Bioscience Methods 2024, Vol.15, No.4, 184-195 http://bioscipublisher.com/index.php/bm 1 86 the conversion of phenylalanine into a variety of anthocyanin compounds (Lee et al., 2023). The key enzymes involved in this pathway include chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) (Zheng et al., 2019; Meng et al., 2021). These enzymes function sequentially to produce anthocyanins, which are responsible for the purple pigmentation observed in various tissues of rice plants (Khan et al., 2020). The dihydroflavonol 4-reductase (OsDFR) plays a pivotal role in the anthocyanin biosynthesis pathway, as it catalyzes the reduction of dihydroflavonols to leucoanthocyanidins (Meng et al., 2021). 3.2 Genetic basis of black or purple pigmentation in rice The genetic basis of black or purple pigmentation in rice is primarily controlled by structural and regulatory genes. The OsC1 gene, which encodes an R2R3-MYB TF, plays a pivotal role in regulating anthocyanin biosynthesis (Meng et al., 2021; Upadhyaya et al., 2021). OsC1 interacts with other tissue-specific genes, including OsPa and OsPs, to activate the expression of OsDFR and other ABP genes, thereby leading to the purple coloration of specific tissues, such as apiculi and stigmas (Figure 1) (Li et al., 2018; Meng et al., 2021). Furthermore, the MYB-bHLH-WD40 complex, which includes OsC1, OsRb, and OsDFR, regulates the expression of ABP genes in rice leaves (Zheng et al., 2019). The hierarchical regulation among these genes ensures the coordinated expression of the entire anthocyanin biosynthesis pathway (Sun et al., 2022). Figure 1 Determinant factors and regulatory systems for anthocyanin biosynthesis in rice apiculi and stigmas (Adopted from Meng et al., 2021) Image caption: Working models of apiculus and stigma coloration in rice. Both OsC1 and OsDFR exhibit high expression levels in apiculi and stigmas, while OsPa and OsPs are more strongly expressed in their respective tissues. Functional OsC1, OsDFR, OsPa, and OsPs form complexes (OsC1-OsPa-OsPAC1 in apiculi and OsC1-OsPs-OsPAC1 in stigmas) that activate OsDFR expression, resulting in purple pigmentation. Nonfunctional OsC1 disrupts complex formation, reducing OsDFR expression and leading to non-pigmented tissues. Nonfunctional OsDFR results in brown apiculi and colorless stigmas. Nonfunctional OsPa or OsPs also lead to non-pigmented tissues. Solid ellipses indicate confirmed protein interactions; dashed ellipses indicate interactions needing confirmation (Adapted from Meng et al., 2021)
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