Genome-wide identification of ADK gene family associated with heat stress tolerance in chili (Capsicum annuum L.)

Abstract

Chili (Capsicum annuum), also known as hot pepper, is a major vegetable crop in the Solanaceae family. It originated from C. annuum var. minimum, native to Mexico, Southern Peru, and Bolivia. High temperatures negatively affect all key development stages of chili, including fruit set, pollen viability, floral aberration, and the number of seeds per fruit. Heat stress tolerance is a crucial trait in chili and may be regulated by the Adenylate Kinase (ADK) gene family. Although ADK genes have been studied in Arabidopsis thaliana, Oryza sativa, Glycine max, Solanum lycopersicum, and Solanum tuberosum, they remain uncharacterized in C. annuum. This study presents the first genome-wide analysis of the ADK gene family in chili, including gene identification, phylogenetics, motif analysis, expression profiling, and stress-related interaction networks. This study identified nine ADK genes in C. annuum and 70 orthologs from four other species using BLASTp. Phylogenetic analysis grouped the genes into four clades, while gene structure analysis revealed gene lengths ranging from 1,204 to 13,304 bp, with 4 to 17 exons. Conserved motifs and ADK domains were identified. Chromosomal mapping placed the genes across nine chromosomes, with segmental duplications aiding expansion. Predicted subcellular localization suggested that most CaADK proteins localize to chloroplasts and mitochondria. In silico expression profiling showed that CaADK1 and CaADK8 had the highest expression under heat stress, indicating their potential role in thermotolerance. These findings provide novel insights into the CDK gene family in chili and offer molecular targets for enhancing heat stress tolerance in C. annuum.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12863-026-01407-9.

Introduction

Chili (C. annuum), also known as red or hot pepper, is a widely cultivated crop valued for its diverse culinary and economic uses [1]. Despite its importance, chili is highly sensitive to heat stress, which adversely affects its growth, development, and productivity. Elevated temperatures disrupt critical physiological processes, including floral development, pollen viability, fruit set, and seed formation [2, 3]. Furthermore, heat stress alters fruit biochemistry, impacting flavor, color, and market value [4]. The optimal temperature range for chili cultivation ranges between 22 °C and 30 °C; however, in regions like Pakistan, temperatures often exceed 40 °C during May and June, posing severe threats to productivity [5].

To mitigate the effects of heat stress, a deeper understanding of the underlying molecular mechanisms is imperative. Among these, the Adenylate Kinase (ADK) gene family plays a crucial role in cellular energy metabolism by catalyzing the reversible phosphorylation of adenosine monophosphate. ADKs are conserved across plant and animal species and are associated with various stress responses [6]. Studies in crops like rice, maize, potato, and tomato have demonstrated the role of ADKs in maintaining cellular homeostasis and enhancing abiotic stress tolerance [7].

Although the ADK gene family has been characterized in model plants A. thaliana [8], O. sativa [9], and G. hirsutum [10], functional insights in economically valuable Solanaceae crops remain limited. Recent work has begun to explore the ADK genes in Solanum lycopersicum [11] and Solanum tuberosum [6], yet no genome-wide identification or characterization of this gene family has been conducted in C. annuum.

Given the global importance of C. annuum and its vulnerability to abiotic stresses such as heat, salinity, and drought, characterizing ADK genes in this species becomes critical. This study addresses this gap by conducting a genome-wide exploration of the ADK gene family in chili pepper, including gene identification, phylogenetic characterization, conserved motif detection, expression profiling, and interaction network prediction under stress conditions.

This study aims to provide functional insights into ADK-mediated heat stress tolerance in chili and offer molecular targets for the development of resilient C. annuum cultivars, contributing to sustainable agriculture and food security in the context of climate change.

Materials and methods

Identification and characterization of CaADK genes

To identify the ADK gene family in C. annuum L., a comprehensive in-silico pipeline was employed. The A. thaliana AtADK1 protein sequence was retrieved from the TAIR10 database (https://www.arabidopsis.org/) and used as a query for BLASTp searches against the C. annum genome in the Sol Genomics Network (https://solgenomics.net/). Domain verification was performed using the InterProScan (https://www.ebi.ac.uk/interpro) and Pfam database. ADK gene sequence from other species (A. thaliana, S. tuberosum, S. lycopersicum, and O. sativa were retrieved from the Phytozome database comparative analysis.

Phylogenetic analysis and sequence logos

Multiple sequence alignment of ADK protein sequences from five species was performed using ClustalW in MEGA11 [12]. A phylogenetic tree was constructed using the maximum likelihood method with 10,000 bootstrap replications. Sequence logos were generated using WebLogo (https://weblogo.berkeley.edu) to identify conserved amino acid motifs.

Gene structure, conserved motifs, and domain analysis

Genomic and CDS sequences of CaADK genes were obtained from the Sol Genomics Network (https://solgenomics.net/). Gene structure (intron-exon organization) was visualized using GSDS 0.2 (Gene Structure Display Server 2.0) (http://gsds.cbi.pku.edu.cn/) [13]. Conserved motifs were identified using MEME Suite (https://meme-suite.org/meme/) with default parameters (maximum 10 motifs). Conserved domains were annotated using NCBI’s CDD tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/), and all graphical outputs were processed in TBtool [14].

Chromosomal mapping, duplication, and divergence analysis

Gene locations were confirmed using the GFF3 file from Sol Genomics (https://solgenomics.net/) and Ensamble Plants (https://plants.ensembl.org/). Chromosomal distribution and visualization were carried out using TBtool. Gene duplication events were identified based on phylogenetic relationships, and Ka/Ks values were calculated to estimate divergence time using TBtools.

Subcellular localization, physicochemical properties, and promoter analysis

Subcellular localization of CaADK proteins was predicted using CELLO (http://cello.life.nctu.edu.tw/) and WoLF-PSORT (https://wolfpsort.hgc.jp/). Physicochemical properties, including the protein length, molecular weight, and isoelectric point, were determined using Expasy Protparam (https://web.expasy.org/protparam/). Promoter regions (2 kb upstream of the start codon) were analyzed for cis-regulatory elements using PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) using TBtool.

Multiple sequence alignment and collinearity analysis

Multiple sequence alignments were conducted in MEGA11, visualized using GeneDoc to identify conserved and divergent regions. Collinearity analysis was performed between C. annuum and A. thaliana genomes using MCScan (One Step) and TBtool.

Protein-protein interaction and protein structure analysis of CaADK

Protein-protein interaction networks were analyzed using STRING v5.11 (https://string-db.org/). 3D protein structures were modeled using trRosetta (https://yanglab.nankai.edu.cn/trRosetta/) based on the predicted peptide sequences of CaADK proteins.

Insilico expression profiling under heat stress

Expression profiles of CaADK genes under heat stress were analyzed using transcriptome data from C. annuum heat stress studies [15]. Transcriptome data for Capsicum annuum under abiotic stress conditions (heat, cold, salt, and osmotic stress) were obtained from Kang et al. (2020). In this study, RNA was extracted from leaf tissues of C. annuum cv. CM334 plants subjected to various stress treatments, and sequencing was performed using the Illumina HiSeq 2500 platform. Heatmaps representing gene expression levels were generated using TBtools, highlighting genes potentially involved in thermotolerance.

Results

Characterization and Identification of the ADK gene family

A systematic approach was used to identify genes encoding the ADK proteins. Known ADK protein sequence from the model plant A. thaliana was used as queries in BLASTp searches against the C. annuum protein database. The adenylate kinase domain in the Arabidopsis reference protein spans amino acid positions 66 to 322, occupying the central region of the 344-residue protein. This positional information serves as a reference for domain localization in CaADK proteins, details are given in supplementary file (Table S1). Specific criteria defined by BLAST analysis were used to select high-confidence matches. Sequences with an E-value ≤ 6e-18 and a bit score ≥ 79 were specifically identified as significant. Strong sequence similarity to known adenylate kinase genes could be assured by these thresholds. Moreover, domain prediction tools (such as Pfam or InterProScan) were used to confirm the putative function of all selected sequences by identifying their presence of conserved adenylate kinase (ADK) domains.

Nine ADK genes (designated CaADK1 to CaADK9) were identified in C. annuum genome via the Sol Genomics network (https://solgenomics.net/). Similarly, ADK genes were retrieved using BLASTp from four other plant species: 16 from A. thaliana, 13 from S. lycopersicum, 21 from S. tuberosum, and 20 from O. sativa using the Phytozome database (https://phytozome-next.jgi.doe.gov/) Detailed information on these non-Capsicum ADK genes is provided in Supplementary Table S1. Comprehensive details, including locus ID, chromosomal locations, strand polarity, gene length, protein length, CDS length, protein weight, isoelectric points, and GRAVY value of CaADK genes are presented in Table 3.

Table 3.

Physio-chemical properties of CaADK genes

Accession number	Gene ID	Chr. No	Location Start-END	Genomic Sequence	CDS sequence	Peptide Sequence	Protein weight (KDa)	PI	Exons, Introns	Sub-cellular localization	GRAVY
CA09g03590	CaADK1	9	10,220,116–10,225,378	5,263	849	282	30.94	5.89	4, 3	Chloroplast	-0.213
CA12g12080	CaADK2	12	140,948,237–140,959,874	11,638	843	280	31.145	6.54	4, 3	Nuclear and Mitochondrial	-0.272
CA04g00190	CaADK3	4	101,169–104,336	3,168	807	268	30.265	6.46	4, 3	Nuclear	-0.498
CA02g30680	CaADK4	2	170,177,084–170,178,569	1,486	795	264	29.401	6.96	4, 3	Cytoplasmic and Mitochondrial	-0.204
CA09g18490	CaADK5	9	252,291,330–252,297,713	6,384	1872	623	70.097	8.8	17, 16	Mitochondrial and chloroplast	-0.357
CA03g24490	CaADK6	3	230,020,064–230,024,195	4,132	732	243	26.487	8.25	6, 5	Cytoplasmic and Mitochondrial	-0.294
CA03g15940	CaADK7	3	181,544,841–181,546,044	1,204	633	210	25.637	7.19	5, 4	Cytoplasmic	-0.22
CA01g27110	CaADK8	1	221,587,349–221,590,067	2,719	729	242	26.796	5.9	6, 5	Chloroplast and cytoplasmic	-0.283
CA01g06710	CaADK9	1	13,862,604–13,865,634	3031	681	226	25.452	7.59	6, 5	Nuclear and cytoplasmic	-0.464

Chr. No. indicates chromosome number, CDS means Coding sequences and PI stands for Isoelectric Point

Sequence logo and phylogenetic analysis

Sequence logos were generated to assess conserved regions and sequence variability across ADK proteins from A. thaliana, C. annuum, S. lycopersicum. These logoes highlighted moderately conserved amino acid residues, especially from N to C terminal regions, including Gly (G), Pro (P), His (H), Arg (R), Asp (D), and Glu (E) (Fig. 1). Although, overall conservation was moderate, the ADK domain region showed strong conservation, consistent with functional importance across species.

Fig. 1.

Sequence logos showing conserved motifs in ADK proteins from Capsicum annuum, Arabidopsis thaliana, and Solanum lycopersicum. (Letter height indicates amino acid frequency and conservation at each position.)

The phylogenetic tree was constructed using the Maximum Likelihood method in MEGA11, classifying ADK proteins from C. annuum, A. thaliana, and S. lycopersicum into four major clades (Fig. 2). Each clade consisted of distinct gene members, indicating evolutionary divergence. As expected, C. annuum CDK proteins clustered more closely with those from S. lycopersicum (both Solanaceae), while a subset showed distant relationships with A. thaliana, reflecting functional or sequence divergence.

Fig. 2.

Phylogenetic tree of ADK proteins from C. annuum, A. thaliana, and S. lycopersicum constructed using the Maximum Likelihood method, showing evolutionary relationships among gene family members

Most CaADK genes, including CaADK1, CaADK2, CaADK3, CaADK4, CaADK6, CaADK7, CaADK8, and CaADK9, clustered within Clade I alongside tomato and potato ADKs. This strong grouping within the Solanaceae family supports the idea of functional conservation among these orthologs, likely reflecting shared physiological roles, such as energy metabolism and stress responses.

Interestingly, CaADK5 was placed in Clade IV, which also included fewer and more divergent members from other species, suggesting that this gene may represent a functionally specialized or evolutionarily distinct ADK type within C. annuum. This divergence is further supported by motif variation (Fig. 4) and the lower bit score observed in BLAST analysis (Table 1), hinting at possible subfunctionalization or neofunctionalization.

Fig. 4.

Conserved protein motif analysis of CaADK proteins identified using MEME. Colored boxes represent distinct conserved motifs across different CaADK members

Table 1.

List of ADK genes identified in Chili

Sr. No	Gene Name	Gene IDs	Bit-score	E-Value
1	CaADK1	CA09g03590	369	2e-130
2	CaADK2	CA12g12080	336	1e-117
3	CaADK3	CA04g00190	158	5e-48
4	CaADK4	CA02g30680	152	2e-45
5	CaADK5	CA09g18490	125	4e-33
6	CaADK6	CA03g24490	106	3e-28
7	CaADK7	CA03g15940	92.8	4e-23
8	CaADK8	CA01g27110	84.7	6e-20
9	CaADK9	CA01g06710	79.0	6e-18

The presence of CaADKs in multiple clades implies that gene duplication followed by functional divergence might have played a key role in the expansion of the ADK gene family in Capsicum. This diversification could enable C. annuum to fine-tune its cellular energy regulation under various developmental and environmental conditions.

Intron-exon structure, conserved motif, and domain analysis

Gene structure analysis using GSDS2.0 revealed variation in gene lengths (767–13304 bp) and exon-intron structures among CaADK. Exon numbers ranged from 4 to 17: CaADK1 to CaADK4 had 4 exons each, CaADK5 had 17 exons, while the rest varied from 5 to 7 exons (Fig. 3). These differences suggest structural divergence within the family.

Fig. 3.

Exon Intron analysis (The blue color indicates the upstream, yellow color indicates exons and empty lines between the two exons indicate introns)

MEME analysis identified nine conserved motifs across CaADK proteins (Fig. 4). Among them, CaADK1, and CaADK2, contained eight distinct motifs, suggesting a conserved domain structure. In contrast, CaADK5 to CaADK9 contained five motifs, indicating potential functional divergence or specialization within this gene subgroup.

NCBI’s CDD analysis confirmed the presence of canonical ADK domains in all CaADK proteins, such as ADK, ADK superfamily, Cytidylate_kin, or Thymidylate_kin, supporting their functional classification as aldehyde kinases. Some genes also contained additional domains (e.g., AAA_17, DEAD-like helicase N-terminal domain), suggesting possible multifunctional roles or divergent regulation. (Fig. 5).

Fig. 5.

Conserved domain analysis of Capsicum annuum ADK proteins. Illustrating the functional domains present in each CaADK protein, highlighting structural similarities and differences

Chromosomal distribution, gene duplication, and divergence

Nine ADK genes were mapped to six out of twelve C. annuum chromosomes, with some chromosomal loci harboring multiple genes (Fig. 6). Chromosome Ca01, Ca03, and Ca09 contains 2 genes each while, Ca02, Ca04 and Ca12 contains single gene. Ca01 contains CaADK8 and CaADK9 which is larges chromosome of chili genome with size nearly 337mb. Ca02 contains CaADK4 while Ca03 contains CaADK6 and CaADK7. Ca04 contains CaADK4 while Ca09 contains CaADK1, and CaADK5, and at last Ca12 contains CaADK2 gene.

Fig. 6.

Chromosomal distribution of CaADK genes. The figure shows the physical locations of CaADK genes across different chromosomes

Ka/Ks analysis revealed evolutionary constraints on these gene pairs. Most pairs exhibited a Ka/Ks ratio < 1, indicating purifying selection. CaADK5 and CaADK6 had the highest Ks value (2.53), suggesting an ancient duplication event (~ 192.83 MYA). Duplication time for other pairs ranged from ~ 69.91 to 192.83 MYA (Table 2), supporting a history of long-term divergence and functional retention.

Table 2.

Nonsynonymous and synonymous (Ka/Ks) substitution rate

Gene I	Gene II	Ka	Ks	Ka/Ks	Duplication type	Time (MYA)
CaADK8	CaADK9	0.250892	1.780732	0.140893	Tendem	135.72
CaADK5	CaADK6	0.741107	2.530022	0.292925	Segmental	192.83
CaADK1	CaADK2	0.276428	1.81015	0.15271	Segmental	137.96
CaADK3	CaADK4	0.162167	0.917247	0.176798	Segmental	69.91

Subcellular localization, physicochemical properties, and promoter analysis

WoLF PSORT predicted that CaADK proteins are localized in various organelles, including mitochondria (CaADK2, CaADK4, CaADK5, and CaADK6), chloroplasts (CaADK1, CaADK8), nucleus (CaADK3, CaADK9), and cytoplasm (CaADK4, CaADK7, CaADK9) as shown in Fig. 7.

Fig. 7.

Predicted subcellular localization of CaADK proteins

Physicochemical properties further revealed functional diversity (Table 3). Molecular weights ranged from 25.45 to 70.10 kDa, with isoelectric points between 5.89 and 8.8. All proteins had negative GRAVY values, indicating a hydrophilic nature. CaADK5 stood out with a large size and complex exon-intron structure, hinting at a potentially regulatory role.

Promotor analysis (3.0 kb upstream) identified various cis-acting elements, involved in hormonal, environmental and stress responses (Fig. 8). These include hormones responsive motifs (ABA, MeJA, GA, AUX), abiotic stress related elements (LTR, DRE) and core promoter elements, suggesting that CaADK gene are regulated by diverse signaling pathways crucial for development and stress adaptation. Elements such as ABRE (ABA responsive), DRE (drought responsive), HSE (heat shock), and LTR (low temperature) were most prominent in CaADK1 and CaADK8, which also had the greatest transcript levels under stress.

Fig. 8.

Predicted cis-acting regulatory elements in the promoter regions of CaADK genes. The elements are associated with stress response, hormone signaling, and developmental processes

Multiple sequence alignment

Multiple sequence alignments through MEGA11 and GeneDoc revealed considerable sequence divergence, particularly in the N-terminal region (positions 1–60), where insertions and deletions were evident. Despite this, conserved catalytic residues and binding motifs crucial for ADK activity were preserved across the family, supporting functional conservation in energy metabolism and stress signaling (Fig. 9).

Fig. 9.

Multiple sequence alignment of Capsicum annuum (CaADK) proteins showing conserved amino acid residues and regions across the gene family

Protein-protein interaction analysis

Protein-protein interaction (PPI) analysis indicated that CaADK proteins interact in a complex network (Fig. 10). Most proteins showed weak or indirect interactions, suggesting participation in diverse biological processes. However, a strong interaction was observed between CaADK8 and CaADK9, implying involvement in a shared protein complex. Or pathways, potentially linked to stress responses or ATP/AMP regulations.

Fig. 10.

Protein-protein interaction (The thickness of lines between the colored dots indicates the magnitude of interaction)

Protein structure analysis

Predicted 3D structure of CaADK proteins revealed structural diversity, indicative of functional specialization (Fig. 11). While some proteins (e.g., CaADK3–CaADK6) exhibited compact and stable structure, others (e.g., CaADK4–CaADK5) had extended or disordered regions, likely reflecting regulatory roles or structural flexibility needed for interaction with diverse molecular patterns.

Fig. 11.

Predicted three-dimensional structures of CaADK proteins, illustrating structural features and similarities among family members

In-silico expression analysis

Transcriptomic data under heat stress (40°C) at multiple time points (3 H, 6 H, 12 H, 24 H and 72 H) showed dynamic expression patterns (Fig. 12). CaADK1, and CaADK8 showed constantly high expression across all time points, suggesting a key role in heat stress response and potential involvement in thermotolerance mechanisms in C. annuum.

Fig. 12.

In silico expression analysis of CaADK genes across different temperature stress conditions. Red indicates high expression, while blue represents low expression levels

Discussion

Global warming, primarily driven by greenhouse gas emissions from fuel combustion, deforestation, urbanization, and industrialization [16], is disturbing patterns of solar radiation, temperature, and precipitation patterns [17]. These changes significantly impact water resources, agriculture, coastal zones, freshwater ecosystems, and forest systems and contribute to glacial melting, desertification, and flooding. The long-term consequences include threats to food security and public health [18]. Even a slight climate shift can drastically influence crop yield, exacerbating regional disparities and creating new challenges for the farmers [19]. According to the Intergovernmental Panel on Climate Change [20], the agriculture sector of developing nations is especially vulnerable to extreme weather events like rising temperatures, droughts, and floods.

In this context, identifying genes that enhance heat stress tolerance is crucial for breeding critical hybrids that maintain high productivity. While many heat-responsive genes have been characterized in various crops, ADK genes stand out for their involvement in vital physiological processes, including energy metabolism, cytokinin regulation, and stress signaling. In A. thaliana, O. sativa, and G. max, for instance, ADK genes have been linked to stress response mechanisms such as oxidative stress, drought, and salinity adaptation.

Yet, no functional characterization of ADK genes had been reported in C. annuum. Our genome-wide and in silico analysis identified and characterized the CaADK gene family, revealing both conserved and species-specific features when compared with ADKs from other plants. Expression profiles and predicted protein-protein interactions under abiotic stress conditions suggest that CaADK may play important roles similar to their orthologs in other species, while also exhibiting regulatory adaptations unique to chili. These findings reinforce the evolutionary conservation of ADK genes and open new avenues for functional validation and crop improvement.

ADK is a highly conserved enzyme across life forms, essential for maintaining energy homeostasis by catalyzing the reversible conversion of AMP to ATP [21]. Its presence in multiple cellular compartments, including the nucleus, cytoplasm, mitochondria, and endoplasmic reticulum, underlines its importance in metabolic regulations and stress response.

Phylogenetic analysis using the Maximum Likelihood methods grouped ADK proteins from C. annuum, A. thaliana, S. lycopersicum, S. tuberosum, and O. sativa into four distinct clades. This clustering reflects their evolutionary history and supports the hypothesis of a shared ancestral origin. Understanding these relationships is crucial not only for predicting gene function but also for identifying gene duplication events that contribute to trait diversity and stress resilience [22].

Gene structure analysis revealed variability in introns and exons organization among CaADK genes, with intron numbers from two to nine and exon numbers from three to ten. Intron diversity plays a regulatory role in gene expression and development [23], while exon variations facilitate alternative splicing, leading to protein isoforms with specialized functions and enhanced adaptability [24].

Motifs analysis uncovered conserved sequence patterns associated with regulatory and enzymatic functions. These motifs are crucial for identifying key genes with a role in stress responses, aiding breeding strategies for stress-resilient crops. Differences in motif composition also reflect gene duplication and evolutionary divergence.

Protein domains analysis revealed a wide range of functional domains, including ADK, ADK superfamily, AAA_17, DEAD-like helicase, and NK superfamily domains. Their presence underscores the functional diversity of the ADK family. Notably, domains such as DEAD-like helicase and NK superfamily are associated with stress tolerance [6, 11], further emphasizing their importance in abiotic stress adaptation. Understanding domain distribution aids in selecting candidate genes for improving stress resilience in chili.

Promoter analysis identified numerous stress-responsive cis-regulatory elements (CREs), including elements associated with heat, drought, light, and pathogen response. Elements such as ABRE (ABA responsive), DRE (drought responsive), HSE (Heat shock), and LTR (low temperature) were particularly prevalent in CaADK1 and CaADK8, both of which also showed the highest transcript levels under stress conditions. This suggests that these CREs play an important role in transcript upregulation during abiotic stress, positioning CaADK1 and CaADK8 as key players in the plant stress response network.

Chromosomal localization of ADK genes provides valuable markers for marker-assisted selection (MAS), allowing breeders to select for traits such as stress tolerance, high yield, and improved growth [25]. Given the role of ADK genes in energy metabolism and stress signaling, their physical properties are a critical resource for crop improvement programs.

Protein-protein interaction (PPI) analyses further deepen our understanding of the CaADK functional network, showing how these proteins interact in regulatory and metabolic pathways. Such insights are instrumental in identifying master regulators of stress tolerance and other agronomic traits [26, 27].

In short, this study presents the first comprehensive characterization of the CaADK gene family in C. annuum. The identification of stress-responsive genes such as CaADK1 and CaADK8, supported by promoter CREs, transcriptomic data, and evolutionary conservation, highlights their potential utility in breeding programs. These genes represent promising candidates for further functional validation and could contribute significantly to developing cultivars with improved tolerance to abiotic stresses, ensuring productivity and quality in the face of climate change.

Conclusion

The identification and characterization of genes associated with heat stress tolerance are essential for developing high-yielding and climate-resilient varieties. While several genes have been studied in different crops, this study presents the first comprehensive genome-wide analysis of the ADK gene family in chili. A total of nine ADK genes were identified and subjected to phylogenetic analysis, revealing the evolutionary relationship of ADK genes from A. thaliana, S. lycopersicum, and S. tuberosum. The dispersed placement of CaADK genes across the phylogenetic tree suggests their evolutionary divergence and potential functional diversity. Gene structure analysis revealed that CaADK genes contain between 2 and 9 introns and 3–10 exons, indicating signification structural variation that may influence gene regulation and functional specialization. Promoter analysis identified multiple Cis-regulatory elements related to light, hormonal, and abiotic stress responses, suggesting that CaADK genes are responsive to a wide range of environmental stimuli. Segmental duplication events were observed, contributing to the expansion of the ADK gene family in chili. Collinearity analysis with A. thaliana confirmed the conservation of ADK genes across species, highlighting their evolutionary importance. Notably, In-silico expression profiling revealed that CaADK1 and CaADK8 showed the highest transcript levels under heat stress, suggesting their prominent role in the heat stress response mechanism. Overall, this study underscores the functional significance of the CaADK gene family, particularly in enhancing heat stress tolerance and regulating energy metabolism, which are crucial for sustaining yield and quality under changing climate conditions. Further research should focus on functional validation of CaADK1 and CaADK8 to incorporate them into breeding programs aimed at developing heat-resilient cultivars.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Saad Farid Usmani: Conceptualization, data analysis, and manuscript writing. Muhammad Abu Bakar Saddique, Sajid Fiaz,: Project administration, methodology, and manuscript revision. Muhammad Abu Bakar Saddique: Supervision, resources, and final approval of the manuscript. Saad Farid Usmani, Muhammad Abu Bakar Saddique, Hafiz Nazar Farid: Data collection, software development, and manuscript editing. Muataz A. Abdalla, Umsalama A. H. Ahmed, Badr Alharthi, Rui Pan and Seung Hwan Yang: reviewed article, manuscript editing, proof-reading, literature review, data re-interpretation. Saad Farid Usmani, Muhammad Abu Bakar Saddique, Ummara Waheed, Sajid Fiaz: Validation, investigation, and manuscript revision.

Funding

No specific funding has been received to conduct present research.

Data availability

All data is present within the manuscript and can be made available on reasonable request with corresponding author.

Declarations

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Competing interests

The authors declare no competing interests.