Bioscience Methods 2024, Vol.15, No.4, 184-195 http://bioscipublisher.com/index.php/bm 1 85 The objective of this study is to overview the functional role of R2R3-MYB genes in the anthocyanin biosynthesis pathway of purple or black rice using CRISPR/Cas9 and overexpression techniques. Specifically, the study focuses on the identification and characterization of the R2R3-MYB genes involved in the anthocyanin biosynthesis pathway in purple rice. Furthermore, it summarizes the effects of CRISPR/Cas9-mediated knockout and overexpression of these genes on anthocyanin accumulation and plant phenotype. The aim is to elucidate the regulatory mechanisms by which R2R3-MYB genes control the expression of anthocyanin biosynthetic genes and to assess the potential of manipulating R2R3-MYB genes to enhance anthocyanin content and improve stress tolerance in rice. The attainment of these objectives will facilitate a comprehensive understanding of the role of R2R3-MYB genes in anthocyanin biosynthesis and their potential applications in crop improvement. 2 Overview of R2R3-MYB Genes 2.1 Structure and function of R2R3-MYB TFs R2R3-MYB TFs constitute a large family of proteins that are distinguished by the presence of two MYB domains (R2 and R3) at their N-terminus. These domains are involved in DNA binding. These TFs play a pivotal role in regulating a multitude of physiological processes in plants, including secondary metabolism, cell fate determination, and responses to environmental stimuli (Wu et al., 2022). It is established that R2R3-MYB proteins form complexes with other TFs, such as basic helix-loop-helix (bHLH) proteins, to regulate gene expression effectively (Chen et al., 2021; Yan et al., 2021; Yang et al., 2022). 2.2 Evolutionary conservation and diversity The R2R3-MYB gene family exhibits high levels of conservation across diverse plant species, which suggests that it plays a pivotal role in plant biology. Notwithstanding this conservation, there is considerable diversity in the number and function of R2R3-MYB genes among species. For example, 146 R2R3-MYB genes have been identified and classified into 19 subfamilies based on domain structures and phylogenetic relationships in carrots (Duan et al., 2022). In rice, a total of 99 R2R3-OsMYB genes were identified from the rice genome and subsequently grouped into 5 subfamilies (Kang et al., 2022). This diversity permits the precise adjustment of diverse metabolic pathways and developmental processes, reflecting the evolutionary adaptation of plants to their environments (Naing and Kim, 2018; Duan et al., 2022). 2.3 Role in secondary metabolism and anthocyanin pathway R2R3-MYB TFs are of great importance in the regulation of secondary metabolism, particularly in the anthocyanin biosynthetic pathway. Anthocyanins are pigments that are responsible for the red, blue, and purple coloration observed in plants. They play a role in attracting pollinators and seed dispersers, and they protect against various stresses. R2R3-MYB proteins regulate the expression of structural genes involved in anthocyanin biosynthesis, including DFR, ANS, and UFGT, by binding to their promoters (Yan et al., 2019; Upadhyaya et al., 2021; Yan et al., 2021). In rice, the R2R3-MYB TF OsC1 has been demonstrated to enhance anthocyanin production (Upadhyaya et al., 2021). Similarly, in tomatoes, the R2R3-MYB TF SlAN2-like activates the transcription of SlMYBATV, thereby regulating the anthocyanin content of the fruit (Yan et al., 2019). These examples illustrate the pivotal function of R2R3-MYB TFs in regulating anthocyanin biosynthesis and their prospective applications in crop enhancement for enhanced nutritional quality and stress resilience (Feng et al., 2018; Bao et al., 2021). In summary, R2R3-MYB TFs play a pivotal role in the regulation of anthocyanin biosynthesis and other secondary metabolic pathways in plants. The structural diversity and evolutionary conservation of these TFs highlight their significance in plant adaptation and development. A comprehension of the functional roles of these TFs can provide valuable insights into plant biology and offer opportunities for genetic engineering to enhance crop traits. 3 Anthocyanin Biosynthesis in Black Rice 3.1 Pathway overview and key enzymes The biosynthesis of anthocyanins in black rice is a complex series of enzymatic reactions that ultimately result in
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