T12:49:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-01T04:39:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-31T04:12:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-28T18:36:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-03-28T18:35:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"22a0e49d-62a8-496d-bf2c-d0770d710f3c","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:08:03+00:00","versionOfRecord":{"articleIdentity":"rs-6320983","link":"https://doi.org/10.1186/s12870-025-06798-y","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-06-05 15:57:52","publishedOnDateReadable":"June 5th, 2025"},"versionCreatedAt":"2025-04-25 12:26:06","video":"","vorDoi":"10.1186/s12870-025-06798-y","vorDoiUrl":"https://doi.org/10.1186/s12870-025-06798-y","workflowStages":[]},"version":"v1","identity":"rs-6320983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6320983","identity":"rs-6320983","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}