1 Introduction

Anthocyanins are natural water-soluble pigments that play a crucial role in plants, providing a range of colors from red to purple and blue to various vegetative tissues and reproductive organs. In addition to attracting pollinators and seed dispersers, these pigments contribute to the plant's defense mechanisms against various environmental stresses (Upadhyaya et al., 2021; Yan et al., 2021). The biosynthesis of anthocyanins is genetically determined by structural and regulatory genes, with MYB transcription factors (TFs) occupying a dominant position within this regulatory network (Shin et al., 2016).

R2R3-MYB TFs play a pivotal role in regulating the anthocyanin biosynthetic pathway (ABP) (Wu et al., 2022). These TFs interact with other proteins, including basic helix-loop-helix (bHLH) and WD-repeat proteins, to form complexes that activate or repress the expression of anthocyanin biosynthetic genes (Wang et al., 2019; Chen et al., 2021; Upadhyaya et al., 2021). For example, the R2R3-MYB protein OsC1 in rice has been demonstrated to regulate anthocyanin biosynthesis and enhance oxidative stress tolerance by modulating the expression of late ABP genes (Upadhyaya et al., 2021). Similarly, other R2R3-MYB proteins, such as TaPL1 in wheat and SsMYB1 in Chinese tallow, have been identified as positive regulators of anthocyanin biosynthesis, underscoring their significance in the regulation of this pathway (Shin et al., 2016).

Dark purple or black rice, distinguished by its elevated anthocyanin concentration, represents an exemplary model for investigating the regulation of anthocyanin biosynthesis. The presence of anthocyanins in dark purple rice not only contributes to its distinctive color but also enhances its nutritional value and stress tolerance (Upadhyaya et al., 2021). The R2R3-MYB TF OsC1 has been identified as a key regulator of anthocyanin biosynthesis in dark purple rice, rendering it a valuable system for elucidating the molecular mechanisms underlying anthocyanin regulation and the role of environmental factors in modulating this process.

This review aims to elucidate the regulatory mechanisms by which R2R3-MYB TFs control anthocyanin biosynthesis in dark purple rice, examine the role of environmental factors in modulating the expression of R2R3-MYB genes and their downstream targets, and assess the impact of altered anthocyanin biosynthesis on the stress tolerance and overall adaptation of dark purple rice. The attainment of these objectives will facilitate the acquisition of invaluable insights into the intricate regulatory networks that govern anthocyanin biosynthesis and the potential for genetic manipulation to enhance crop quality and stress resilience.

2 R2R3-MYB Gene Family in Dark Purple Rice

2.1 Genome-wide characteristics and classification of R2R3-MYB genes

The R2R3-MYB TFs constitute a large family of proteins that play a pivotal role in regulating a multitude of physiological processes in plants, including the biosynthesis of anthocyanins. These TFs are distinguished by the presence of two MYB domains (R2 and R3) at their N-terminus, which are involved in DNA binding. The R2R3-MYB genes are classified into distinct subfamilies based on their sequence homology and functional characteristics (Blanco et al., 2022; Li et al., 2023; Shi et al., 2024). In rice, R2R3-MYB genes have been demonstrated to regulate flavonoid production, including the biosynthesis of anthocyanins and proanthocyanins (Li et al., 2016; Upadhyaya et al., 2021; Yang et al., 2023).

2.2 Identification and characterization of R2R3-MYB genes in dark purple rice

In black rice, several R2R3-MYB genes have been identified and characterized concerning their roles in anthocyanin biosynthesis. For example, the OsC1 gene, which encodes an R2R3-MYB TF, has been demonstrated to regulate the expression of late ABP genes, resulting in the accumulation of anthocyanins, particularly cyanidin 3-glucoside, in the panicle stage of black rice (Upadhyaya et al., 2021). OsMYB3/OsKala3, an MYB TF, was identified as the R2R3-MYB gene that plays a pivotal role in anthocyanin biosynthesis in rice pericarps (Chen et al., 2023; Kim et al., 2021; Zheng et al., 2021). Comprehensive bioinformatic analyses have identified numerous R2R3-MYB genes in various plant species, including rice, and have provided insights into their gene structure, motif conservation, and chromosomal localization (Yang et al., 2023).

2.3 Phylogenetic analysis and evolutionary insights

A phylogenetic analysis of R2R3-MYB genes in rice and other plant species has revealed that these genes are highly conserved and can be grouped into distinct subfamilies. For example, a study on Ananas comosus var. bracteatus identified 99 R2R3-MYB genes, which were classified into 33 subfamilies. The gene structures and protein motifs within each subfamily were found to be conserved (Yang et al., 2023). Such analyses indicate that segmental duplication events have played a significant role in the expansion of the R2R3-MYB gene family in plants. Furthermore, phylogenetic studies have demonstrated that R2R3-MYB genes involved in anthocyanin biosynthesis are evolutionarily conserved across diverse plant species, suggesting their indispensable role in plant adaptation and survival (Albert et al., 2011).

2.4 Expression patterns of R2R3-MYB genes in different tissues and developmental stages

The expression patterns of R2R3-MYB genes in dark purple rice exhibit variability across different tissues and developmental stages. For example, the OsC1 gene is highly expressed during the panicle stage, which correlates with increased anthocyanin accumulation (Upadhyaya et al., 2021) (Figure 1). In other plants, such as Arabidopsis thaliana, R2R3-MYB genes like PAP1, PAP2, and MYB113 demonstrate differential expression in juvenile and adult leaves, indicating stage-specific regulation of anthocyanin biosynthesis (Koo and Poethig, 2021). Similarly, in Ananas comosus var. bracteatus, ten R2R3-MYB genes have been observed to exhibit tissue-specific expression patterns, with some genes being highly expressed in flowers, bracts, and leaves. This suggests their potential involvement in the spatial and temporal regulation of anthocyanin biosynthesis (Yang et al., 2023). The expression patterns are frequently influenced by environmental factors, including light, temperature, and stress conditions, which serve to modulate the activity of R2R3-MYB TFs (Albert et al., 2011; Shin et al., 2016; Zhang et al., 2019; Yang et al., 2022).

Figure 1 Expression analysis of anthocyanin biosynthesis pathway genes in black and white rice panicles at various developmental stages (Adopted from Upadhyaya et al., 2021). Image caption: (A) Generalized anthocyanin biosynthesis scheme in plants. Genes encoding enzymes are indicated in uppercase letters: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavanone 3’-hydroxylase; DFR, Dihydroflavonol 4-reductase; ANS, Anthocyanidin synthase; GT, UDP-glucose:flavonoid-3-O-glycosyltransferase. Structural and regulatory genes have been labeled in the scheme for clarity. (B-H) qRT-PCR expression analysis in panicle tissue of black and white rice for anthocyanin biosynthesis genes CHS (B), CHI (C), F3H (D), F3’H (E), DFR (F), ANS (G), and C1 (H). The developmental stages are 0, 3, 6, 9, 12, and 15 DAP, where DAP represents days after pollination. ACTIN was used as an internal control. Data are represented as relative expression level in black rice panicle with respect to white rice at corresponding stages and genes. Data are depicted as mean±SD of three independent experiments (n=3 biological replicates, each containing six plants); *P<0.01, **P<0.001, and ***P<0.0001 show statistically significant differences with control that is, white rice panicle using one-way ANOVA followed by Dunnett's test. (I) Heatmap showing the expression level of each gene in both rice varieties at variable seed developmental stages. The expression profile of enzymes was represented in terms of relative expression as compared to 0 DAP, obtained from qRT-PCR experiment. The upper panel shows different color phenotypes of pericarp tissue at successive developmental stages of grain filling (Adopted from Upadhyaya et al., 2021)

A comprehensive understanding of the characteristics, classification, phylogenetic relationships, and expression patterns of R2R3-MYB genes enables researchers to gain valuable insights into the regulatory mechanisms underlying anthocyanin biosynthesis and plant adaptation in dark purple rice.

3 Environmental Factors Affecting Anthocyanin Biosynthesis

3.1 Light intensity and quality

The influence of light on anthocyanin biosynthesis is a critical factor in environmental studies (Song et al., 2022). It has been demonstrated that light intensity and quality can markedly upregulate the expression of R2R3-MYB TFs, which are pivotal regulators of anthocyanin biosynthesis (Ma et al., 2021). For example, the expression of TaPL1 in wheat coleoptiles is markedly enhanced by light, resulting in elevated anthocyanin accumulation (Shin et al., 2016). Similarly, the regulation of anthocyanin biosynthetic mechanisms by MYB TFs is influenced by light, which modulates the complex formation of anthocyanins (Yan et al., 2021). The multilevel regulation of R2R3-MYB TFs in response to light has been extensively documented, underscoring its pivotal role in the transcriptional control of anthocyanin biosynthesis (Yang et al., 2022).

3.2 Temperature variations

Temperature fluctuations have been demonstrated to exert a considerable influence on the regulation of anthocyanin biosynthesis. For instance, evidence indicates that cold stress can lead to an increase in the expression of R2R3-MYB genes, which in turn enhances the accumulation of anthocyanins. In wheat, TaPL1 expression is markedly elevated in response to cold stress, which is known to induce anthocyanin accumulation (Shin et al., 2016). The regulatory network of anthocyanin biosynthesis, which is mediated by R2R3-MYB activators, is subject to influence by temperature. This, in turn, affects the transcriptional regulation of anthocyanin biosynthetic genes (Yang et al., 2022).

3.3 Soil composition and nutrient availability

The biosynthesis of anthocyanins is contingent upon the composition of the soil and the availability of nutrients. Nutrient stress, such as salt stress, has been demonstrated to induce the expression of R2R3-MYB TFs, which in turn has been shown to result in increased anthocyanin production. In wheat, the expression of TaPL1 is markedly elevated in response to salt stress, leading to elevated anthocyanin levels (Shin et al., 2016). The role of soil nutrients in the regulation of anthocyanin biosynthesis is also underscored in the context of metabolic engineering, where the availability of specific nutrients can influence the expression of anthocyanin-related genes (Zhu et al., 2017).

3.4 Drought and oxidative stress

The availability of water and the effects of drought stress are significant environmental factors that influence the biosynthesis of anthocyanins. Drought stress can result in the up-regulation of R2R3-MYB TFs, which, in turn, enhances anthocyanin accumulation. The expression of OsC1, an R2R3-MYB transcriptional regulator in rice, is associated with increased anthocyanin production under oxidative stress conditions (Figure 2), which can be linked to drought stress (Upadhyaya et al., 2021). Nevertheless, the question of whether dark purple rice is more drought-resistant than white rice remains unanswered. The modulation of anthocyanin biosynthesis by water availability is of great consequence for the adaptation and stress tolerance of plants.

3.5 Biotic factors: pathogens and symbiotic relationships

The biosynthesis of anthocyanins can be influenced by biotic factors, including pathogens and symbiotic relationships, through the regulation of R2R3-MYB TFs. A pathogen attack can prompt the expression of these TFs, resulting in augmented anthocyanin production as a defensive mechanism. The function of MYB TFs in the control of anthocyanin biosynthesis in response to biotic stress has been elucidated in numerous studies (Naing and Kim, 2018; Bao et al., 2021). Furthermore, symbiotic relationships can modulate the expression of genes involved in anthocyanin biosynthesis, thereby contributing to the overall regulation of this process (Yang et al., 2022). However, there is no empirical evidence to suggest that dark purple rice is more resistant to pests than white rice in paddy fields.

Figure 2 Figure 2 Effect of OsC1 overexpression in ABP and corresponding oxidative stress tolerance (Adopted from Upadhyaya et al., 2021). Image caption: (A) Semi-quantitative expression analysis of OsC1 in overexpressed rice plants along with NT and VT plants. ACTIN has been used as an internal reference control. (B) Total anthocyanin content estimation, (C) antioxidant activity measurement of isolated anthocyanin by DPPH radical scavenging assay of the OsC1-transformed (L1-L3), NT and VT plants (*P<0.01 and **P<0.001 show statistically significant differences with NT plants using one-way ANOVA followed by Dunnett's test). (D) Phenotypes of OsC1, VT, and NT seedlings grown under exogenously applied oxidative stress (10 μM MV) 3 and 5 days treatment. Unstressed nontransformed seedlings have been referred to as control (C). (E,F) Measurement of shoot length (E) and root elongation (F) of the OsC1-transformed, NT, and VT plants 3 and 5 days after oxidative stress application. At least five seedlings were measured for each individual parameters. (G-M) Effect of OsC1 overexpression and subsequent oxidative stress exposure in the transcript level of ABP structural genes. qRT-PCR expression analyses in seedlings before stress (BS) and after 3 days of stress (AS) for OsC1 gene (G) and the anthocyanin biosynthesis genes CHS (H), CHI (I), F3H (J), F3’H (K), DFR (L), ANS (M). The relative expression level of the genes in stressed NT and OsC1-transformed seedlings (L1, L2, and L3) were determined with respect to unstressed NT seedlings. ACTIN has been used an internal reference control. Data are represented as mean ± SD of three individual experiments (n=3 biological replicates, each containing 10 seedlings); *P<0.01 and ***P<0.0001 show statistically significant differences between before and after stress conditions of transformed plants using one-way ANOVA followed by Dunnett's test. Different alphabets above the bars represent significant differences (P<0.01) among the different transformed lines (i.e., L1, L2, and L3) and NT seedlings using one-way ANOVA followed by Tukey's HSD test. Alphabets without any prime represent the differences among seedlings before stress, while alphabets with primes (’) represents the differences among seedlings after stress (Adopted from Upadhyaya et al., 2021).

4 Regulation of R2R3-MYB Gene Expression by Environmental Factors

4.1 Light

The expression of R2R3-MYB genes is subject to regulation by light, which in turn affects the biosynthesis of anthocyanins. In the case of petunia, the R2R3-MYB TFs DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) are induced by high light conditions, which results in increased anthocyanin production in vegetative tissues (Albert et al., 2011). Conversely, under conditions of shade, the expression of PhMYB27, a putative R2R3-MYB active repressor, is upregulated, thereby reducing anthocyanin synthesis (Albert et al., 2011). Similarly, in wheat, the expression of TaPL1, an R2R3-MYB gene, is significantly upregulated by light, which enhances anthocyanin accumulation in coleoptiles (Shin et al., 2016).

4.2 Temperature

Temperature has been demonstrated to exert a considerable influence on the expression of R2R3-MYB genes. In wheat, TaPL1 expression is responsive not only to light but also to cold stress, which further promotes anthocyanin biosynthesis (Shin et al., 2016). Conversely, elevated temperatures have been demonstrated to impede anthocyanin biosynthesis by diminishing the expression of pivotal MYB activators (Yang et al., 2022). This indicates that R2R3-MYB genes may be involved in a more expansive stress response mechanism, whereby temperature fluctuations induce anthocyanin synthesis as a protective strategy.

4.3 Nutrients

The expression of R2R3-MYB genes can be influenced by soil nutrients, particularly the availability of essential minerals. In Ananas comosus var. bracteatus, the expression of multiple R2R3-MYB genes is regulated by hormonal treatments, which are frequently associated with nutrient availability. For example, the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) has been demonstrated to induce the expression of specific R2R3-MYB genes, thereby enhancing anthocyanin biosynthesis (Yang et al., 2023). This suggests that soil nutrient status, via its impact on hormonal levels, may regulate R2R3-MYB gene expression indirectly.

4.4 Water stress

Another environmental factor that affects R2R3-MYB gene expression is water stress. In rice, the R2R3-MYB gene OsC1 has been demonstrated to enhance oxidative stress tolerance by regulating anthocyanin biosynthesis (Figure 2). In plants subjected to water stress, the elevated anthocyanin levels observed in OsC1-overexpressing plants appear to mitigate oxidative damage, indicating a protective role for anthocyanins in stressful conditions (Upadhyaya et al., 2021). This underscores the significance of R2R3-MYB genes in plant acclimation to water stress through the modulation of anthocyanin concentrations.

4.5 Biotic factors

The expression of R2R3-MYB genes can also be influenced by biotic factors, such as pathogen attacks. In the species Capsicum annuum, the insertion of a non-long terminal repeat retrotransposon in the promoter region of the CaAn2 gene, which encodes an R2R3-MYB TF, results in its activation. This activation is probably mediated by the recruitment of TFs to the retrotransposon sequence, which enhances anthocyanin biosynthesis and provides a defense mechanism against biotic stress (Jung et al., 2019). This example demonstrates how biotic interactions can result in genetic modifications that upregulate R2R3-MYB gene expression, thereby enhancing plant resilience.

In summary, the expression of R2R3-MYB genes in dark purple rice and other plants is subject to intricate regulation by a multitude of environmental factors, including light, temperature, soil nutrients, water stress, and biotic interactions. These factors collectively influence the biosynthesis of anthocyanins, thereby contributing to the adaptation and survival of plants in diverse environmental conditions.

5 Molecular Mechanisms Linking R2R3-MYB Genes to Anthocyanin Biosynthesis

5.1 Signal transduction pathways involved in R2R3-MYB regulation

R2R3-MYB TFs play a crucial role in the regulation of anthocyanin biosynthesis, responding to a multitude of environmental and internal signals. It has been demonstrated that light, temperature, and hormonal signals, including MeJA and ethylene, can influence the activity of R2R3-MYB proteins. For example, the expression of R2R3-MYB genes in petunia is subject to strict regulation by light conditions, with high-light environments inducing the expression of MYB factors that promote anthocyanin synthesis (Albert et al., 2011). Similarly, in wheat, environmental stresses such as cold and salt have been demonstrated to upregulate the expression of R2R3-MYB genes, resulting in increased anthocyanin accumulation (Shin et al., 2016). These findings underscore the intricate interplay between environmental cues and R2R3-MYB-mediated anthocyanin biosynthesis.

5.2 Interaction with other TFs and co-regulators

R2R3-MYB TFs frequently act in conjunction with other TFs and co-regulators to regulate anthocyanin biosynthesis. In petunia, the R2R3-MYB factors DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) interact with the basic helix-loop-helix (bHLH) factor ANTHOCYANIN1 (AN1) and the WD-repeat protein AN11 to regulate anthocyanin production in vegetative tissues (Albert et al., 2011). Similarly, in Chinese tallow, the R2R3-MYB factor SsMYB1 interacts with the bHLH protein SsbHLH1 to enhance the transcription of anthocyanin biosynthetic genes, thereby promoting anthocyanin accumulation (Chen et al., 2021). These interactions highlight the significance of transcriptional complexes in the precise regulation of anthocyanin biosynthesis.

5.3 Post-translational modifications (PTMs) and stability of R2R3-MYB proteins

PTMs are of great consequence for the stability and activity of R2R3-MYB proteins. These modifications can include phosphorylation, ubiquitination, and sumoylation, which can either enhance or repress the activity of MYB proteins. For example, the stability of the OsC1 protein in rice, an R2R3-MYB TF, is of critical importance for its role in regulating anthocyanin biosynthesis and oxidative stress tolerance (Upadhyaya et al., 2021). While the specific PTMs of OsC1 were not delineated, the general significance of PTMs in regulating MYB protein function is well documented in the literature (Yang et al., 2022).

5.4 Epigenetic modifications influencing R2R3-MYB gene expression

Epigenetic modifications, including DNA methylation and histone modifications, have been demonstrated to exert a considerable influence on the expression of R2R3-MYB genes. In Capsicum annuum, the insertion of a non-long terminal repeat retrotransposon in the promoter region of the CaAn2 gene, an R2R3-MYB TF, results in its activation by recruiting TFs to the 3' untranslated region of the retrotransposon (Jung et al., 2019). This example demonstrates how epigenetic alterations can result in the activation of MYB genes and subsequent anthocyanin biosynthesis. Furthermore, natural variations in the promoter regions of R2R3-MYB genes can result in differential expression and anthocyanin accumulation in a range of plant species (Yang et al., 2022).

In conclusion, the regulation of R2R3-MYB genes and their role in anthocyanin biosynthesis is a complex process involving signal transduction pathways, interactions with other TFs, PTMs, and epigenetic modifications. These mechanisms collectively ensure the precise control of anthocyanin production in response to environmental and developmental cues.

6 Significance of Anthocyanin Modulation in Dark Purple Rice

6.1 Role of anthocyanins in stress tolerance

The anthocyanins present in dark purple rice play a pivotal role in enhancing the stress tolerance of this particular variety. The R2R3-MYB TFs, such as OsC1, are of great importance in the regulation of the anthocyanin biosynthesis pathway (ABP) (Lin et al., 2022). The overexpression of OsC1 in rice has been demonstrated to elevate anthocyanin levels, which subsequently enhances the plant's capacity to withstand oxidative stress by reducing the concentration of reactive oxygen species (ROS). This results in enhanced photosynthetic efficiency and reduced membrane damage under stress conditions, thereby strengthening the plant's overall resilience to oxidative stress (Upadhyaya et al., 2021; Yang et al., 2022).

6.2 Impact on plant growth and development

The influence of anthocyanins on plant growth and development is a significant area of research. The presence of these pigments, which are regulated by MYB TFs, contributes to the visual appeal and nutritional quality of the plant. The accumulation of anthocyanins, particularly cyanidin 3-glucoside, in dark purple rice is associated with the expression of late ABP genes during the panicle stage. This not only enhances the plant's aesthetic value but also supports its growth by improving photosynthetic efficiency and reducing oxidative damage (Feng et al., 2018; Upadhyaya et al., 2021; Yan et al., 2021; Kavas et al., 2022).

6.3 Contribution to reproductive success and seed quality

Anthocyanins play a role in the reproductive success and seed quality of dark purple rice. The pigmentation provided by these compounds attracts pollinators and seed dispersers, which is essential for the plant's reproductive processes. Furthermore, the increased stress tolerance afforded by anthocyanins facilitates superior seed development and quality in dark purple rice. The regulatory role of MYB TFs in anthocyanin biosynthesis is of great importance for the maintenance of these benefits, as they control the expression of genes involved in both the early and late stages of anthocyanin production (Shin et al., 2016; Kim et al., 2021; Yan et al., 2021) (Figure 3). However, in general, dark purple rice is understood to exhibit lower quantitative and quality characteristics than white rice. It is plausible that the pigment composition of the pericarp exerts a direct influence on these traits on rice yield and eating quality.

Figure 3  Proposed model of anthocyanin biosynthesis of seed and vegetative tissue in rice (Adopted from Kim et al., 2021) Image caption: (A) Heat map diagram of expression level for anthocyanin regulators consisting of MYB (M), bHLH (B), and WD40 (W) from rice pericarp and leaf, respectively. Color scale indicates fold changes in gene expression. (B) Working models of anthocyanin coloration in different tissues of rice. Both OsKala3 and OsKala4 had extremely high expression levels in rice pericarp, while OsC1 and OsRb were more strongly expressed in respective rice leaf. When all of OsDFR was functional, it forms the functional MBW complex consisting of OsKala3-OsKala4-OsTTG1 and OsC1-OsRb-OsTTG1 activating anthocyanin biosynthesis in rice (Adopted from Kim et al., 2021)

6.4 Ecological implications and potential advantages in natural habitats

The ecological implications of anthocyanin modulation in dark purple rice are of considerable significance. The presence of anthocyanins confers a competitive advantage in natural habitats, enhancing the plant's ability to withstand a range of environmental stresses, including cold, salt, and light. The up-regulation of anthocyanin biosynthesis genes in response to these stresses, which is mediated by R2R3-MYB TFs, indicates that anthocyanin-rich plants are better adapted to fluctuating environmental conditions. This adaptive advantage may result in enhanced survival and proliferation in diverse ecological niches (Shin et al., 2016; Karppinen et al., 2022; Yang et al., 2022).

A deeper comprehension of the multifaceted roles of anthocyanins in dark purple rice will enable researchers to more fully recognize the significance of these compounds in plant adaptation and resilience. The modulation of anthocyanin biosynthesis through genetic and environmental factors presents a promising avenue for enhancing crop quality and stress tolerance in agricultural practices.

7 Applications and Future Perspectives

7.1 Breeding strategies for improved anthocyanin production

Strategies for the breeding of dark purple rice may be enhanced by the exploitation of the natural variations in R2R3-MYB gene promoters and their regulatory networks (Li et al., 2020). The selection and cross-breeding of rice varieties with high expression levels of R2R3-MYB genes may facilitate the development of new cultivars with increased anthocyanin content. For example, the OsC1 gene in black rice has been demonstrated to have a positive correlation with anthocyanin biosynthesis, indicating that breeding programs could prioritize this gene to enhance anthocyanin production (Upadhyaya et al., 2021). Furthermore, an understanding of the genetic basis of anthocyanin accumulation, as observed in the pl6 mutant, can provide valuable insights into breeding strategies (Khan et al., 2020).

7.2 Genetic engineering approaches targeting R2R3-MYB genes

Genetic engineering provides precise tools for the manipulation of R2R3-MYB genes, which can be employed to enhance anthocyanin production (Wang et al., 2010). Techniques such as CRISPR/Cas9 can be employed to edit the promoters or coding regions of these genes, thereby enhancing their expression. The overexpression of R2R3-MYB genes, such as OsC1, has been demonstrated to increase anthocyanin levels and improve oxidative stress tolerance in rice (Upadhyaya et al., 2021). Moreover, the integration of multiple R2R3-MYB activators into the rice genome could result in a synergistic enhancement of anthocyanin biosynthesis, as these TFs often function in concert with other regulatory proteins (Zhang et al., 2020; Yan et al., 2021; Yang et al., 2022).

7.3 Potential for developing stress-resistant rice varieties

It is established that anthocyanins possess antioxidant properties, which enable plants to mitigate oxidative stress. The manipulation of R2R3-MYB genes can enhance anthocyanin production, thereby developing rice varieties that are more resistant to environmental stresses, including high light intensity, temperature fluctuations, and oxidative damage. For example, the OsC1 gene has been demonstrated to not only increase anthocyanin content but also to improve photosynthetic efficiency and reduce membrane damage under stress conditions (Upadhyaya et al., 2021). Similarly, the pl6 mutant demonstrates that elevated anthocyanin levels can augment the plant's antioxidant capacity, thereby providing a robust defense against reactive oxygen species (ROS) (Khan et al., 2020).

7.4 Future research

Future research should concentrate on elucidating the intricate regulatory networks involving R2R3-MYB genes and their interactions with other TFs and environmental signals. While considerable progress has been made in elucidating the function of R2R3-MYB genes in anthocyanin biosynthesis, numerous questions remain unanswered. For example, further investigation is required to elucidate the specific upstream regulators and epigenetic modifications that control R2R3-MYB gene expression (Naing and Kim, 2018; Yang et al., 2022; Zuo et al., 2023). Furthermore, it is essential to investigate the potential trade-offs between high anthocyanin production and other agronomic traits, such as yield and stress tolerance, to develop commercially viable rice varieties. Ultimately, the integration of multi-omics methodologies, encompassing genomics, transcriptomics, and metabolomics, can facilitate a comprehensive elucidation of the anthocyanin biosynthetic pathway and its regulatory mechanisms in rice (Yan et al., 2021).

8 Concluding Remarks

The research on the environmental modulation of R2R3-MYB gene expression in dark purple rice has yielded significant insights into the regulation of anthocyanin biosynthesis and plant adaptation. Notable findings include the identification of R2R3-MYB TFs as pivotal regulators of anthocyanin biosynthetic genes (ABGs). These TFs are subject to influence from a range of environmental factors, including light, temperature, and internal signals such as sugar and ethylene, which modulate their activity at multiple levels. Specifically, the OsC1 gene in rice has been demonstrated to enhance anthocyanin production and improve oxidative stress tolerance, thereby underscoring its role in both pigmentation and stress response. Furthermore, the OsKala3 gene in rice has been identified as a positive regulator of anthocyanin biosynthesis, particularly under environmental stress conditions.

A comprehensive understanding of the environmental modulation of R2R3-MYB genes is of paramount importance for several reasons. Firstly, it provides insights into the complex regulatory networks that control anthocyanin biosynthesis, which is of great importance for understanding plant coloration and adaptation. This knowledge can facilitate the development of strategies to enhance the nutritional and aesthetic qualities of crops by manipulating anthocyanin levels. Secondly, the capacity of R2R3-MYB genes to respond to environmental stresses, including oxidative stress, cold, and salt, underscores their role in enhancing plant resilience and survival. This understanding can facilitate the development of crop varieties that are better adapted to changing environmental conditions, thereby ensuring food security.

The findings from this paper have significant implications for the fields of agriculture and plant biology. By elucidating the role of R2R3-MYB genes in anthocyanin biosynthesis and stress response, researchers can develop genetically modified crops with enhanced pigmentation and improved stress tolerance. This could result in the production of crops with enhanced nutritional value and superior market appeal. Moreover, the insights gained from studying the environmental modulation of these genes can inform breeding programs aimed at developing crop varieties that are more resilient to environmental stresses, thereby contributing to sustainable agriculture. Further research should concentrate on investigating the interactions between R2R3-MYB genes and other regulatory pathways to gain a comprehensive understanding of their role in plant adaptation and development.

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), 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

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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