Carotenoids, these fascinating isoprenoid compounds, have captivated scientific curiosity ever since their serendipitous discovery in the 19th century (Stafsnes et al., 2010). Their enigmatic diversity unfolds through the analysis and identification of more than 700 distinct types over the years, revealing a rich tapestry of potential benefits waiting to be unraveled (Gitelson and Merzlyak, 1998). These compounds have various biological functions across different organisms, such as bacteria and plants (Misawa, 2011). Carotenoids serve as important secondary pigments in photosynthetic organisms, protecting them from photooxidation and helping to capture light more efficiently (Hashimoto et al., 2016). Additionally, certain apocarotenoids, produced through the cleavage of carotenoids, aid in regulating plant growth and development, as well as their response to biotic and abiotic stresses (Felemban et al., 2019). The carotenoid cleavage oxygenase’s (CCOs) are a series of enzymes involved in the breakdown of carotenoids which are naturally occurring pigments that contribute color to many fruits, vegetables, and flowers (Liang et al., 2018). CCOs comprise two types of enzymes carotenoid cleavage dioxygenases (CCDs) and 9-cis-epoxycarotenoid dioxygenases (NCEDs). These enzymes are essential for turning carotenoids into apocarotenoids, which have degraded (Yue et al., 2022). CCOs use oxygen to split the carotenoid molecule and produce apocarotenoids, which have a variety of physiological roles such as serving as plant hormones, enticing pollinators, and giving defense against environmental stress (Shi et al., 2023).

9-Cis-epoxycarotenoid dioxygenase (NCED) is a major enzyme in the ABA production pathway found in numerous plant species (Sun et al., 2012). NCED is evolutionarily conserved throughout plant lineages, with little divergence within its subfamily and excellent exon conservation (Liu et al., 2023). This demonstrates the significance of NCED in preserving the structural integrity of the protein for its function. NCED genes change somewhat slowly, which may contribute to the functional divergence of NCED subfamilies across tissues (Wan et al., 2021). Various expression patterns and activity levels of NCED subfamilies in various plant tissues may contribute to controlling ABA biosynthesis and plant responses to environmental signals (Pu et al., 2020).

The identification of the NCED gene marked a significant milestone in advancing our comprehension of ABA production and its role in plant growth and development. The NCED gene was initially cloned in the maize ABA-deletion mutant Vp14, leading to a better understanding of NCED’s involvement in ABA synthesis (Fei et al., 2023). Further research in Arabidopsis revealed nine CCOs genes, five of which (AtNCED2, AtNCED3, AtNCED5, AtNCED6, and AtNCED9) are involved in ABA synthesis (Hu et al., 2021). Manipulation of AtNCED3 increased drought tolerance by increasing endogenous ABA accumulation (Seki et al., 2007). Identifying NCED genes in additional plant species, including Tamatim, cowpea, and rice, increased our understanding of NCED evolution and function across plant taxa (Wang and Guo, 2023). These findings show that NCED is a conserved and essential component of the ABA biosynthesis pathway that governs plant responses to environmental stress and developmental stimuli (Tan et al., 2003). The NCED gene family has been identified and investigated in many plant species, such as Arabidopsis  (Tan et al., 2003), citrus (Xian et al., 2014), cotton (Li et al., 2021), cucumber (Zhang et al., 2022), wheat, and tobacco (Zhang et al., 2014). However, the function of NCED genes in rice has not been clearly defined. Carotenoid cleavage dioxygenases (CCDs) are an enzyme that uses molecular oxygen to cleave carotenoids, a group of pigments commonly found in plants, animals, and microorganisms (Daruwalla and Kiser, 2020). CCDs are responsible for various biological functions such as vision, stress response, and reproduction, producing apocarotenoids with multiple biological activities (Wei et al., 2022). Arabidopsis now has nine CCO gene members, five NCED genes, and four CCD genes. The CCD gene family is divided into five subfamilies based on differences in cleavage sites and substrates: CCD1, CCD4, CCD7, CCD8, and NCED (Cheng et al., 2023). For the first time, CCD-like (CCDL), a new member of the CCD family, was found in tomatoes (Cheng et al., 2022). The retinal pigment epithelium membrane protein (RPE65) domain is found in all CCD genes (Kiser, 2022), which is typical of enzymes involved in carotenoid cleavage (Kim et al., 2016). The CCD gene family has been found and studied in several plant species, including Arabidopsis (Chernys and Zeevaart, 2000). CCD gene family is described in grass species maize (Zea mays), rice (O. sativa), and sorghum (Sorghum bicolor) (Zhou et al., 2020). CCD research has also been conducted in wheat (Takezawa, 2000), watermelon (Cheng et al., 2022), pepper (Yao et al., 2022), tobacco (Zhou et al., 2019), rapeseed (Zhou et al., 2020), and maize (Sun et al., 2022). However, the function of CCD genes in rice has not been precisely defined. Extensive research on CCO genes has been conducted on Arabidopsis thaliana. Still, due to the absence of CCDL proteins, it is not an ideal candidate for investigating CCD enzymes and their functions. Fortunately, CCDL genes have been discovered in Citrullus lanatus and Cucumis melo, providing opportunities for further exploration (Xu et al., 2023). Carotenoids provide vibrant colors to fruits and vegetables, and their production and degradation are facilitated by CCD enzyme family (Kiokias et al., 2016). C. lanatus and C. melo have CCDL genes, making them good candidates for future research into CCD enzymes and their functions.

Rice (O. sativa) is a cereal crop grown in many parts of the world (Sami et al., 2023). It is a staple food for over half of the world’s population and is Asia’s most important food crop (Fukagawa and Ziska, 2019). The International Rice Genome Sequencing Project (IRGSP), a cooperation of research institutes from ten nations, sequenced the genome of rice (O. sativa) for the first time in 2002 (Project and Sasaki, 2005). However, research on rice CCO genes is sparse (He et al., 2022). The functional role of the CCO family in rice development remains unclear. To address this issue, we performed an extensive bioinformatics analysis of the complete rice genome in order to identify all potential members of the OsCCO gene family. We discovered several genes involved in the change in ABA content during rice seedling growth and development under environmental stress. The RNA-seq data analysis has identified promising candidates in OsNCED6 and OsNCED10 genes, offering potential avenues for the development of salt stress-resistant rice varieties to enhance crop yield in saline conditions. Meanwhile, OsCCD7 and OsCCD8 genes display higher activity in inflorescences, implying their potential involvement in grain development, although further investigations, such as gene cloning and functional analysis, are essential to validate their roles in diverse physiological and biological processes. This in-silico analysis expands our comprehensive understanding of the rice OsCCO gene family across the genome. The results of this study shed new light on the functional diversity and evolutionary aspects of the CCO gene family in plants. Our findings will serve as a valuable resource for further investigations, enabling the functional analysis and cloning of these genes through the comprehensive genome-wide identification and characterization conducted in this study.

Rice is a major crop for many poor countries, but because of many environmental stressors, both abiotic and biotic, it is vulnerable to yield reductions (Seah et al., 2023). To help minimize these losses, transcription factors (TFs) play a vital role in regulating plant responses to these stresses. The investigation of OsCCOs gene family is essential in plant research particularly in relation to its role in environmental stress such as drought, seed dormancy, salinity, cold and heat stress. The role of the OsCCOs gene family in rice is yet to be fully comprehended. Knowledge of CCO genes’ functions and their association with these activities opens the door for novel approaches to improve crop production and increase the resilience of rice plants against environmental stressors (Dhankher and Foyer, 2018).

A genome-wide investigation was carried out to find and understand the CCO genes in rice (Griffiths et al., 2003). The physicochemical properties of sixteen OsCCO genes in the rice genome were investigated to observe their distinctions within a clade of proteins. It was determined that all identified OsCCO proteins exhibited hydrophilic characteristics as evidenced by negative GRAVY values, reflecting a tendency to interact with water and possess electrical charges dependent on pH levels (Wilkins et al., 1998). Upon examination using the instability index, it was observed that ten distinct proteins possessed features suggestive of instability. The examination of subcellular localization demonstrated that OsCCO proteins were dispersed among multiple organelles, such as the chloroplast, mitochondria, cytoplasm, cytosol, endoplasmic reticulum nucleus and plasma membrane (Tan et al., 2003). It is significant to observe that more than half of the proteins, precisely 47.76% and 23.98%, were correspondingly identified in the chloroplast and cytoplasm. This suggests that the OsCCO proteins could serve vital roles within these particular organelles.

By examining the genome of diverse species, substantial insight can be gained regarding the history and arrangement of genes. Moreover, such analyses can enable the transfer of genomic data from a well-researched taxonomic group to one that is not as extensively studied (Auldridge et al., 2006). In the current research, 56 paralogous genes were detected within OsCCO, indicating genes replicating via gene duplication (Xue et al., 2023). This occurrence of duplication grants particularly valuable knowledge into the expansion of gene families, a phenomenon frequently observed in the realm of plants as a result of both tandem and segmental duplications (Fatima et al., 2023).

Comparative analysis of members of similar subgroups can provide significant insights into their functional behavior. This study, 37 CCO proteins were discovered and classified into three subfamilies based on their sequence structures and evolutionary relationships (Xue et al., 2023). Twelve OsNCED proteins, seventeen OsCCD proteins, and eight OsCCDL proteins were discovered using phylogenetic analysis. These findings imply that the OsNCED and OsCCD proteins in their respective subgroups may perform roles similar to AtNCED and AtCCD in Arabidopsis. In contrast, the OsCCDL proteins may perform functions similar to ClCCDLa, ClCCDLb, and CmCCDL proteins (Zhao et al., 2022).

Previous studies have suggested that positioning exons and introns within gene families is important for evolution (Zhao et al., 2022). This study’s investigation of gene structure and motifs revealed that the distribution of exons, introns, and motifs among members of the same population and clade was compatible with the shape of the phylogenetic tree (Wei et al., 2022). Every CCO gene contained exons and introns ( Figure 4 ). Notably, in keeping with the traits of plants, the motifs of the NCED subfamily were found to be more maintained than those of the CCD subfamily.

Cis-regulatory elements are frequently located in the promoter region of genes and are essential for controlling gene expression at the transcriptional level (Zhou et al., 2019). Research on cis-regulatory elements revealed that a significant portion of the largest group, constituting 28 elements (equivalent to 37.833%), had a stress response and contained motifs such as ABRE, MYB, STRE, TGACG-motif, and WRE3. Meanwhile, the second-largest group, composed of 19 elements (or 25.67%), focused on metabolism and development and contained motifs such as A-box, Box 4, and TCCC-motif (Zhou et al., 2019). In addition, OsCCO contains various motifs responsible for different responses, such as CGTCA-motif and TGACG-motif for the MeJA response, TCA-element for the SA response, GARE-motif, TATC-box, and P-box for the GA response, and the ABRE TGA-element for the Auxin response.

Plants can undergo transpiration and photosynthetic processes, which can be affected by drought and salinity, resulting in reduced crop yields. The role of stomata is crucial in this process (Ahluwalia et al., 2021). Rice plants under salt stress were examined for the functions of various genes, including OsCCO genes, using transcriptome data (Liang et al., 2021). Interestingly, while CCD genes did not show any impact during salt stress, OsNCED6 and OsNCED10 genes were identified as possible candidates for developing salt-resistant rice varieties based on the RNA-seq data analysis. By producing ABA hormone, these genes facilitate stomata closure, resulting in reduced water loss due to transpiration and improved water-use efficiency under salt-stress conditions, thereby impacting rice Physiology and development.

The Go enrichment analysis of OsCCO genes highlights their significant involvement in carotene metabolic and terpene catabolic processes (Bao et al., 2021), pointing to their roles in pigment synthesis, vitamin A production and plant defense mechanisms. Additionally, their association with carotenoid dioxygenase activity underscores their potential in carotenoid cleavage reactions. The high enrichment of OsCCO gene in the chloroplast stroma suggests their crucial participation in diverse metabolic processes within this organelle, particularly related to photosynthesis and pigment biosynthesis (Chengcheng et al., 2022).

OsCCD11 stands out with the highest number of associations in our network analysis. This protein, along with OsNCED4, OsNCED6, OsNCED16, and OsCCD1 forms a distinct cluster of interconnected proteins, implying their potential involvement in shared cellular processes. Conversely, OsCCDL12 and OsCCDL3 exhibit a broader range of interactions, suggesting their roles in diverse biological contexts. These results hint at a specific functional relationship or concerted involvement in a particular biological process within the larger interaction network (Shimada et al., 2017).

Through a comprehensive transcriptome analysis utilizing RNA sequencing data from the NCBI GEO database (GSE227706), the importance of the CCD group in rice has been elucidated (Zhao et al., 2021). An in-depth investigation unveiled that CCD7 and CCD8 exhibited elevated expression levels during the critical floral development phase in rice. This significant discovery not only opens doors for further research aimed at enhancing rice productivity and quality but also establishes a potential foundation for the development of novel rice varieties with enhanced traits (Yue et al., 2022).

The analysis of RNA-seq data of various part of rice gene expression investigations (available at https://www.ebi.ac.uk/gxa/experiments?experimentType=differential/) has revealed important information about the expression patterns of OsCCOs gene in rice (Priya et al., 2019). The results show that most CCD genes are expressed in seed and shoot tissues, implying that they are involved in important activities associated to these organs. Furthermore, the CCDL group expresses specifically in the seed and carpel, whereas the NCED group expresses specifically in the anther, carpel, and seed. This extensive analysis of gene expression data demonstrates the importance of the OsCCOs gene family in rice crop improvement. The discovery of their tissue-specific expression patterns opens the door to future breeding programs to produce new rice varieties with improved characteristics and attributes.

MicroRNAs (miRNAs) are crucial regulatory molecules in plants that play a significant role in almost all biological processes, including plant growth, development, and responses to biotic and abiotic stress (Ding et al., 2020). They are highly conserved and exhibit specific functions (Millar et al., 2019). The investigation showed that the OsCCOs has a variety of miRNAs with various roles, including by focusing on genes involved in nitrogen uptake and assimilation, Osa-miR169 controls how effectively the plant uses nitrogen (Peng et al., 2020). It is believed that Osa-miR5802 regulates rice grain yield and size (Kumar and Sanan-Mishra, 2022). By targeting genes associated with stress signaling and cellular homeostasis, Osa-miR1430 aids rice plants in surviving drought stress. Osa-miRN2366 is thought to be involved in controlling the growth and development of rice plants, while its exact role is still unclear (61). By focusing on genes involved in stress signaling and ion transport, Osa-miR1432 controls how rice plants react to salt stress (Zhai et al., 2020). Osa-miR2275 is an OsCCD1 microRNA that has been connected to the production of siRNAs, small interfering RNAs that are unique to the inflorescence of rice (Fei et al., 2021). The possible implication from the observed results conveys that miRNAs could potentially govern the post-transcriptional control relating to OsCCO genes in rice progression.

This study’s outcomes provide fresh insights into the functional diversity and evolutionary dimensions of the CCO gene family in plants. These discoveries will serve as a valuable asset for future research, facilitating the functional examination and cloning of these genes. This comprehensive genome-wide identification and characterization, as undertaken in this study, will enable further investigations.

Sixteen OsCCO genes were discovered in O.sativa in this study. Based on structural analyses, the number of introns in OsCCO genes ranged from one to twelve. The presence of cis-regulatory elements related to light responsiveness, development-related response, developmental and hormone responsiveness, and certain abiotic stress in the promoter of OsCCO genes suggested their function in the abiotic stress of rice. OsNCED6 and OsNCED10 genes identified by RNA-seq data analysis can potentially be utilized to develop salt stress-resistant rice varieties for enhanced crop yield under salt conditions. The OsCCD7 and OsCCD8 genes are more active in inflorescences than in other organs, which suggests that they might play a role in grain development. However, additional research, including gene cloning and functional analysis, is required to confirm the significance of these genes in various physiological and biological processes. This in-silico analysis reveals the extensive genome-wide knowledge of rice OsCCO genes.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

MZ: Data curation, Formal Analysis, Writing – original draft. AS: Conceptualization, Data curation, Methodology, Writing – original draft. MS: Data curation, Formal Analysis, Software, Supervision, Writing – original draft. WA: Data curation, Investigation, Methodology, Writing – review & editing. SAli: Funding acquisition, Methodology, Resources, Writing – original draft. QA: Formal Analysis, Funding acquisition, Resources, Software, Writing – review & editing. SM: Writing – review & editing, Methodology, Resources. MS: Data curation, Funding acquisition, Investigation, Writing – review & editing. DA: Formal Analysis, Funding acquisition, Project administration, Supervision, Visualization, Writing – review & editing. SAla: Data curation, Funding acquisition, Methodology, Project administration, Resources, Writing – review & editing. IM: Writing – review & editing, Methodology, Formal Analysis, Software.