Bioscience Methods 2024, Vol.15, No.4, 184-195 http://bioscipublisher.com/index.php/bm 1 84 Feature Review Open Access Unraveling the Functional Role of R2R3-MYB Genes in Black Rice Anthocyanin Pathway: A CRISPR/Cas9 and Overexpression Study Mengmeng Yin 1,2,4, Fan Luo 1,2,4, Xiaoli Zhou 1,2,4, Changhe Wei 5, Qian Zhu 1,2,3, Huirong Dong 1,2,3, Lijuan Chen 1,2,3 , Dong Sun Lee 1,2,3 1 State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, Yunnan, China 2 The Key Laboratory for Crop Production and Smart Agriculture of Yunnan Province, Yunnan Agricultural University, Kunming 650201, Yunnan, China 3 Rice Research Institute, Yunnan Agricultural University, Kunming, 650201, Yunnan, China 4 College of Agricultural Science, Xichang University, Liangshan, 615013, Sichuan, China 5 Panxi Crops Research and Utilization Key Laboratory of Sichuan Province, Xichang University, Liangshan, 615013, Sichuan, China Corresponding author: jing.he@cuixi.org Bioscience Methods, 2024, Vol.15, No.4 doi: 10.5376/bm.2024.15.0019 Received: 05 Jul., 2024 Accepted: 17 Aug., 2024 Published: 29 Aug., 2024 Copyright © 2024 Yin et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Yin M.M., Luo F., Zhou X.L., Wei C.H., Zhu Q., Dong H.R., Chen L.J., and Lee S.D., 2024, Unraveling the functional role of R2R3-MYB genes in black rice anthocyanin pathway: a CRISPR/Cas9 and overexpression study, Bioscience Methods, 15(4): 184-195 (doi: 10.5376/bm.2024.15.0019) Abstract R2R3-OsMYBs play a pivotal role in the regulation of anthocyanin biosynthesis, which is responsible for the black pigmentation observed in rice. This paper is aimed to overview the functional mechanisms of these genes through the application of CRISPR/Cas9 gene editing and overexpression techniques. Recent studies have demonstrated the effectiveness of CRISPR/Cas9 in targeting key genes within the anthocyanin biosynthesis pathway, including OsF3'H, OsDFR, and OsLDOX, leading to significant alterations in anthocyanin content and seed color in rice. Moreover, the overexpression of R2R3-MYB genes, such as OsC1, has been demonstrated to increase anthocyanin production and enhance oxidative stress tolerance in rice. Comparative studies in other plant species, including tomato and Nitraria sibirica, have further illuminated the diverse regulatory roles of R2R3-MYB TFs in anthocyanin biosynthesis. This review summarizes current findings to provide a comprehensive understanding of the genetic and molecular basis of anthocyanin regulation in black rice, offering insights into potential applications for crop improvement and functional food development. Keywords Black rice; R2R3-MYB; Anthocyanin biosynthesis; CRISPR/Cas9; Overexpression 1 Introduction Anthocyanins are a class of flavonoid compounds that are responsible for the red, purple, and blue pigmentation observed in various plant tissues, including flowers, fruits, and leaves. These pigments play a pivotal role in plant physiology, including attracting pollinators and seed dispersers and providing protection against various biotic and abiotic stresses (Naing and Kim, 2018; Piatkowski et al., 2020; Yan et al., 2021). The biosynthesis of anthocyanins is a complex process, regulated by a network of structural and regulatory genes (Wang et al., 2023). Among these, the MYB TFs, particularly the R2R3-MYB subgroup, are pivotal regulators that control the expression of genes involved in the anthocyanin biosynthetic pathway (Naing and Kim, 2018; Upadhyaya et al., 2021; Yan et al., 2021). R2R3-MYB (2R-MYB) TFs play a crucial role in the regulation of secondary metabolism in plants, including the biosynthesis of anthocyanins. These TFs bind to specific DNA sequences in the promoters of target genes, thereby modulating their expression (Naing and Kim, 2018; Yan et al., 2021; Kavas et al., 2022). In rice, the R2R3-MYB gene OsC1 has been demonstrated to regulate anthocyanin biosynthesis and enhance oxidative stress tolerance by upregulating the expression of late anthocyanin biosynthetic pathway (ABP) genes (Upadhyaya et al., 2021). Similarly, in other plant species, such as Rehmannia glutinosa and Nitraria sibirica, R2R3-MYB genes have been identified as key regulators of anthocyanin accumulation, influencing traits such as fruit color and antioxidant activity (Bao et al., 2021; Zuo et al., 2023). The functional diversity and regulatory capacity of R2R3-MYB genes render them valuable targets for genetic engineering to improve crop quality and stress resilience (Naing and Kim, 2018; Yan et al., 2021).
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