Genomics and Applied Biology 2024, Vol.15, No.4, 200-211 http://bioscipublisher.com/index.php/gab 204 5.2 Stage-specific expression The stage-specific expression of R2R3-MYB genes is a crucial element in their functional characterization. These genes are involved in the regulation of diverse processes throughout the plant life cycle, encompassing germination, flowering, and grain filling. The expression levels of Os2R_MYB genes exhibit significant variation across different developmental stages, indicating their involvement in regulating developmental processes. For example, Kang et al. (2022) analyzed the transcription levels of 20 Os2R_MYB genes under different stress conditions. Their findings revealed that some genes exhibited altered expression patterns at specific developmental stages, underscoring their pivotal role in developmental regulation. 5.3 Responses to environmental stimuli R2R3-MYB genes have also been demonstrated to respond to a range of environmental stimuli, including abiotic and biotic stresses. The expression of these genes in response to stress conditions provides insights into their roles in stress tolerance mechanisms. In rice, the Os2R_MYB genes contain multiple stress-responsive elements within their promoter regions, including ABRE, TGACG, CGTCA, and MBS motifs. These elements are associated with responses to abiotic stresses, such as drought and heavy metal exposure. For example, the transcription levels of Os2R_MYB genes were found to be significantly altered under conditions of polyethylene glycol (PEG) and cadmium chloride (CdCl2) stress, indicating their involvement in stress response pathways (Kang et al., 2022). 6 Functional Roles of R2R3-MYBGenes in Anthocyanin Biosynthesis 6.1 Known functions of R2R3-MYBgenes in other species R2R3-MYB TFs play a crucial role in regulating anthocyanin biosynthesis across a diverse range of plant species. In Nitraria sibirica Pall., the R2R3-MYB gene NsMYB1 has been identified as a key regulator of anthocyanin biosynthesis, influencing fruit color differentiation by promoting the transcription of structural genes involved in the anthocyanin biosynthetic pathway (Bao et al., 2021). Similarly, in wheat (Triticum aestivum), the R2R3-MYB protein TaPL1 has been demonstrated to act as a positive regulator of anthocyanin biosynthesis, particularly in response to environmental stresses such as cold, salt, and light, which are known to induce anthocyanin accumulation (Shin et al., 2016). In Freesia hybrida, the R2R3-MYB regulator FhPAP1 has been demonstrated to activate anthocyanin biosynthetic genes and interact with other TFs to form the MYB-bHLH-WD40 (MBW) complex, which is essential for anthocyanin production (Li et al., 2020). These examples illustrate the conserved role of R2R3-MYBgenes in anthocyanin biosynthesis across different plant species. 6.2 Potential roles in purple rice R2R3-MYBgenes probably play a significant role in the biosynthesis of anthocyanins in purple rice, as they do in other species. The OsC1 gene, an R2R3-MYB transcription factor in rice, has been demonstrated to regulate anthocyanin biosynthesis by modulating the expression of late anthocyanin biosynthetic pathway (ABP) genes. The overexpression of OsC1 in white rice plants has been demonstrated to induce anthocyanin production, which in turn results in enhanced photosynthetic efficiency and a reduction in oxidative stress (Upadhyaya et al., 2021). Furthermore, the OsKala3 gene, another R2R3-MYB transcription factor in rice, interacts with the bHLH transcription factor OsKala4 to activate anthocyanin biosynthetic genes, thereby contributing to the pigmentation of the rice pericarp (Kim et al., 2021). These findings indicate that R2R3-MYBgenes in purple rice may similarly regulate anthocyanin biosynthesis, thereby enhancing the plant’s antioxidant properties and stress tolerance. 6.3 Experimental approaches for functional validation To validate the functional roles of R2R3-MYB genes in anthocyanin biosynthesis in colored rice, several experimental approaches may be employed. 1) Gene overexpression and knockout studies: The overexpression of candidate R2R3-MYB genes, such as OsC1 and OsKala3, in transgenic rice plants can facilitate the determination of their impact on anthocyanin production. Conversely, the knockout or knockdown of these genes can be achieved using CRISPR/Cas9 or RNA interference (RNAi), which will reveal their necessity in the biosynthetic pathway (Upadhyaya et al., 2021; Kim et al., 2021).
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