The CCO gene family has been extensively studied in various species in the past few years through bioinformatics analysis. However, research on the HaCCO genes family in sunflower has been relatively limited compared to other species, leaving a gap in our knowledge about CCOs in sunflower (Priya et al., 2019).
Twenty-one HaCCO genes in sunflower genome were studied based on their physicochemical parameters to see how they differed among proteins in the same clade (Cheng et al., 2022). All identified HaCCO proteins were hydrophilic based on negative GRAVY values, indicating a preference for interaction with water with net electrical charges at different pH levels (Priya et al., 2019). The instability index indicated that six proteins were unstable. Subcellular localization analysis revealed that HaCCO proteins were distributed in various organelles, including the chloroplast, mitochondria, cytoplasm, cytosol, endoplasmic reticulum, nucleus, and plasma membrane (Ji et al., 2023). Interestingly, more than half of the proteins were localized in the chloroplast (86.5) and cytoplasm (80.5) (52.2%, 167 of 302.5), suggesting that HaCCO proteins may play crucial roles in the function of these organelles.
Comparing genomes between different species can provide valuable information about the evolution and organization of genes (Yao et al., 2022). It also helps to transfer genomic data from a well-studied taxon to a less-studied one (Xu et al., 2013). In this case, we identified 73 pairs of paralogous genes in HaCCO, which are genes that have arisen through gene duplication events. This duplication can provide useful information about expanding gene families, a common occurrence in plants due to tandem and segmental duplications (Wei et al., 2022).
Members with comparable subgroups tend to have similar behaviors (Zhou et al., 2019). As a result, phylogenetic analysis might aid in advancing functional genomics. The current study, 43 CCO proteins were identified with full-length domain sequences and grouped into three subfamilies based on their sequence structures and phylogenetic relationships. The phylogenetic trees revealed the presence of seven HaNCED proteins, ten HaCCD proteins and four HaCCDL proteins. These results suggest that the HaNCED and HaCCD proteins within this subgroup may have functions similar to those of AtNCED and AtCCD, respectively (Zhang et al., 2021). Similarly, the HaCCDL proteins may have functions similar to ClCCDLa, ClCCDLb, and CmCCDL proteins (Zhao et al., 2021).
Previous research suggests that positioning exons and introns within gene families is crucial to evolution (Yue et al., 2022). In this study, the analysis of gene structure and motifs indicated that members of the same population and clade had similar numbers and locations of exons, introns, and motifs, consistent with the topology of phylogenetic trees (Wei et al.). Exons and introns were present in all CCO genes (Fig. 3). It's a characteristic of plants that the motifs of the NCED subfamily were discovered to be more preserved than those of the CCD subfamily. Additionally, cis-regulatory elements are critical in controlling gene expression at the transcriptional level. These elements are found in the promoter region of genes (Wei et al.).
A study of the cis-regulatory elements revealed that a significant proportion of cis-elements involved in the largest group, consisting of 213 (51.82%) elements, was responsive to light and contained motifs like Box 4, MRE, and G-box. The second-largest group, with 103 (25.06%) elements, was related to plant hormones and contained motifs like CGTCA-motif and TGACG-motif for MeJA response, TCA-element for SA response, GARE-motif, TATC-box, and P-box for GA response, ABRE for ABA response, and TGA-element for Auxin response.
Drought and salinity stresses can reduce photosynthetic rates and transpiration in plants, ultimately resulting in crop yield losses. Stomata are crucial in plant photosynthetic activities and transpiration (Liang et al., 2011). To investigate the potential functions of HaCCO genes, the expression patterns of HaCCO genes were studied using transcriptomic data from sunflower plants exposed to water deficit. The RNA-seq data analysis (GEO accession: GSE145709) identified HaNCED16 and HaNCED19 genes as potential candidates for developing drought stress-resistant sunflower varieties, while the CCD genes were not involved during drought stress. This suggests that these genes may be crucial in regulating sunflower physiology and development, specifically under drought stress conditions (Dhar et al., 2020). These genes aid in the production of ABA, a hormone that helps in the closure of stomata, reducing water loss through transpiration and improving water-use efficiency under drought-stress conditions (Bouvier et al., 2003).
Similarly, the RNA-seq data analysis of organ-specific gene expression (GEO accession: GSE221055) has revealed useful insights into putative pigmentation candidates in leaves. These genes are involved in chloroplast-related functions and light response in plants (Zhu et al., 2010). The differential expression analysis of RNA-seq data revealed that the HaCCD12 and HaCCD20 genes have higher expression levels in leaves than in other organs implying that they play particular functions in leaf pigmentation. This suggests that these genes may help synthesize and accumulate carotenoids in leaves, which leads to colour (Cárdenas-Conejo et al., 2023). They could regulate chlorophyll production, chloroplast biogenesis or form photosynthetic pigment-protein complexes (Liang et al., 2020).
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 (Y. Wang et al., 2023), (Mazhar et al., 2023). They are highly conserved and exhibit specific functions. In our study, 16 (76.19%) HaCCO genes were found to have 60 miRNA target sites predicted based on previously described sunflower miRNAs. These findings suggest that miRNAs may play a role in the post-transcriptional regulation of HaCCO genes during sunflower development.