Unravelling the expression patterns of the CCO gene family in Barley (Hordeum vulgare) under various stress conditions

Abstract

Background

Carotenoid cleavage oxygenases (CCOs) are non-heme iron-dependent enzymes that catalyze the oxidative cleavage of carotenoids and play important roles in the biosynthesis of apocarotenoids involved in plant growth, development, and stress responses. However, a comprehensive genome-wide characterization of the CCO gene family in barley (Hordeum vulgare L.) has not been reported.

Methods and Results

In this study, a genome-wide analysis was performed to identify and characterize CCO genes in barley using comparative genomics and bioinformatics approaches. A total of 11 HvCCO genes were identified and classified into three subfamilies: carotenoid cleavage dioxygenases (CCDs), carotenoid cleavage dioxygenase-like proteins (CCDLs), and 9-cis-epoxycarotenoid dioxygenases (NCEDs). Gene structure and conserved motif analyses revealed high conservation within subfamilies, while promoter analysis identified 190 cis-regulatory elements associated with light, phytohormone, stress, and developmental regulation. Chromosomal mapping, duplication, and synteny analyses indicated that segmental duplication contributed to HvCCO gene family expansion, while Ka/Ks analysis suggested purifying selection of duplicated gene pairs. Prediction of miRNA targets identified 12 barley miRNAs potentially regulating HvCCO genes. Publicly available RNA-seq datasets revealed tissue-specific expression patterns. Under abiotic and biotic stresses, HvCCD4 and HvCCD6 showed relatively consistent expression, whereas HvNCED1 and HvNCED2 exhibited increased expression under drought conditions in tolerant genotypes.

Conclusions

These findings provide a comprehensive genomic resource for the barley CCO gene family and identify HvCCD4 and HvCCD6 as potential candidate genes for future functional validation and genetic improvement of stress tolerance in barley.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s11033-026-12775-x.

Introduction

Carotenoids are isoprenoid pigments that play an essential role in photosynthesis, photoprotection, and antioxidant defence in plants. The oxidative cleavage of carotenoids is catalyzed by carotenoid cleavage oxygenases (CCOs), a family of non-heme iron-dependent enzymes that generate biologically active apocarotenoids involved in plant growth, development, signaling, and adaptation to environmental stresses [1, 2]. According to sequence similarity and catalytic function, the CCO family is classified into two major subfamilies: carotenoid cleavage dioxygenases (CCDs) and 9-cis-epoxycarotenoid dioxygenases (NCEDs) [3]. The CCO proteins possess a highly conserved retinal pigment epithelium-specific 65 kDa (RPE65; PF03055) domain, which is essential for carotenoid cleavage activity. These CCO gene family plays a central role in carotenoid metabolism by generating diverse apocarotenoids that function as phytohormones, signaling molecules, and volatile compounds. In particular, NCED enzymes catalyze the rate-limiting step in abscisic acid (ABA) biosynthesis, thereby regulating seed dormancy, stomatal closure, drought tolerance, and other abiotic stress responses [4]. Members of the CCD subfamily participate in the biosynthesis of strigolactones, which regulate shoot branching, root architecture, and beneficial interactions with arbuscular mycorrhizal fungi, while several CCDs also produce volatile apocarotenoids that contribute to plant aroma, pollinator attraction, and defense against pathogens and herbivores [2, 5]. Consequently, the CCO gene family has emerged as an important target for understanding plant adaptation to environmental stresses and improving crop performance.

To date, CCO genes have been identified and characterized in several plant species, including Arabidopsis thaliana, where the CCO family comprises nine members involved in carotenoid metabolism and abscisic acid (ABA) biosynthesis [4]. The CCO family comprises 16 members in rice [6], 10 in cucumber [7], 21 in sunflower [8], and 19 in Cucumis melo [9]. In wheat, functional characterization of TaNCED1 demonstrated that NCED-mediated ABA biosynthesis plays an important role in drought and salinity responses, and heterologous expression of TaNCED1 significantly enhanced drought tolerance in tobacco [10]. These studies collectively demonstrate the evolutionary conservation and biological importance of CCO genes across both model plants and economically important crop species.

Barley (Hordeum vulgare L.) is one of the world’s most important cereal crops, serving as a major source of food, animal feed, and malt for the brewing industry [11, 12]. Although barley possesses relatively high levels of tolerance to several environmental stresses, grain yield and quality are still substantially affected by drought, salinity, waterlogging, and pathogen infection. These stresses represent some of the most important production constraints in barley-growing regions worldwide and therefore provide important targets for investigating stress-responsive genes. Since ABA biosynthesis and apocarotenoid-mediated signaling are closely associated with plant responses to both abiotic and biotic stresses, genome-wide characterization of the barley CCO gene family may facilitate the identification of candidate genes associated with stress responses and provide a foundation for future functional studies. Despite the biological significance of CCO genes, a comprehensive genome-wide characterization of this gene family has not yet been reported in barley. Therefore, the present study aimed to identify and characterize the HvCCO gene family through genome-wide analyses, including phylogenetic relationships, gene structure, conserved motifs, chromosomal distribution, duplication events, synteny, cis-regulatory elements, and miRNA target prediction. Furthermore, publicly available RNA-seq datasets representing drought, salt, waterlogging, and yellow mosaic disease were analyzed to investigate the in-silico expression profiles of HvCCO genes across different tissues and stress conditions. The findings of this study provide a valuable genomic resource and identify potential candidate genes for future functional validation and genetic improvement of stress tolerance in barley.

Materials and methods

Identification and characterization of HvCCO genes in Barley

The amino acid sequences of the CCO proteins from Arabidopsis thaliana were retrieved and used as query sequences to identify putative HvCCO genes in the barley (Hordeum vulgare L.) genome. Candidate proteins were screened for the presence of the conserved RPE65 domain (Pfam accession: PF03055), which is the characteristic catalytic domain of carotenoid cleavage oxygenases and is essential for carotenoid cleavage activity [13, 1]. A BLASTP search was performed against the barley genome available in Phytozome v13 using the default search parameters. The retrieved candidate sequences were subsequently verified for the presence of the RPE65 domain using the Conserved Domain Database (CDD) at NCBI [14]. Gene information, including gene IDs, chromosomal locations, genomic sequences, coding DNA sequences (CDS), and protein sequences, was retrieved from the Phytozome v13 database. The physicochemical properties of HvCCO proteins, including amino acid length, molecular weight, theoretical isoelectric point (pI), grand average of hydropathicity (GRAVY), and instability index, were predicted using ProtParam [15]. Subcellular localization was predicted using WoLF PSORT with default parameters [16].

Analysis of gene structure and conserved cis-regulatory elements

Gene structures were analyzed using Gene Structure Display Server (GSDS v2.0) [17] by aligning the genomic DNA sequences with their corresponding coding sequences (CDS). Default parameters of GSDS were used for exon-intron structure visualization. To identify putative cis-regulatory elements (CREs), the 2,000 bp upstream promoter sequences of each HvCCO gene were retrieved from the barley genome and analyzed using the PlantCARE database [18] with default parameters.

Analysis of conserved motif and domain prediction

Conserved motifs were identified using the MEME Suite v5.5.4 [19]. The analysis was performed using the following parameters: maximum number of motifs = 15, optimum motif width = 6–50 amino acids, and other parameters were kept at their default settings. Conserved domains of HvCCO proteins were confirmed using the Conserved Domain Database (CDD) at NCBI with default search parameters [20].

Comparative phylogenetic analysis

To compare the CCO gene family across H. vulgare, A. thaliana, T. aestivum, Z. mays, and O. sativa, amino acid sequences were retrieved from the Phytozome database using BLAST-p. Multiple sequence alignment was performed using MUSCLE implemented in MEGA11. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with the Poisson substitution model, pairwise deletion, and 1,000 bootstrap replications to evaluate branch support.

Gene duplication and synteny analysis

To determine the divergence time, we calculated the Ka/Ks ratios for paralogous gene pairs by using TB tools software [21]. Subsequently, divergence time (DT) was estimated using the formula T = Ks/2λ, where λ represents the neutral substitution rate (6.1 × 10⁻⁹ substitutions per synonymous site per year), a value that has been widely adopted in genome-wide evolutionary studies for estimating gene duplication times [22, 23]. Gene duplication and synteny analyses were performed using MCScanX v1.0 [24] implemented in TBtools with default parameters. Syntenic relationships among barley and other plant genomes were visualized using the Dual Synteny Plot module of TBtools.

MiRNA analysis

The micro-RNA (miRNA) sequences associated with 11 CCO genes in barley were identified using the psRNATarget tool [25]. This was achieved by inputting the CDS sequences of all the CCO genes and using the default scoring schema with an expected value cutoff of ≤ 3.0, while all remaining parameters were retained as default.

Gene ontology enrichment analysis

For Gene ontology analysis Uniprot (Online database) was used to find out the molecular functions of genes as well as the various biological processes. Gene ontology (GO) enrichment analysis was performed using ShinyGO v0.77 [26], with GO terms considered significantly enriched at FDR-adjusted P < 0.05.

In Silico expression analysis of HvCCO genes in eight barley tissues from different developmental stages

To investigate tissue-specific expression patterns, publicly available RNA-seq data generated by the International Barley Genome Sequencing Consortium (IBGSC) were retrieved from the BARLEX expression database [27]. No new experiments were performed in this study. Normalized transcript abundance values (TPM) were obtained directly from the public database and used to visualize expression profiles of HvCCO genes across eight barley tissues representing different developmental stages. Heatmaps were generated using TBtools after log₂ transformation of TPM values for visualization purposes only.

In Silico expression analysis of HvCCO genes in response to abiotic and biotic stresses in Barley

To investigate the stress-responsive expression patterns of HvCCO genes, publicly available RNA-seq datasets were retrieved from the NCBI Gene Expression Omnibus (GEO) and Expression Atlas. No RNA-seq experiments were performed in the present study. The datasets included GSE225255 (drought stress; [28], GSE230751 (salt and waterlogging stress; [29], GSE225976 (yellow mosaic disease; [30], together with publicly available transcriptomic datasets associated with inflorescence meristem size and yield-related traits. Normalized TPM/FPKM expression values generated by the original studies were downloaded and log₂-transformed [log₂(TPM/FPKM + 1)] solely for visualization of gene expression patterns. Heatmaps were generated using TBtools to illustrate the relative expression profiles of HvCCO genes under different stress conditions. Since the analyses were based entirely on previously processed public transcriptomic datasets, no additional statistical analyses or differential expression tests were performed in this study. Where available (e.g., Expression Atlas dataset E-MTAB-4634), differential expression statistics, including log₂ fold change and statistical significance, had already been computed using the original RNA-seq data by the corresponding database pipeline.

Results

Identification of the CCO Genes in H. vulgare and their localization

Using the conserved RPE65 (PF03055) domain as a query, a blastp search of the barley genome identified a total of 11 HvCCO genes. The results revealed that these genes encode proteins with lengths varying from 324 to 644 amino acids, and molecular weights ranging from 36.209 to 71.963 kDa; with HvCCD5 was found to be the shortest protein while HvCCD3 was the longest one. All the HvCCO proteins were identified as hydrophilic, as indicated by the negative GRAVY scores. The isoelectric points of HvCCO ranged from 5.34 (HvNCED1) to 7.98 (HvCCD7). Subcellular localization analysis predicted that most of the HvCCO proteins were located in the cytoplasm, while others were localized in the chloroplast (Figure S1; Table 1). These results provide the foundation for subsequent analyses of gene structure, evolutionary relationships, and expression patterns analysis of the HvCCO gene family in barley.

Table 1.

Physiochemical properties of CCO gene family

CCO Gene name	TXN ID	Chr no.	Start	End	Direction	No. of aa	CDS Seq	Molecular weight	PI value	GRAVY	Inst-Index	Exon	Intron
HvCCD1	HORVU3Hr1G071170	3	538,296,855	538,299,286	F	478	1437	53087.11	5.49	-0.265	35.8	5	4
HvCCD2(CCDL)	HORVU3Hr1G108100	3	674,152,811	674,164,162	R	596	1791	66563.95	5.7	-0.132	48.47	13	12
HvNCED1	HORVU5Hr1G008050	5	18,037,868	18,039,692	R	607	1824	65985.17	5.34	-0.257	40.9	1	0
HvNCED2	HORVU5Hr1G092850	5	589,662,188	589,664,852	F	644	1935	70044.2	5.51	-0.23	43.32	1	0
HvCCD3(CCDL)	HORVU5Hr1G055740	5	437,153,572	437,157,796	F	643	1932	71963.53	5.85	-0.301	45.62	13	12
HvCCD4	HORVU5Hr1G000320	5	1,313,686	1,319,573	F	439	1317	50036.33	5.81	-0.323	26.8	11	10
HvCCD5(CCDL)	HORVU1Hr1G000550	1	1,505,981	1,510,426	R	324	975	36209.11	5.93	-0.331	29.67	7	6
HvCCD6	HORVU6Hr1G068720	6	476,864,534	476,870,198	R	643	1932	69174.5	6.24	-0.201	47.1	1	0
HvCCD7	HORVU7Hr1G111580	7	634,156,077	634,158,668	F	428	1287	46101.37	7.98	-0.026	59.28	6	5
HvCCD8	HORVU4Hr1G006300	4	14,973,851	14,978,888	F	555	1668	62730.81	5.8	-0.345	33.72	14	13
HvCCD9(CCDL)	HORVU4Hr1G077550	4	604,897,887	604,910,615	F	572	1716	63608.39	5.93	-0.223	36.17	13	12

Exon introns analysis

The results of gene structure prediction revealed that three CCO genes, including HvNCED1, HvNCED2, and HvCCD6 (27.27%), possess a simple structure, which only has one exon with no intron, indicating no interruption in coding sequences. Different from others, HvCCD8 owns the most complex structure owning 14 exons, followed by HvCCD2 and HvCCD3, which possess 13 exons individually, and HvCCD4 owns 11 exons (Table 1; Fig. 1A). The NCED subfamily was characterized by a simple gene structure, which only contains one exon and no intron. The difference of exon-intron organization among HvCCO genes indicated that there might be the functional diversity of this gene family during evolution, while the conservative exon organization within the NCED subfamily may imply that this subfamily may have highly conserved protein functions.

Fig. 1.

Visualization of Intron-Exon Structures in all HvCCO genes along with the Phylogenetic Tree (A), HvCCO gene distribution on barley chromosomes indicates potential gene duplication on distinct chromosomes (B), Timing of HvCCO gene duplication in barley as determined by Ka and Ks values (C)

Conserved cis-elements

As shown in Fig. 2 and Table S1, we identified totally 190 cis-elements, excluding common elements such as TATA-box and CAAT-box, including 84 kinds of light responsive, 68 kinds of hormone responsive cis-elements and 38 kinds of stress, development and metabolism-responsive elements. The G-Box was the most common cis-element responsive to light, while ABRE was the most common cis-element responsive to plant hormones and some motif, such as MBS, LTR, CAT-box, and WUN-motif were abundant in stress responsive cis-regulatory elements.

Fig. 2.

Cis-Regulatory Elements in the Promoters of HvCCO Genes in Barley. (A) hormone-responsive elements. (B) Light-responsive elements and (C) stress and development metabolism-responsive elements

Conserved motif analysis and domain prediction

Conserved domain analysis confirmed that all HvCCO proteins contained the characteristic RPE65 superfamily domain, confirming their classification as carotenoid cleavage oxygenases (Fig. 3; Table S2). All 15 conserved motifs were found in the barley NCED1 and NCED2 proteins, including a conservation of their relative locations. All CCO proteins have motif 1, 3, 5, and 10, with the exception of HvCCD7. HvCCD7 has the lowest motif count with only 3 motifs, while HvCCD2, HvCCD3, HvCCD8, HvNCED1, and HvNCED2 include all 15 motifs across all CCOs. Overall, the NCED subfamily exhibited a higher degree of motif conservation than the CCD subfamily, suggesting greater structural conservation within this group. The conservation of individual motifs within individual subfamilies indicates that these genes are closely related evolutionarily and have similar functions.

Fig. 3.

A) Phylogenetic grouping of 11 HvCCO genes. B) Total 20 motifs identified in all HvCCO genes, with motifs represented by numbers for better understanding. C) Conserved domains in all HvCCO genes in barley, highlighting key functional regions

Phylogenetic analysis of the HvCCO gene family

A neighbor-joining phylogenetic tree was constructed to investigate the evolutionary relationships between CCO proteins by using 79 protein sequences, including 11 from barley (Hordeum vulgare), 9 from Arabidopsis thaliana, 14 from rice (Oryza sativa), 22 from wheat (Triticum aestivum), and 23 from maize (Zea mays) sequences (Fig. 4). Based on the obtained results, three distinct clades of CCO proteins were classified into three distinct clades: (CCD), carotenoid cleavage dioxygenase-like proteins (CCDL) and (NCED). Within the same subfamily, the proteins were clustered together, irrespective of species they were derived from, indicating that the CCO family is evolutionarily conserved in monocot and dicot plants. Within the three clades, the CCD clade was the largest one, whereas the NCED clade contained only two barley members (HvNCED1 and HvNCED2) and the CCDL clade included four barley proteins. The observed pattern of CCO gene distribution among plant species reflects their conserved domain composition and can be used to define their evolution and functional divergence.

Fig. 4.

Neighbor-joining phylogenetic tree showing the evolutionary relationships among carotenoid cleavage oxygenase (CCO) proteins from barley (Hordeum vulgare), Arabidopsis thaliana, rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays). The HvCCO proteins are indicated by red stars. The phylogenetic tree was constructed using MEGA11 based on protein sequence alignment and subsequently visualized and annotated using the Interactive Tree of Life (iTOL) software

Chromosomal distribution and duplication time analysis of HvCCO genes

Chromosomal distribution analysis demonstrated that the 11 HvCCO genes were unevenly distributed on a total of six barley chromosomes (1, 3, 4, 5, 6, and 7), while, no HvCCO gene was detected on chromosome 2 (Fig. 1B). Analysis of duplicated gene pairs showed that the Ka/Ks ratios ranged from 0.0999 for the HvCCD4/HvCCD8 gene pair to 0.4859 for the HvCCD6/HvCCD7 and were less than 1, indicating that these duplicated genes have been subjected to purifying selection (Fig. 1C; Table S3). Based on synonymous substitution rates, the estimated duplication time varied among the gene pairs. The HvCCD6/HvCCD7 gene pair showed the oldest estimated duplication time (approximately 135.95 MYA), whereas the HvNCED1/HvNCED2 gene pair exhibited a comparatively more recent duplication time estimation (approximately 34.95 MYA). These findings suggest that HvCCO genes have undergone duplication at different evolutionary periods, contributing to the diversification of the barley CCO gene family.

Gene duplication and synteny analysis

Synteny analysis detected a total of five duplicated HvCCO gene pairs distributed across the barley genome, indicating that segmental duplication has contributed to the expansion of the HvCCO gene family (Fig. 5A). The 11 HvCCO genes were located within syntenic blocks on all chromosomes except chromosome 2. Chromosome 5 contained the highest number of duplicated regions, harboring both HvNCED1 and HvNCED2. In contrast, HvCCD1 was identified as a singleton gene, with no detectable duplicated counterpart. Comparative collinearity analysis revealed five orthologous CCO gene pairs between barley and maize (Fig. 5B), four between barley and rice (Fig. 5C), and six between barley and Brachypodium (Fig. 5D), while no orthologous gene pair was detected between barley and Arabidopsis. Collectively, these results indicated a higher degree of genomic conservation of the HvCCO gene family among grasses compared with the dicot species Arabidopsis.

Fig. 5.

A) The advanced circos diagram illustrates the chromosomal locations of HvCCO genes in barley. Connecting lines between genes on different chromosomes indicate potential gene duplication events. B) Dual synteny analysis of barley CCO genes with maize. C) Rice and D) Brachypodium. Red and green lines highlight the syntenic relationships between pairs of CCO genes

MiRNA analysis

A total of 12 miRNAs were predicted to target the HvCCO genes (Table S4). The identified miRNAs ranged from 20 to 24 nucleotides (nt) in length. Among the HvCCO genes, HvCCD6 was targeted by two miRNAs, hvu-miR6207 and hvu-miR6182, whereas each of the remaining HvCCO genes was predicted to be targeted by a single miRNA. These predicted miRNA–target interactions suggest that post-transcriptional regulation may contribute to the regulation of HvCCO genes.

Functional annotation of genes (GO analysis)

Gene Ontology (GO) Term Analysis was conducted to annotate and classify the functions of the identified CCO genes based on their molecular functions, biological processes, and cellular components. The role of CCO genes in related biological processes includes primary involvement in carotene catabolic process (GO:0016121), terpene catabolic process (GO:0046247), hydrocarbon catabolic process (GO:0120253), carotene metabolic process (GO:0016119), and isoprenoid catabolic process (GO:0008300). Molecularly, CCO genes are involved in carotenoid dioxygenase activity (GO:00010436), oxidoreductase activity (GO:0016702), dioxygenase activity (GO:0051213), and metal ion binding (GO:0046872). Analysis of cellular components reveals that the majority of HvCCO genes are located in the chloroplast stroma (GO:0009570) (Fig. 6). Additionally, the orthologs and their expression and function in Arabidopsis were identified and are presented in (Table S5), providing comprehensive insights into the functional annotation and expression patterns of orthologous genes.

Fig. 6.

Gene ontology lollipop plot of HvCCO genes. The length of the bar indicates fold enrichment, the color represents −log10 (false discovery rate), and the size of the circle at the tip shows the number of genes involved

In Silico expression analysis of HvCCO genes in eight barley tissues at different developmental stages

Analysis of publicly available RNA-seq datasets revealed distinct, tissue-specific expression profiles among the HvCCO genes (Figure S2). HvCCD4 was continuously expressed across all tissues and developmental stages examined, with maximal transcript abundance in the seedling shoot. HvCCD6 expression was predominantly restricted to the internode, while HvNCED1 transcripts accumulated most strongly in the germinating embryo and during inflorescences development (1 cm and 5 mm). HvCCD1 displayed a comparatively low expression level, detectable exclusively in the seedling root, whereas HvCCD3 transcripts were present in both the germinating embryo and the seedling root. HvCCD8 displayed detectable expression in the caryopsis at15 days post-anthesis and in the seedling shoot. The remaining HvCCO family members showed low or negligible expression across all tissues and developmental stages analyzed. The corresponding orthologous genes in Arabidopsis thaliana, along with their previously reported expression patterns and functional annotations, are summarized in Table S5.

Analysis of HvCCO genes in response to abiotic and biotic stresses

Analysis of publicly available RNA-seq datasets unveiled differential expression levels of HvCCO genes under the influence of abiotic and biotic stress conditions (Fig. 7). Upon drought, salt, and waterlogging stress, HvCCD4 and HvCCD6 genes showed fairly consistent expression among the analyzed genotypes, with generally higher transcript abundance in leaves than in roots. For HvNCED1 and HvNCED2 genes, transcript levels were elevated under drought stress in the tolerant genotype (BCS-24DR) and the drought-treated sensitive genotype (BCS-8DR), whereas HvCCD5 expression was comparably high in BCS-24CK and BCS-24DR. In contrast, HvCCD2 was more prominent in the drought-treated sensitive genotype (BCS-8DR) (Fig. 7A). In case of biotic stress, HvCCD4 and HvCCD6 genes consistently showed higher transcript accumulation in both healthy and diseased plants, while HvCCD2 and HvCCD3 transcripts demonstrate genotype-specific expression pattern in the diseased UD variety (Fig. 7D). These trends hint at a putative differential response of HvCCO genes to different stress conditions.

Fig. 7.

Heatmap showing the in-silico expression profiles of HvCCO genes under different stress conditions and developmental traits based on publicly available transcriptomic datasets. (A) Drought stress, (B) Salt stress, (C) Waterlogging stress, (D) Yellow mosaic disease, and (E) Inflorescence meristem size and yield-related traits. Heatmaps were generated using normalized expression values obtained from the original published RNA-seq datasets and are presented to visualize the relative expression patterns of HvCCO genes across different conditions. The color scale represents relative gene expression levels, with higher and lower expression indicated by contrasting colors. Statistical analyses of differential gene expression were performed in the respective original studies

Discussion

Barley (Hordeum vulgare L.) is an agronomically vital cereal crop, valued globally for its versatility and versatility and adaptability across diverse cropping systems. Despite substantial research on various gene families in barley have been extensively characterized, the carotenoid cleavage oxygenase (CCO) gene family has received comparatively little attention.

In the present study, 11 HvCCO genes were identified and comprehensively characterized at the genomic and protein levels. Analysis of their physicochemical properties showed that all HvCCO proteins possessed negative grand average of hydropathicity (GRAVY) values, indicative of a hydrophilic character, consistent with previous findings reported for the CCO gene family in Liriodendron chinense [31]. The instability index analysis predicted HvCCD1, HvCCD4, HvCCD5, HvCCD8, and HvCCD9 to be stable proteins, suggesting that these members may retain structural stability under normal physiological conditions. Subcellular localization prediction further indicated that most HvCCO proteins localized to the cytoplasm, with a smaller subset to localize to the chloroplast. This distribution pattern is consistent with previous findings in cucumber [7] and aligns with the established biological roles of CCO proteins, given that carotenoid biosynthesis occurs predominantly within plastids, whereas several downstream metabolic and signaling processes are mediated by cytoplasmic components. Collectively, these findings indicate that the identified HvCCO proteins possess conserved physicochemical characteristics and subcellular localization patterns comparable to those reported in other plant species.

Conserved motif analysis showed that all HvCCO proteins had contained motifs 1, 3, 5, and 10, except HvCCD7, which had possessed only three conserved motifs. This might be attributed to evolutionary divergence or the loss of conserved motif regions during gene evolution. The number of conserved motifs in the two subfamilies was higher for NCED than for CCD, implying stronger conservation of the former subfamily (Fig. 3a. Similar conserved motif distribution patterns have also been reported for Rosaceae species [32]. Thus, these data suggest that motif conservation might be general characteristic of the CCO gene family evolution.

Gene structure analysis revealed that members of the NCED subfamily members had a simple exon–intron organization with only one exon without introns. Such a conserved gene structure is reported for tobacco [33] and seems to be characteristic of NCED subfamily evolution in different plant species (Fig. 3b). The simplicity of the exon–intron organization may have implications for the functional conservation of these genes. In particular, this finding might help in elucidating the roles of these genes in plant responses to abiotic and biotic stresses.

Promoter analysis identified a total of 190 cis-regulatory elements, including light-responsive (G-box), hormone-responsive (ABRE), and stress- and development-related elements such as the MYB-binding site (MBS), which is associated with drought responsiveness. The distribution pattern of these diverse cis-elements suggests that HvCCO genes may be regulated by multiple environmental and hormonal signals. Similar cis-regulatory element distributions have been reported for the CCO gene family in Liriodendron chinense [31] and banana (Musa acuminata) [34], indicating that promoter architecture is highly conserved among plant species. These conserved regulatory elements provide a basis for the differential expression patterns of HvCCO genes observed across tissues and under abiotic and biotic stresses.

Phylogenetic analysis of 79 CCO proteins from barley, Arabidopsis thaliana, rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays) classified the proteins into three major clades: carotenoid cleavage dioxygenases (CCD), carotenoid cleavage dioxygenase-like proteins (CCDL), and 9-cis-epoxycarotenoid dioxygenases (NCED). A similar phylogenetic classification has been reported for the CCO gene family in cucumber [7] and sunflower (Helianthus annuus) [8], indicating that the CCO family is evolutionarily conserved across plant species. The clustering of HvCCO proteins with homologous genes from other cereals, particularly wheat and rice, suggests a high degree of sequence conservation among monocot species. This conserved phylogenetic relationship supports the structural conservation and potential functional conservation of the HvCCO gene family.

Analysis of the duplicated HvCCO gene pairs revealed Ka/Ks ratios of less than 1 for all paralogous pairs, suggesting that each of these genes have likely evolved under purifying selection. Five paralogous pairs of HvCCO genes were identified that were unevenly distributed across the barley genome, indicating that segmental duplication has played an important role in the expansion of the HvCCO gene family. Similar patterns of segmental duplication have been reported for the CCO gene family in cotton and apple [21]. The estimated divergence times suggest, however, that HvCCO gene duplication events occurred at different evolutionary periods. The HvCCD6/HvCCD7 gene pair was estimated to be diverged approximately 135.95 MYA, representing an ancient duplication event, whereas HvNCED1/HvNCED2 diverged approximately 34.95 MYA, indicating a comparatively more recent duplication event. These estimates broadly correspond to major evolutionary periods in the angiosperm evolution, but should be interpreted with caution due to the reliance on the assumed neutral substitution rate, they should be interpreted with caution. Together, these findings suggest that the HvCCO gene family has experienced both ancient and relatively recent duplication events, contributing to its evolutionary diversification in barley.

Comparative synteny analysis identified five orthologous CCO gene pairs between barley and maize, four between barley and rice, and six between barley and Brachypodium, while, none were found between barley and Arabidopsis. The higher synteny conservation observed between barley and other grass species reflects the greater conservation of CCO genes within the monocots compared with the dicot species Arabidopsis.

In the current study, 12 miRNAs were predicted to target the HvCCO genes. Of these, hvu-miR6176 was predicted to target HvCCD1 and HvNCED1, hvu-miR6207 targeted HvCCD6, and hvu-miR168-5p targeted HvCCD8. These predicted miRNA–target interactions suggest that post-transcriptional regulation plays a role in the regulation of HvCCO gene expression in barley. Of particular interest is the miR168 that is a highly conserved plant miRNA that regulates ARGONAUTE1 (AGO1) and has been linked to abscisic acid (ABA)-mediated signalling, as well as abiotic stress responses, including drought and salinity, in plants [35]. In contrast, limited functional information is currently available for barley-specific miRNAs, such as hvu-miR6176 and hvu-miR6207, regarding stress responses. consequently, these predicted miRNA–target interactions offer promising avenues for future functional studies to elucidate their roles in regulating HvCCO genes during stress adaptation. However, the predicted interactions identified in the current study require further experimental validation.

Analysis of publicly available RNA-seq dataset revealed that HvCCD4 and HvCCD6 exhibited relatively high expression level under drought, salt, and waterlogging stress and their expressions were relatively higher under yellow mosaic disease conditions, suggesting that these two genes might be involved in stress responsive pathways in barley. Earlier reports have also shown that CCO family members are involved in carotenoid metabolism and abiotic stress responses [13, 36]. Moreover, the relatively higher expression level of HvCCD4 and HvCCD6 in leaves and comparatively higher expression level in roots of HvCCD2, HvCCD3, HvCCD9 suggested their differential regulation in the tissues. Expression analysis using publicly available RNA sequence datasets indicated that HvCCD4 and HvCCD6 showed relatively higher expression in yellow mosaic disease condition than other HvCCO genes, suggesting that these genes may also participate in the barley response to biotic stress. However, these observations are based on publicly available RNA-seq datasets, which need to be validated experimentally to conclude their roles in stress response.

Conclusion

To conclude, this genome-wide study reports the first comprehensive characterization of the carotenoid cleavage oxygenase (CCO) gene family in barley and identified a total of 11 HvCCO genes. The study highlights the comparative and differential gene structure, motifs, cis-acting regulatory elements, phylogenetic relationships, gene duplication, synteny, and miRNA-target interactions revealed that the HvCCO gene family is evolutionarily conserved and possesses diverse regulatory features. Analysis of publicly available RNA-seq datasets demonstrated distinct tissue-specific and stress-responsive expression patterns, with HvCCD4 and HvCCD6 exhibiting relatively higher expression under drought, salt, waterlogging, and yellow mosaic disease conditions. These findings suggest that HvCCD4 and HvCCD6 may play important roles in barley stress responses. Overall, this study provides a valuable genomic resource for future functional investigations and identifies HvCCD4 and HvCCD6 can be considered as potential candidates for improving stress tolerance in barley by molecular breeding and future functional validation approaches.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, methodology, formal analysis, investigation, data curation, writing-original draft preparation, P.K.S., S.K.S., M.S. and R.S. Investigation, data curation, supervision, writing-review and editing, and M.S.S. All authors have read and agreed to the published version of the manuscript.

Funding

Open access funding provided by Sultan Qaboos University. This research received no external funding.

Data availability

All data supporting the findings of this study are available within the paper.

Declarations

Conflict of interest

None to declare.

Declarations

We used the ChatGPT tool to improve the language and readability of the manuscript. However, the entire manuscript was later manually reviewed and edited by the authors.