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

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.

Keywords: Capsicum annuum; HD-Zip gene family; Abiotic stress; Gene expression.

Fig. 1

Multiple sequence alignment and conserved motif analysis of CaHD-Zip proteins. 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

Fig. 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

Fig. 3

Genomic synteny analysis of CaHD-Zip genes. (a) Intraspecific synteny analysis within the Capsicum annuum genome. Red lines indicate syntenic relationships among CaHD-Zip genes, while blue trapezoids and red curves represent gene density distribution along the chromosomes. (b) Comparative genomic synteny analysis between Capsicum annuum and five other plant species. C. annuum represents pepper, A. thaliana represents Arabidopsis, while S. lycopersicum (tomato), O. sativa (rice), S. tuberosum (potato), and Z. mays (maize) serve as reference species in the synteny analysis. Gray lines connect all syntenic gene pairs, while red lines specifically highlight syntenic relationships involving HD-Zip genes

Fig. 4

Phylogenetic Relationships, Conserved Motifs, Gene Structures, and Domain Architectures of Capsicum HD-Zip Genes. a Phylogenetic tree of HD-Zip 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)

Fig. 5

Prediction and Functional Categorization of Cis-Regulatory Elements in the CaHD-Zip Gene Family. a Distribution of cis-regulatory elements identified within the 2000 bp upstream promoter regions of CaHD-Zip genes. 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

Fig. 6

Relative Expression Analysis of CaHD-Zip Genes in Different Capsicum Tissues. This figure illustrates the transcripts per million (TPM) based expression profiles of CaHD-Zip genes across 11 distinct tissues in Capsicum annuum. 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

Fig. 7

Spatiotemporal Expression Analysis of CaHD-Zip Genes in Response to Hormonal Treatments. (a) Heatmap showing TPM-based expression profiles of CaHD-Zip genes 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 CaHD-Zip genes (DEGs; |log2 FC|≥ 1, adjusted p < 0.05) under ABA, MeJA, ET, and SA treatments

Fig. 8

Expression Profiles and Differentially Expressed Genes (DEGs) Analysis of CaHD-Zip Genes Under Different Abiotic Stress. Conditions (a–d) TPM-based expression profiles of CaHD-Zip genes under cold (a), heat (b), drought (c), and salt (d) stress at 0, 3, 6, 12, 24, and 72 h. 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 CaHD-Zip genes responsive to cold, heat, drought, and salt stress

Fig. 9

Stress-responsive expression validation of CaHD-Zip genes. qRT-PCR analysis of eight CaHD-Zip genes 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 < 0.05, **P < 0.01