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). 

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 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).

3.3 Regulatory networks involving R2R3-MYB genes

R2R3-MYB TFs play a pivotal role in the regulatory networks that control anthocyanin biosynthesis in rice. OsMYB3, an R2R3-MYB protein, is a pivotal regulator that modulates the expression of late ABP genes, thereby influencing anthocyanin accumulation (Zheng et al., 2021). The MYB-bHLH-WD40 (MBW) complex, which includes OsC1, plays a pivotal role in this regulatory network. In this complex, the bHLH protein S1 activates the expression of OsC1, which in turn activates the expression of the WD40 protein WA1, thereby establishing a cascading regulatory hierarchy (Sun et al., 2022). This hierarchical regulatory system ensures the efficient coordination of anthocyanin biosynthesis genes, thereby enabling precise control over anthocyanin production in different rice tissues. 

The regulatory role of R2R3-MYB genes is not limited to pigmentation. For example, the overexpression of OsC1 in white rice has been demonstrated to enhance oxidative stress tolerance by increasing anthocyanin content, which facilitates the reduction of reactive oxygen species and improves photosynthetic efficiency (Upadhyaya et al., 2021). This underscores the multifaceted function of R2R3-MYB genes in both anthocyanin synthesis and stress response pathways in rice.

4 CRISPR/Cas9 Technology in Functional Genomics

4.1 Principles of CRISPR/Cas9 genome editing

CRISPR/Cas9 represents a revolutionary advance in the field of genome editing, offering the potential for highly precise modifications to the DNA of a diverse range of organisms. The system is comprised of two principal components: the Cas9 nuclease, which performs the cutting of the DNA, and a guide RNA (gRNA), which directs the Cas9 to the specific genomic location that is to be edited. The gRNA is designed to be complementary to the target DNA sequence, thereby ensuring specificity. Following the induction of a double-strand break by Cas9, the cell's intrinsic repair mechanisms, including non-homologous end joining (NHEJ) and homology-directed repair (HDR), are activated to repair the break, resulting in targeted mutations or insertions (Klimek-Chodacka et al., 2018; Kim et al., 2019).

4.2 Applications in plant research

CRISPR/Cas9 has become a widely utilized tool in plant research, particularly in the fields of functional genomics and crop improvement (Mackon et al., 2023). The technique enables researchers to inactivate genes to elucidate their functions, introduce advantageous traits, and enhance resistance to diseases. For example, CRISPR/Cas9 has been employed to modify genes associated with anthocyanin biosynthesis in black rice, resulting in alterations in seed color and anthocyanin content (Jung et al., 2019; Mackon et al., 2024). Furthermore, it has been utilized to impart resistance to bacterial blight in rice by knocking out the Os8N3 gene, which enhanced resistance to Xanthomonas oryzae pv. oryzae without affecting other agronomic traits (Kim et al., 2019). 

4.3 Case studies: successful CRISPR/Cas9 edits in rice

Several successful applications of CRISPR/Cas9 in rice have been documented, demonstrating its efficacy and versatility:

Anthocyanin Biosynthesis Pathway: A study was conducted to investigate the potential of CRISPR/Cas9 to alter the OsF3'H, OsDFR, and OsLDOX genes in black rice. This involved introducing mutations that were shown to affect seed color and anthocyanin content. The mutations were stably inherited, and the edited plants exhibited no discernible differences from non-GMO plants under the strictest GMO regulations (Jung et al., 2019).

Disease Resistance: The Os8N3 gene in rice was inactivated using the CRISPR/Cas9 system to enhance resistance to bacterial blight caused by Xanthomonas oryzae pv. Oryza. The edited plants demonstrated stable transmission of the mutations across generations and exhibited no significant differences in agronomic traits when compared to non-transgenic controls (Kim et al., 2019).

Expanded Targeting Scope: The development of Cas9-NG, a variant that recognizes a broader range of PAM sequences, has broadened the scope for targeting in rice using CRISPR/Cas9. The variant was employed to edit a range of NGN PAM sites, thereby illustrating its potential for more flexible and efficient genome editing in plants (Ren et al., 2019).

The case studies presented here demonstrate the potential of CRISPR/Cas9 technology to facilitate advancements in functional genomics and crop improvement in rice, thereby paving the way for future innovations in plant biotechnology.

5 Functional Analysis of R2R3-MYB Genes Using CRISPR/Cas9

5.1 Target selection and design of CRISPR constructs

The selection of target genes for CRISPR/Cas9-mediated knockout or knockdown is of paramount importance for elucidating the functional roles of R2R3-MYB genes in the anthocyanin biosynthesis pathway. In black rice, the R2R3-MYB gene OsMYB3 has been identified as a key regulator of anthocyanin biosynthesis, exerting a particularly pronounced influence on the expression of late ABP genes (Zheng et al., 2021). Similarly, other studies have underscored the significance of R2R3-MYB genes in regulating anthocyanin production in a range of plant species, including OjMYB1 in Oenanthe javanica and ASR genes in Petunia inflata (Feng et al., 2018; Zhang et al., 2019). These findings provide a foundation for the selection of specific R2R3-MYB genes for CRISPR/Cas9 targeting, thereby facilitating the investigation of their roles in black rice.

5.2 Generation of knockout and knockdown lines

To generate knockout and knockdown lines, CRISPR/Cas9 constructs were designed to target the coding regions of selected R2R3-MYB genes. The constructs were introduced into rice plants via Agrobacterium-mediated transformation. The successful integration and expression of the CRISPR/Cas9 system were confirmed through molecular techniques, including polymerase chain reaction (PCR) and sequencing. Prior research has substantiated the efficacy of CRISPR/Cas9 in introducing targeted mutations in MYB genes, resulting in altered anthocyanin production in a range of plant species (Jung et al., 2019; Duan et al., 2022). These methodologies were adapted for the generation of knockout and knockdown lines in black rice, with a particular focus on the OsC1 gene and other candidate R2R3-MYB genes identified through comparative genomics and expression analysis.

5.3 Phenotypic and molecular characterization

The phenotypic and molecular characterization of the generated knockout and knockdown lines entailed the assessment of anthocyanin content, expression levels of ABP genes, and other related phenotypic traits. Phenotypic assessments were conducted in the knockout (KO) and knockdown (KD) lines to determine the impact of specific R2R3-MYB gene disruptions on anthocyanin biosynthesis and related physiological traits (Figure 2) (Zheng et al., 2021). Molecular characterization was conducted through quantitative real-time polymerase chain reaction (qRT-PCR) analysis of ABP gene expression and high-performance liquid chromatography (HPLC) quantification of anthocyanins, as previously described in studies on other plant species (Naing and Kim, 2018; Yang et al., 2022).

5.4 Insights into the role of specific R2R3-MYB genes

The functional analysis of R2R3-MYB genes using CRISPR/Cas9 provided valuable insights into their roles in the anthocyanin biosynthesis pathway in black rice. The disruption of OsC1 and other targeted R2R3-MYB genes resulted in significant alterations in anthocyanin content and the expression patterns of ABP genes, thereby confirming their regulatory roles (Zheng et al., 2019; Upadhyaya et al., 2021). Furthermore, the study demonstrated the involvement of these genes in broader physiological processes, such as the oxidative stress response and photosynthetic efficiency, as observed in OsC1-overexpressed plants (Upadhyaya et al., 2021). These findings contribute to a more profound comprehension of the molecular mechanisms that underpin anthocyanin biosynthesis and the potential for metabolic engineering of anthocyanin production in rice and other crops (Zhang et al., 2019; Yang et al., 2023).

6 Overexpression Studies of R2R3-MYB Genes

6.1 Methodology for gene overexpression

The overexpression of R2R3-MYB genes in plants is achieved through the insertion of the target gene into the plant genome, typically via Agrobacterium-mediated transformation. This method has been widely employed to elucidate the functional roles of these TFs in a range of species. For example, the overexpression of the OsC1 gene in white rice plants was achieved through Agrobacterium-mediated transformation, resulting in increased anthocyanin production and enhanced oxidative stress tolerance (Upadhyaya et al., 2021). Similarly, the OjMYB1 gene from Oenanthe javanica was overexpressed in Arabidopsis thaliana, resulting in elevated anthocyanin content and the up-regulation of anthocyanin biosynthesis genes (Feng et al., 2018).

6.2 Development of transgenic black rice lines

To develop transgenic black or purple rice lines, the R2R3-MYB genes are cloned into a suitable expression vector under the control of a strong promoter, such as the CaMV 35S promoter. Subsequently, the vector is introduced into rice calli via Agrobacterium tumefaciens. The transformed calli are selected on a medium containing an appropriate antibiotic or herbicide, and the regenerated plants are screened for the presence of the transgene using polymerase chain reaction (PCR) and Southern blot analysis (Figure 3) (Sun et al., 2022). For example, the overexpression of the OsC1 gene in rice resulted in the development of transgenic lines exhibiting markedly elevated anthocyanin levels (Upadhyaya et al., 2021).

6.3 Phenotypic changes and anthocyanin accumulation

The overexpression of R2R3-MYB genes in transgenic plants frequently gives rise to discernible phenotypic alterations, including modifications in pigmentation resulting from elevated anthocyanin accumulation. In rice, the overexpression of OsC1 resulted in purple pigmentation in various tissues, including leaves and panicles, due to the up-regulation of late ABP genes (Upadhyaya et al., 2021). Similarly, transgenic Arabidopsis plants overexpressing OjMYB1 exhibited enhanced anthocyanin content and up-regulation of structural genes related to anthocyanin biosynthesis (Feng et al., 2018). These phenotypic alterations are indicative of the successful modulation of the anthocyanin biosynthesis pathway by the overexpressed R2R3-MYB genes.

6.4 Comparative analysis with CRISPR/Cas9 studies

A comparative analysis between overexpression and CRISPR/Cas9 studies provides insights into the functional roles of R2R3-MYB genes. While overexpression studies enhance the expression of target genes, CRISPR/Cas9 technology allows for precise gene editing, including knockout and knock-in mutations. For example, the overexpression of OsMYB3 in rice has been shown to result in increased anthocyanin production in the rice pericarp. Conversely, the CRISPR/Cas9-mediated knockout of specific R2R3-MYB genes could prove invaluable in identifying their contributions to the anthocyanin pathway (Figure 2) (Zheng et al., 2021). Such comparative studies are of great importance for the comprehension of the regulatory networks and potential redundancies among R2R3-MYB genes involved in anthocyanin biosynthesis.

7 Regulatory Mechanisms of R2R3-MYB Genes in Anthocyanin Pathway

7.1 Transcriptional regulation and promoter analysis

The transcriptional regulation of R2R3-MYB genes in the anthocyanin pathway is a complex process, the outcome of which is influenced by several environmental and internal factors. R2R3-MYB TFs play a pivotal role in regulating the expression of ABP genes. These TFs can be activated by upstream TFs and are subject to natural variations in their promoter regions, which can significantly impact anthocyanin accumulation (Zhang et al., 2020; Yan et al., 2021; Yang et al., 2022). Furthermore, the transcriptional regulation of these genes can be influenced by environmental factors, including light, temperature, and internal signals such as sugar and ethylene. These factors interact at multiple levels to regulate anthocyanin accumulation (Zhou et al., 2018; Peng et al., 2020; Yang et al., 2022).

7.2 Protein-protein interactions and co-factors

R2R3-MYB proteins frequently operate as components of larger protein complexes, engaging with other TFs and co-factors to regulate anthocyanin biosynthesis (Karppinen et al., 2021). These interactions are crucial for the formation of the MYB-bHLH-WD40 (MBW) complex, which represents a pivotal regulatory module in the anthocyanin pathway. For example, in the case of Petunia, the ASR proteins (a type of R2R3-MYB) interact with AN1 and AN11 TFs to form the MBW complex, which is indispensable for anthocyanin synthesis (Zhang et al., 2019). Similarly, in wheat, the TaPL1 protein, an R2R3-MYB TF, interacts with bHLH proteins to activate ABP genes in response to environmental stresses (Shin et al., 2016). These protein-protein interactions are essential for the precise regulation of anthocyanin production, ensuring that the pathway is activated only under appropriate conditions.

7.3 Epigenetic modifications and post-translational regulation

Epigenetic modifications and post-translational regulation play a pivotal role in the regulation of R2R3-MYB gene activity. Epigenetic alterations, including DNA methylation and histone modifications, can modify the expression of R2R3-MYB genes, thereby influencing anthocyanin biosynthesis. For example, the expression of R2R3-MYB genes can be regulated by epigenetic mechanisms that respond to environmental cues, resulting in alterations in anthocyanin accumulation (Yang et al., 2022). Post-translational modifications (PTMs), including phosphorylation, ubiquitination, and sumoylation, can also influence the stability, localization, and activity of R2R3-MYB proteins. These modifications can facilitate the precise regulation of R2R3-MYB TFs, ensuring a balanced production of anthocyanins and preventing excessive accumulation that could be detrimental to the plant (Zhou et al., 2018). The interplay between these regulatory mechanisms underscores the intricate nature of anthocyanin biosynthesis and the pivotal function of R2R3-MYB genes in this process.

Anyhow, the regulation of R2R3-MYB genes in the anthocyanin pathway is a complex process involving a multitude of factors, including transcriptional control, protein-protein interactions, and epigenetic and PTMs. These mechanisms function in concert to guarantee the precise and context-dependent activation of anthocyanin biosynthesis, thereby contributing to the diverse pigmentation patterns observed in plants.

8 Challenges and Future Prospects

8.1 Technical challenges in CRISPR/Cas9 and overexpression studies

The application of CRISPR/Cas9 and overexpression techniques in the study of the R2R3-MYB genes involved in the anthocyanin pathway in black or purple rice presents several technical challenges. One significant challenge is achieving high efficiency and specificity in gene editing. Despite the successful application of CRISPR/Cas9 to edit genes such as OsF3'H, OsDFR, and OsLDOX in rice, concerns remain regarding the stability and heritability of these edits, as well as the potential for off-target effects (Jung et al., 2019). Furthermore, the integration of vector backbone sequences and the potential for unintended mutations can complicate the identification of true gene function and phenotypic outcomes (Jung et al., 2019). Furthermore, overexpression studies present additional challenges, including the need to achieve consistent and high levels of gene expression across different tissues and developmental stages. This is crucial for understanding the full impact of R2R3-MYB genes on anthocyanin biosynthesis (Upadhyaya et al., 2021; Zuo et al., 2023).

8.2 Potential for enhancing anthocyanin content in rice

The genetic manipulation of rice to enhance its anthocyanin content shows great promise for both nutritional and aesthetic improvements (Zhou et al., 2022). The overexpression of R2R3-MYB genes, such as OsC1, has been demonstrated to markedly enhance anthocyanin production, which in turn augments oxidative stress tolerance and photosynthetic efficiency in rice plants (Upadhyaya et al., 2021). This indicates that the targeted overexpression of specific R2R3-MYB genes may represent a viable strategy for the development of rice varieties with higher anthocyanin content. Furthermore, the use of CRISPR/Cas9 to knock out repressive genes or modify regulatory elements can facilitate additional precision in the fine-tuning of anthocyanin biosynthesis pathways, as evidenced in other crops such as tomatoes and Rehmannia (Yan et al., 2019; Zhi et al., 2020; Zuo et al., 2023). These approaches have the potential to result in the development of rice varieties with enhanced nutritional value and improved stress resilience.

8.3 Broader implications for crop improvement and nutritional enhancement

The successful manipulation of R2R3-MYB genes in the anthocyanin pathway has broader implications for crop improvement and nutritional enhancement. In addition to their role in plant coloration, anthocyanins possess notable antioxidant properties, conferring significant health benefits. By capitalizing on the regulatory functions of R2R3-MYB TFs, it is feasible to augment the anthocyanin concentration in a multitude of crops, consequently enhancing their nutritional profiles (Feng et al., 2018; Naing and Kim, 2018; Yang et al., 2022). This could result in the creation of functional foods with enhanced health benefits, addressing consumer demand and public health concerns simultaneously. Moreover, an understanding of the multilevel regulation of these TFs in response to environmental and internal signals can facilitate the optimization of crop performance under varying conditions (Yang et al., 2022). This knowledge can be applied to other horticultural plants, with the potential for widespread improvements in crop quality and resilience.

9 Concluding Remarks

The study on the functional role of R2R3-MYB genes in the anthocyanin pathway of black or purple rice has yielded several significant findings. The CRISPR/Cas9 system was successfully employed to target and mutate pivotal genes involved in the ABP, including OsF3'H, OsDFR, and OsLDOX. This resulted in discernible alterations in seed color and anthocyanin content in rice. Furthermore, the overexpression of R2R3-MYB TFs, such as OsC1, OsMYB3, has been demonstrated to enhance anthocyanin production and improve oxidative stress tolerance in rice. Studies in other plant species, such as N. sibirica and R. glutinosa, have demonstrated that R2R3-MYB TFs like NsMYB1 and RgMYB41 can regulate anthocyanin biosynthesis and influence fruit color differentiation and overall anthocyanin content. Moreover, the hierarchical interactions between MYB activators and repressors, as observed in peach and tomato, underscore the intricate regulatory networks that maintain equilibrium between anthocyanin and proanthocyanidin accumulation.

The results of previous study provide a foundation for further investigation in several areas. Firstly, the high efficiency and specificity of the CRISPR/Cas9 system in editing anthocyanin-related genes indicate that this technology may be further explored for the enhancement of other desirable traits in rice and other crops. Secondly, the function of R2R3-MYB TFs in regulating anthocyanin biosynthesis across diverse plant species suggests that these genes may serve as potential targets for genetic engineering to enhance the nutritional and aesthetic qualities of various crops. Further research should concentrate on elucidating the precise mechanisms of MYB activators and repressors and their interactions with other TFs, such as bHLH proteins, to achieve precise control over anthocyanin and proanthocyanidin levels. Furthermore, investigating the broader impact of MYB overexpression on the transcriptome and metabolome could provide insights into the pleiotropic effects of these TFs and their potential applications in crop improvement. 

In conclusion, the study has successfully elucidated the functional role of R2R3-MYB genes in the anthocyanin pathway of black or purple rice using CRISPR/Cas9-mediated mutagenesis and overexpression approaches. The results illustrate the potential of these genetic tools to enhance anthocyanin content and improve stress tolerance in rice, with broader implications for other crops. The intricate regulatory networks involving MYB TFs underscore the complexity of anthocyanin biosynthesis and highlight the necessity for further research to fully exploit these pathways for agricultural and nutritional benefits. The combination of advanced genetic techniques with conventional breeding approaches offers promise for the development of crops with enhanced characteristics, which could contribute to food security and human health. 

Acknowledgments

We appreciate the anonymous peer reviewers for their revision suggestions on the manuscript.

Funding

This work was supported by the Scientific Research Foundation of Panxi Crops Research and Utilization Key Laboratory of Sichuan Province (Grant No. XNFZ2203), and the Ph.D. Programs Foundation of Xichang University (Grant No. YBZ202340), and the Key and Major Science and Technology Projects of Yunnan (Grant nos. 202202AE09002102).

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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