Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 98-107 http://genbreedpublisher.com/index.php/tgmb 102 when the fruits turn red, and they directly determine how much anthocyanin can accumulate (Yang et al., 2015; Yang et al., 2021). In addition, the expression of some regulatory genes, such as MYB and bHLH, is also closely related to these structural genes. Some regulatory factors are expressed only in specific sites, such as the endopeel of kiwifruit, which can control the synthesis of local anthocyanins (Wang et al., 2019). 5.2 Hormonal and environmental modulation The synthesis of anthocyanins is not only determined by genes, but also influenced by plant hormones and the external environment. Hormones such as abscisic acid (ABA) can enhance the expression of regulatory genes and thereby promote the accumulation of anthocyanins (Karppinen et al., 2021). In the environment, light is one of the most important factors. It can induce the expression of MYB transcription factors, thereby activating structural genes and increasing anthocyanin synthesis. For instance, after apples are exposed to light, the expression of MdMYB1 increases, and anthocyanins also rise accordingly. In addition, temperature changes and some adverse conditions, such as drought or cold damage, can also indirectly affect anthocyanin production by influencing these regulatory factors (Ma et al., 2021; Yan et al., 2021). 5.3 Co-expression networks and transcriptional hierarchies The process of regulating anthocyanin synthesis is very complex and requires the cooperation of many transcription factors. The three proteins, MYB, bHLH and WD40, can form a complex, and MYB plays a major role in this process. They jointly control the expression of structural genes (Wang et al., 2019; Sharma et al., 2024). Studies using co-expression network analysis have found that transcription factors such as PacbHLH13, PacbHLH74, and PacMYB308 are expressed together with structural genes and can directly activate the promoters of key enzymes (Yang et al., 2021). Moreover, because genes have the processes of replication and differentiation, members of the MYB family have gradually formed different functional branches, such as controlling the synthesis of cyanidin or delphinidin (Karppinen et al., 2021). In addition, the expression of anthocyanin genes is also influenced by epigenetics. For example, the interaction between H2A.Z and H3K4me3 also regulates the process of anthocyanin synthesis (Cai et al., 2018). 6 Functional Analysis and Verification Approaches 6.1 Heterologous expression in model plants Heterologous expression is a common method used to verify whether a certain gene is useful. Researchers often transfer regulatory genes from Morella rubra, such as MrMYB1, MrbHLH1 and MrWD40-1, into model plants like tobacco to see if they increase anthocyanins. The experiment found that when MrMYB1 and MrbHLH1 were overexpressed in tobacco, anthocyanins significantly increased, and the expression of related structural genes also enhanced (Niu et al., 2010; Liu et al., 2013b). In addition, if MrMYB1, MrbHLH1 and MrWD40-1 are expressed together, the effect will be stronger. This also indicates that these three genes are very crucial in regulating anthocyanins (Liu et al., 2013a). 6.2 Virus-induced gene silencing (VIGS) and overexpression studies Virus-induced gene silencing (VIGS) and overexpression experiments are two commonly used methods in molecular biology. By using VIGS to “turn off” a certain gene, one can observe whether anthocyanins have decreased, thereby determining whether this gene is involved in anthocyanin synthesis. Conversely, if certain genes (such as MrMYB1 and MrMYB6) are “turned on”, changes in anthocyanins or other related substances can also be observed. For example, after MrMYB6 is overexpressed in tobacco, the contents of anthocyanins and proanthocyanidins decrease instead, indicating that it plays an inhibitory role (Shi et al., 2021). These methods can directly indicate which genes are promoting and which are inhibiting anthocyanin synthesis (Niu et al., 2010; Liu et al., 2013b). 6.3 Subcellular localization and protein-protein interactions Scientists also conducted some cell localization experiments to see exactly where the proteins work within the cells. Whether proteins will bind to each other can also be verified through experiments such as yeast two-hybrid and BiFC. For instance, the yeast two-hybrid experiment confirmed that MrMYB1 can interact with MrbHLH1 and MrWD40-1. These three proteins can assemble into a complex specifically for regulating anthocyanin
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