Introduction

Flower color is one of the most prominent traits in plants and is a decisive factor in assessing ornamental value. The expression of flower color in plants is influenced by several factors, including the types of pigments, the intracellular environment of flower cells, metal ions, and external conditions, with the types and content of pigments being the primary determinants1,2. Plant pigments can be classified into four main categories: flavonoids, carotenoids, chlorophylls, and betacyanins. Betacyanins can give flowers yellow, orange, and red hues, existing only in the order Caryophyllales. Flavonoids primarily consist of anthocyanins, flavonol glycosides, and flavones3. Among them, compounds such as flavones, dihydroflavones, and chalcones lead to light yellow to yellow flower colors4.

Anthocyanins are compounds formed by the glycosidic bond between anthocyanins and sugars, representing a class of natural water-soluble pigments that are widely present in the cell sap of flower, fruit, stem, leaf, and root organs. The six most common anthocyanins are pelargonidin, cyanidin, delphinidin, peonidin, malvidin, and petunidin, which exhibit various colors from red, purple-red to blue. These colors are largely related to the number of hydroxyl groups on the B ring: the greater the number, the bluer the color. Furthermore, methylation of anthocyanins can lead to a slight reddening effect. The biosynthetic pathway of anthocyanins has been extensively studied, regulated by structural and regulatory genes. Structural genes are responsible for encoding the enzymes involved in the biosynthesis of anthocyanins, while regulatory genes mainly include those encoding three types of transcription factors: MYB, bHLH, and WD405,6.

MYB transcription factors (TFs) are present in all eukaryotes studied to date and are among the largest members of the plant transcription factor family. R2R3-MYB TFs are extensively involved in the regulation of various biological processes7. R2R3-MYB proteins form MBW complexes with basic helix-loop-helix (bHLH) proteins and WD-repeat proteins to co-regulate anthocyanins8,9,10. Christian Dubos classified Arabidopsis R2R3-MYB into 23 subfamilies, with genes in subfamilies S4-S7 mainly controlling the synthesis of flavonols, anthocyanins, and proanthocyanidins (PAs)11. The S6 subfamily gene AtMYB75 in Arabidopsis enhances secondary metabolites (anthocyanins and flavonols); the S6 subfamily gene Ze MYB9 in zinnia positively regulates anthocyanin synthesis; the S5 subfamily gene LcsMYB123 in Schima superba has a potential role in flavonol biosynthesis; and the S4 subfamily gene CsMYB4a in tea plants inhibits the biosynthesis of phenylpropanoids and flavonoids12,13,14,15. In addition, in Purple pepper, overexpression of CaMYB5 not only induced the accumulation of delphinidin and anthocyanin, but also improved the expression level of anthocyanin biosynthesis structural gene in fruit16. In roses, RcMYB114, RcbHLH and RcWD40 promote anthocyanin accumulation and determine the color of roses by forming MBW (MYB-bHLH-WD40) complex17. In bletilla striata, the over-expression of BsMYB36 increased the content of flavonoids, and the over-expression of BsMYB51 promoted the accumulation of flavonoids, anthocyanins and proanthocyanidins18.

The investigation into the molecular mechanisms governing flower color synthesis in H macrophylla is essential for understanding the complex biological processes that influence its ornamental traits. This species, a woody plant belonging to the family Hydrangeaceae, is primarily characterized as an evergreen or deciduous shrub, valued for its vibrant flower colors and large flower clusters. Its applications span potted plants, cut flowers, and landscape greening, highlighting its popularity in horticulture19,20. In addition to its ornamental appeal, H. macrophylla is rich in flavonoids, isoflavones, and cyclic ether terpenes, which are noted for their health benefits, including heat-clearing and detoxifying properties, as well as diuretic and anti-swelling effects21. Within Chinese traditional culture, this plant is emblematic of hope, reunion, and love, thereby solidifying its role in various festivals and celebrations. The convergence of its aesthetic, medicinal, and cultural values positions H. macrophylla as a significant subject of research, particularly in understory floral studies. The synthesis of flower color in H. macrophylla is intricately influenced by a myriad of molecular mechanisms, which merit thorough investigation, especially through genetic engineering approaches. Research has elucidated key genes involved in anthocyanin biosynthesis, which are critical for the development of blue flower coloration. This has been demonstrated by the finding that the anthocyanin biosynthesis pathway is pivotal, whereas the carotenoid pathways play a comparatively minimal role in color formation22. Moreover, it has been shown that aluminum ions interact with anthocyanins to regulate color changes; under aluminum stress, the downregulation of various anthocyanin biosynthesis-related genes adversely affects flower pigmentation23. Transcriptomic analyses have identified several hub genes, including F3′5′H and DFR, which are integral to the color variations observed across different pH levels, thereby underscoring the significance of environmental factors in the regulation of flower color24. Additionally, epigenetic modifications, particularly DNA methylation, have been implicated in pH-dependent color changes, suggesting that such mechanisms contribute further to the observed variations in flower color25. The transport of aluminum ions into flower tissues has also been investigated to elucidate how these ions enhance color intensity, presenting potential avenues for breeding practices aimed at improving ornamental traits26. Notably, while traditional breeding methods face challenges in producing novel colors, molecular techniques offer a means to enhance breeding efficiency and enable more targeted modifications27. Therefore, comprehensive insights into these molecular mechanisms not only enrich the understanding of flower color synthesis in H. macrophylla but also facilitate innovative approaches in the breeding of new ornamental varieties.

In summary, this study comprehensively analyzes the MYB gene family in H macrophylla, identifying 72 MYB genes that exhibit diverse physicochemical properties and subcellular localizations, including predominance in the nucleus. Phylogenetic analysis reveals that several MYB transcription factors cluster into specific subfamilies associated with anthocyanin biosynthesis, suggesting potential roles in color formation. Expression profiling indicates that genes such as HmMYB15 and HmMYB54 correlate positively with anthocyanin accumulation during flowering, while HmMYB42 and HmMYB57 respond significantly to external stressors, including Pb exposure. Moreover, the expression analysis highlights key correlations between R2R3-MYB transcription factors and structural genes in anthocyanin synthesis, proposing a regulatory model that delineates the interplay between these factors in flower color development. Collectively, these findings underscore the critical roles of MYB transcription factors in regulating color and stress responses in H. macrophylla, paving the way for further investigations into their functional mechanisms.

Materials and methods

Experimental materials

The sterile flower bud stage (FSF1), flowering stage (FSF2) and flowering stage (FSF3) of hydrangea variety ‘Forever Summer’, sterile flower flowering stage (EMF3) of ‘emilia’ and sterile flower flowering stage (HMF3) of ‘Fendai’ were used as experimental materials.

Data sources

The genomic sequence and annotation files of H macrophylla were obtained from databases, while MYB transcriptome data (SRP061814, SRP451343, SRP463671) were sourced from the ENA Browser (ENA Browser (ebi.ac.uk)).

Identification of MYB gene family members in hydrangea and analysis of physicochemical properties

The HMM file for the MYB domain (PF00249) was downloaded from the InterPro website (InterPro (ebi.ac.uk)). Using HMMER 3.0, members of the MYB gene family were screened from the H macrophylla protein sequence library, with a retrieval threshold of E < 1e-5. This initial screening yielded 114 candidate MYB sequences. These sequences were then submitted to iTAK for secondary screening, resulting in the identification of 72 genes containing the MYB domain, which were subsequently renamed HmMYB1 to HmMYB72. The physicochemical properties of the 72 MYB proteins were analyzed using the Protein Parameter Calc window in TBtools. Subcellular localization predictions for the MYB proteins were conducted utilizing the online tool WoLF PSORT (WoLF PSORT: Protein Subcellular Localization Prediction (hgc.jp)).

Analysis of conserved motifs and gene structure in hydrangea MYB proteins

The protein sequences of the H macrophylla MYB family were submitted to MEME Suite 5.5.6 (MEME - Submission form (meme-suite.org)), with motif parameters set to 7, allowing for the identification of conserved motifs among MYB family members. To obtain domain information, the protein sequences were submitted to CDD (NCBI Conserved Domain Database (nih.gov)), and visual analysis was performed using TBtools. The Visualize Gene Structure function in TBtools was employed to submit the genomic GFF file of H macrophylla in order to acquire the coding sequence (CDS) location information for the MYB genes.

Phylogenetic analysis of hydrangea MYB proteins

Using MEGA X version 10.0.3, a phylogenetic tree was constructed for the 72 identified MYB family members from H macrophylla in comparison with 133 MYB genes from Arabidopsis. The neighbor-joining (NJ) method was employed for tree construction, with a bootstrap setting of 1000 replicates. The resulting phylogenetic tree was further refined using the online tool evolview 3.0 (https://www.evolgenius.info/evolview/#/treeview), and the tree was classified according to the subfamilies of Arabidopsis MYB family members.

Data analysis of hydrangea MYB transcriptome

The expression HeatMap of hydrangea MYB gene under different conditions can be obtained by putting the transcriptome data into the Heat Map tool of TBtools. Besides, correlation heat map between hydrangea MYB gene and anthocyanin and the correlation heat map with the key structural genes of anthocyanin synthesis pathway can be obtained by uploading data on the online website (https://www.omicshare.com/tools/).

Real-time fluorescence quantitative PCR

Using the sterile flower cDNA of hydrangea as a template, according to the specific reaction system and steps of reagent ChamQUniversal SYBR qPCR Master Mix, the cDNA was diluted 10 times with sterile water, and the reaction system and procedure were set up according to the instructions for quantitative experiments. The internal reference gene is ACTIN gene with stable expression in hydrangea, which was screened by our research group. After the reaction, the experimental data were analyzed.

Results and analysis

Identification and physicochemical properties of MYB gene family members in H macrophylla

Using the HMM (Hidden Markov Model) method, a total of 114 MYB gene family members were extracted from the H macrophylla genome. After screening for genes containing the MYB domain, 72 MYB genes were identified. The physicochemical properties and subcellular localization of the 72 H. macrophylla MYB proteins were analyzed (Table S1). The results indicated that the number of amino acids ranged from 65 to 770, with the largest protein being HmMYB29 and the smallest protein being HmMYB35. The theoretical isoelectric point varied, with a maximum of 10.89 for HmMYB40 and a minimum of 4.12 for HmMYB69. Among the proteins, six (HmMYB3, HmMYB23, HmMYB25, HmMYB35, HmMYB36, and HmMYB61) exhibited instability coefficients less than 40, classifying them as stable proteins, while the remaining 66 proteins had instability coefficients greater than 40, categorizing them as unstable proteins. Furthermore, all 72 H. macrophylla MYB proteins had hydrophilicity coefficients less than 0, indicating that they are hydrophilic proteins. According to the subcellular localization results, most of the identified genes were localized in the nucleus. Additionally, HmMYB29 and HmMYB36 were localized to the plasma membrane, HmMYB43 and HmMYB44 were found in the mitochondria, HmMYB61, HmMYB69, and HmMYB70 were localized to the cytoskeleton, while HmMYB24, HmMYB32, HmMYB49, HmMYB65, and HmMYB72 were found in the chloroplasts.

Phylogenetic tree analysis of MYB protein system in hydrangea

To investigate the evolutionary relationships among the MYB gene members, a phylogenetic tree was constructed using 72 MYB genes from H macrophylla and 133 MYB genes from Arabidopsis thaliana with the MEGA software (Fig. 1). Based on the classification criteria of the Arabidopsis MYB gene family11it was found that 13 MYB transcription factors from H. macrophylla are distributed across five subfamilies of the Arabidopsis R2R3-MYB transcription factors. Specifically, HmMYB4 clusters within subfamily 2, while HmMYB3, HmMYB11, HmMYB42, HmMYB61, HmMYB54 and HmMYB57 cluster in subfamily 5, suggesting that these genes may be related to the formation of anthocyanins or proanthocyanidins. HmMYB2, HmMYB7, HmMYB8, and HmMYB19 belong to subfamily 13, where the Arabidopsis S13 member AtMYB60 is known to influence lignin deposition, mucilage production, and stomatal aperture. Additionally, HmMYB1 clusters in subfamily 15, potentially relating to trichome formation, while HmMYB15 is positioned in subfamily 23. The phylogenetic tree analysis of 13 MYB proteins from hydrangea and 13 protein sequences with high homology in other plants showed that (Fig. 2) HmMYB11 was clustered with VcMYB4; HmMYB42, HmMYB61, HmMYB54 and HmMYB57 have the closest genetic relationship with PsMYB114L (Paeonia Suffruticosa) and CsMYB114L (Camellia sinensis). Studies have shown that overexpression of PsMYB114L in Arabidopsis thaliana can significantly increase the expression level of AtDFR and AtANS genes, and the accumulation of anthocyanins is significantly increased, resulting in purple leaves28. HmMYB3 is clustered with CjMYB1 and DkMYB4, in which DkMYB4 acts as a regulator of proanthocyanidins biosynthesis in persimmon. CjMYB1 gene can regulate the expression of anthocyanin synthesis genes downstream, thus playing an important role in the synthesis of anthocyanins and proanthocyanidins, flavonoids in camellia petals29,30.

Fig. 1
figure 1

Phylogenetic tree of hydrangea HmMYB and Arabidopsis AtMYB gene family. Note: The phylogenetic tree was divided into S1-S25 subfamilies according to the classification standard of AtMYB gene family in Arabidopsis thaliana.

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Fig. 2
figure 2

Phylogenetic tree analysis of MYB protein between hydrangea and other plants Note: GenBank accession number of protein sequences of other species is PsMYB114L (QBK15079.1), CsMYB114L (XP_028068989.1), LsMYB111(XP_023741633.1), LjMYB12 (QER90717.1), PsMYB12L (QBK15080.1), RvMYB12 (XP_058189634.1), CfMYB6 (XP_059634405.1), CjMYB1 (UZK92551.1), DkMYB4 (BAI49721.1), PhMYB27 (AHX24372.1), VcMYB4 (ALP43794.1), EpMYB4 (WCJ25261.1), VvMYB6 (XP_002273328.1).

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Analysis of the conserved motifs and gene structure of the MYB family proteins in H macrophylla

The analysis of 6 R2R3-MYB gene sequences from H macrophylla was conducted using the MEME online tool, which facilitated the identification of conserved motifs and amino acid sequences within the MYB proteins (Figs. 3 and 4). The results revealed that these 13 R2R3-MYB gene sequences contain five conserved motifs, designated as motif 1 through motif 5. Among these, motif 1, motif 2, and motif 3 represent typical R2R3 domains. Notably, with the exception of HmMYB1, all other 12 MYB proteins exhibit the presence of both motif 2 and motif 3. Additionally, members of the MYB family in H. macrophylla that reside within the same phylogenetic branch display identical or similar conserved motifs, as exemplified by HmMYB4 and HmMYB19. These proteins contain typical MYB gene family structural domains, indicating a close relationship between the exons and the evolutionary history of the gene family. To further elucidate the structural characteristics of the MYB gene family in H. macrophylla, an analysis of the exons in the MYB genes was performed (Fig. 4). Among the 13 MYB genes analyzed, HmMYB3 and HmMYB15 were found to contain only one exon, while the remaining genes possess two to three exons.

Fig. 3
figure 3

Motif amino acid sequence of MYB protein.

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Fig. 4
figure 4

Protein conserved motif, conserved domain and gene structure of Hydrangea R2R3-MYB.

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Expression analysis of 13 R2R3-MYB transcription factors related to color in hydrangea

R2R3-MYB transcription factors (TFs) play a crucial role in plant metabolism. To further investigate the functions of R2R3-MYB TFs in H macrophylla, samples were collected from the following developmental stages of the infertile flowers of ‘Forever Summer’: flower bud stage (FSF1), discoloration stage (FSF2), flowering stage (FSF3), flowering stage of ‘Emilia’ (EMF3), and flowering stage of ‘Fendai’ (HMF3). Transcriptome sequencing was conducted, resulting in a gene expression heat map (Fig. 5A). HmMYB1, HmMYB4 and HmMYB19 have no difference in expression, so they are not shown in the figure. The expression levels of HmMYB15 and HmMYB54 in Forever Summer increased with the accumulation of anthocyanins during flowering, with expression levels at FSF3 being higher than those at EMF3 and HMF3. In contrast, the expression level of HmMYB61 in Forever Summer decreased as the flowering period progressed. Additionally, the expression heat map for green stems at an early developmental stage, normal purple stems, and colorless stems subjected to light deprivation treatment with aluminum foil was generated (Fig. 5B). HmMYB61 exhibited higher expression levels in both green and purple stems, gradually declining with the light deprivation treatment. Meanwhile, HmMYB54 showed the highest expression level in the purple stems, with expression decreasing following the light deprivation treatment. To explore the regulatory mechanism of Al³⁺ in altering the flower color of hydrangeas, AlCl₃ was applied to the roots and leaves of H. macrophylla (Fig. 5C). After treatment, the expression levels of HmMYB42, HmMYB57, and HmMYB61 significantly increased. It can be inferred that HmMYB15, HmMYB54, HmMYB61, HmMYB42 and HmMYB57 may be related to the formation of anthocyanins.

Fig. 5
figure 5

Expression thermogram of R2R3-MYB transcription factor in hydrangea77. Note: A hydrangea ‘Forever Summer’ bud stage (FSF1), discoloration stage (FSF2), flowering stage (FSF3), ‘Emilia’ flowering stage (EMF3) and ‘FenDai’ flowering stage (HMF3); B green stems, purple stems and stems treated in darkness for 21 days; C roots and leaves treated with AlCl3.

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Correlation between R2R3-MYB transcription factors and anthocyanin content in H macrophylla

The expression levels of R2R3-MYB transcription factors in H macrophylla were analyzed for their correlation with anthocyanin content (Fig. 6A). The results indicated significant positive correlations for HmMYB8, HmMYB15, HmMYB42, HmMYB54, and HmMYB57, while HmMYB8 displayed significant correlations with nearly all anthocyanins, except for delphinidin-3-O-glucoside and cyanidin-3-O-(2’’-O-glucosyl) glucoside, with the highest correlation coefficient observed for delphinidin-3,5,3’-O-glucoside. HmMYB15 was positively correlated with delphinidin − 3-O- glucoside at the level of P < 0.05. HmMYB54 showed a highly significant positive correlation with delphinidin-3-O-glucoside. at the level of P ≤ 0.001. Additionally, HmMYB42 was positively correlated with delphinidin − 3-O- rutinoside − 7-O- glucoside and cyanidin − 3-O- sophoricoside − 5-O- glucoside at the level of P < 0.01. HmMYB57 was positively correlated with delphinidin − 3-O- rutinoside − 7-O- glucoside and delphinidin − 3,5,3’- tri -O- glucoside. HmMYB7 was negatively correlated with cyanidin − 3-O- glucoside and cyanidin − 3-O- rutinoside at the level of P < 0.05. HmMYB61 was negatively correlated with delphinidin − 3-O- glucoside and positively correlated with cyanidin − 3-O- rutinoside. In addition, the correlation between the expression levels of R2R3-MYB transcription factors and key structural genes involved in anthocyanin synthesis was analyzed (Fig. 6B). The results showed that HmMYB15 was positively correlated with HmDFR and HmANS at P < 0.05. HmMYB54 was correlated with HmF3H at p < 0.05, with HmCYP75B1 at P < 0.01, with HmC3’5’H and HmANS at P ≤ 0.001, with HmMYB61 negatively correlated with HmANS at P < 0.05 and with HmF3H and HmDFR at P ≤ 0.001.

Fig. 6
figure 6

Correlation thermogram of R2R3-MYB transcription factor with anthocyanins and key structural genes in hydrangea. Note: A key structural genes of anthocyanin synthesis pathway; B delphinidin-3-O- glucoside and other seven anthocyanins. * means 0.01 < P < 0.05; ** means 0.001 < P < 0.01; *** means P ≤ 0.001.

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Expression analysis of 6 R2R3-MYB transcription factors related to color in hydrangea

In order to deeply explore the role of S5 subfamily genes in hydrangea flower color regulation, the genes HmMYB3, HmMYB11, HmMYB42, HmMYB54, HmMYB57 and HmMYB61 expression was verified in Hydrangea ‘Forever Summer’ bud stage (FSF1), discoloration stage (FSF2), flowering stage (FSF3), ‘Emilia’ flowering stage (EMF3) and ‘Fendai’ flowering stage (HMF3) by qPCR experiment. The qPCR detection data and the analysis results of RNAseq were integrated and compared (Fig. 7). The experimental results show that HmMYB11, HmMYB42 and HmMYB54 in the two groups of data have a correlation at P < 0.05, while HmMYB61 has a significant correlation at p < 0.0001.

From the expression level, six genes at different developmental stages are highly consistent with the previous transcriptome sequencing data. Specifically, the expression levels of qPCR and RNAseq of HmMYB3 in three hydrangea varieties are very low, with little difference, and the expression of gene HmMYB11 is the highest at the flowering stage (EMF3) in Emilia. In addition, three genes, HmMYB42, HmMYB54 and HmMYB57, have the highest expression at the flowering stage of hydrangea (FSF3). It is worth noting that the expression level of gene HmMYB54 in Forever Summer sterile flowers showed a significant upward trend with the continuous accumulation of flower color substances, which probably participated in the positive regulation process of flower color synthesis in Forever Summer sterile flowers; On the contrary, the expression of HmMYB11 and HmMYB61 decreased gradually with the accumulation of flower color, which may play a negative regulatory role in the process of flower color formation in Forever Summer infertility.

Fig. 7
figure 7

Gene expression of S5 subfamily in hydrangea. Note: hydrangea Forever Summer bud stage (FSF1), discoloration stage (FSF2), flowering stage (FSF3), Emilia flowering stage (EMF3) and Fendai flowering stage (HMF3). The bar chart represents qpcr, and the line chart represents RNAseq.

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Prediction formation of the regulatory model of R2R3-MYB transcription factors in hydrangea

According to the correlation between transcription factor R2R3-MYB of hydrangea and anthocyanin expression, as well as the correlation analysis of key structural genes of anthocyanin synthesis, the flower color formation mechanism of hydrangea is predicted (Fig. 8). Taking hydrangea Forever Summer as an example, during the flower color formation process, HmMYB61 negatively regulates HmF3H and HmDFR to influence anthocyanin synthesis; HmMYB15 positively regulates the synthesis of HmDFR and HmANS and affects the accumulation of delphinidin 3-O- glucoside; HmMYB54 positively regulates the synthesis of HmF3H, HmCYP75B1, HmC3’5’H, and HmANS, with the greatest influence on HmC3’5’H and HmANS, and then affects the accumulation of anthocyanins in hydrangea, especially the accumulation of delphinidin − 3-O- glucoside, which makes the sterile flowers of hydrangea appear blue.

Under Al3+ stress, the expression of HmMYB54 and HmMYB3 in the root of hydrangea increased to adapt to ions. A large number of studies have confirmed that aluminum ions play a key role in the formation of blue pigment in hydrangea sepals, which is closely related to the unique physiological and biochemical characteristics and complex molecular regulatory network of hydrangea. The bluing process of hydrangea sepals is essentially a dynamic process coordinated by environmental factors and gene expression. As an important environmental signal molecule, Al3+ can affect the synthesis and stability of anthocyanins through a series of complex physiological and biochemical reactions26. HmMYB54 is positively regulated in the process of endless summer flower color formation of hydrangea, and Al3+ may promote the expression of HmMYB54 gene through many potential ways, and finally regulate the accumulation of blue anthocyanins in hydrangea.

Fig. 8
figure 8

Formation process of sterile flower color of hydrangea regulation mode. Note: The upper part of the picture shows that MYB transcription factor affects the formation of color by regulating the key genes of anthocyanin synthesis; The second part shows the response mechanism of roots under Al3+ stress, which can enhance ion tolerance and promote flower color formation by increasing the expression of MYB transcription factor.

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Discussion

The R2R3-MYB transcription factors, as the largest subfamily within the MYB family, are recognized for their multifaceted roles in plant biology, encompassing diverse functions related to primary and secondary metabolism, growth and development, and responses to environmental stresses. These transcription factors are involved in the biosynthesis of critical metabolites, including glucosinolates (GSL)31 anthocyanins32,33,34,35 and phenylpropanoids36,37 as well as the synthesis of epicuticular wax38 carotenoids39 and capsaicinoids40. Additionally, they play vital roles in cellular processes such as leaf senescence41 root development42,43 and pollen maturation44. Current studies have documented the presence of R2R3-MYB transcription factors in various species, including Arabidopsis thaliana45 cucumber46 Japanese morning glory47 and mung bean48. Notably, Arabidopsis possesses 125 identified R2R3-MYB members45 while 55 members exist in cucumber46 127 in Japanese petunia47 and 168 in mung bean48. These transcription factors are characterized by their unique molecular structures, which include distinct R2 and R3 repeats essential for their DNA-binding capabilities. The functional properties of R2R3-MYB proteins are governed by specific activation and repression domains, which modulate their regulatory functions across various biosynthetic pathways, particularly those associated with flavonoids and secondary metabolites49.

Recent structural studies have elucidated the interactions of R2R3-MYB proteins with target DNA, highlighting specific residues that are critical for binding affinity and regulatory activity. These interactions are influenced by methylation states, which can inhibit DNA binding50. Furthermore, R2R3-MYB proteins engage in complex interactions with other transcription factors, such as bHLH and WD40, forming regulatory complexes that intricately fine-tune gene expression patterns through both direct and indirect mechanisms51. The regulatory effects exerted by R2R3-MYB proteins include direct activation or repression of target genes, as well as indirect modulation via protein-protein interactions52,53. Insights gained from molecular dynamics simulations have further revealed the importance of hydrophobic and hydrogen bonding interactions in maintaining the stability and functional integrity of these complexes during DNA binding50. The evolutionary expansion and diversification of the R2R3-MYB family across multiple plant species underscore their significance and suggest that these transcription factors play critical roles in the adaptation and innovation of various plant traits51,52. This synthesis of findings emphasizes the vital contributions of R2R3-MYB transcription factors to plant biology and their potential applications in understanding plant adaptation and trait enhancement.

The investigation into the evolutionary relationships of R2R3-MYB transcription factors in H macrophylla and Arabidopsis was conducted through the analysis of assembled and annotated genomic data from H. macrophylla. This study identified a total of 72 MYB transcription factors, which were subsequently compared to the 133 MYB members identified in Arabidopsis to construct a comprehensive phylogenetic tree. Based on the established classification criteria for R2R3-MYB transcription factors in Arabidopsis, 13 distinct R2R3-MYB transcription factors were identified within H. macrophylla, distributed among subfamilies 2, 5, 13, 15, and 23. Notably, the R2R3-MYB family in H. macrophylla exhibits a reduced number of members relative to other plant species, suggesting a unique evolutionary trajectory. Extensive research has explored the evolutionary dynamics of R2R3-MYB transcription factors, underscoring the intricate relationships established through gene duplication and functional diversification across a variety of plant taxa. It has been demonstrated that significant expansions of R2R3-MYB genes are primarily attributed to whole-genome duplications, in addition to subsequent smaller duplicative events, which have collectively contributed to their functional diversity54. A pertinent example of this diversification can be observed in Arabidopsis, where paralogous MYB genes, such as WEREWOLF and GLABROUS1, have been shown to define distinct cell types while maintaining functionally equivalent roles. This illustrates that alterations in cis-regulatory sequences can lead to novel functions without necessitating changes in protein structure55. Moreover, the comparative analysis of MYB gene families across different taxa, including bHLH genes, has revealed that these expansions are closely associated with the morphological and metabolic complexities of higher plants. This association suggests that the evolution of these transcription factors is a direct response to selective pressures encountered in their respective environments56. Further studies examining the phylogenetic relationships among various MYB subfamilies, including 1R-MYB, 3R-MYB, and 4R-MYB, have provided insights into the conservation and divergence of these transcription factors57. The evolutionary pathways of these subfamilies imply that they have been shaped by specific adaptive radiations, enabling them to assume distinct functional roles in processes such as trichome morphogenesis and stress responses58. These findings collectively emphasize the significance of gene duplication events and the functional plasticity of MYB transcription factors, which enhance the capacity of plants to adapt to diverse environmental conditions. Ultimately, synthesizing data from an evolutionary perspective on R2R3-MYB transcription factors reveals a consistent pattern of expansion and functional diversification, facilitating the emergence of specialized roles in plant biology. Understanding this ongoing evolutionary process is crucial for elucidating how plants have adapted to their environments over time.

The intricate functions of R2R3-MYB transcription factors, particularly their roles in regulating anthocyanin biosynthesis and other secondary metabolites, highlight their significance within various metabolic pathways. Genes within the same subfamily generally exhibit similar functions59. For instance, AtMYB123 (TT2), the sole representative of subfamily 5 among R2R3-MYB transcription factors, encodes a protein crucial for proanthocyanidin biosynthesis in the seed coat of Arabidopsis60 and is known to enhance flavonoid production in peaches61. Within subfamily 5, H macrophylla genes HmMYB3, HmMYB11, HmMYB42, HmMYB61, HmMYB54, and HmMYB57 cluster together, suggesting their potential roles in anthocyanin or proanthocyanidin synthesis pathways62. Additionally, HmMYB4 and HmMYB19, alongside HmMYB2, HmMYB7, and HmMYB8, are positioned in adjacent branches, possessing similar conserved motifs that indicate possible functional similarities.

Research on flower color formation in H macrophylla suggests that the transcription factor HymWER-like exerts a negative regulatory effect on the coloration of sterile flowers in the blue hydrangea cultivar ‘Forever Summer‘1. Notably, genes such as HmDFR63 and HmF3H64 are thought to play pivotal roles in the color variations observed among different H. macrophylla cultivars. Under specific cultivation conditions, the interaction of aluminum ions (Al³⁺), delphinidin-3-glucoside, and copigments has been shown to facilitate a color transformation in the hydrangea sepals from red to blue65. Despite these findings, there remains a scarcity of research focused on the identification and functional analysis of R2R3-MYB genes in H. macrophylla. This study aimed to bridge this gap by conducting heatmap analyses on transcriptomic data obtained from sterile flowers across various cultivars and developmental stages. The clustering of HmMYB54, HmMYB61, HmMYB42, and HmMYB57 within subfamily 5 indicates their potential association with anthocyanin formation. The investigation sought to elucidate the correlation between H. macrophylla MYB transcription factors, anthocyanin content, and key genes implicated in anthocyanin synthesis, ultimately aiming to predict the regulatory mechanisms that underpin flower color formation.

A comprehensive examination of R2R3-MYB transcription factors reveals their integral role in anthocyanin biosynthesis regulation, emphasizing their interaction with various metabolic pathways. The mechanisms governing anthocyanin production have been characterized extensively, highlighting the activation of R2R3-MYB factors modulated by environmental stimuli and internal signals, thereby influencing color variations in horticultural plants66. These transcription factors engage with other regulatory proteins, such as bHLH transcription factors, to establish a complex regulatory network that balances the accumulation of anthocyanins and proanthocyanidins67. Furthermore, investigations have demonstrated the involvement of MYB transcription factors in the biosynthesis of flavonoids and lignins, elucidating how these secondary metabolic pathways integrate with primary metabolism68.

The functional repertoire of R2R3-MYB transcription factors extends to developmental processes, where they participate in cell cycle regulation and organ-specific developmental functions, interfacing with hormone signaling pathways to modulate growth responses69. Additionally, these transcription factors are essential for enhancing plant resilience against abiotic stressors and mediating plant-pathogen interactions, showcasing their dual capacity to respond to environmental challenges and biotic threats68,70. Comparative analyses among different species illustrate the unique adaptations of R2R3-MYB functions in crop plants versus model organisms, underlining their evolutionary development in response to specific ecological niches71. The complexity of transcriptional and post-transcriptional regulation of R2R3-MYB genes encompasses various mechanisms, including epigenetic modifications that influence protein stability and turnover, thereby affecting metabolic outcomes69. In the context of biotechnological applications, R2R3-MYB transcription factors have emerged as crucial tools in genetic engineering aimed at enhancing crop traits such as stress tolerance, nutritional quality, and ornamental characteristics, signifying substantial potential for advancements in synthetic biology and molecular breeding strategies68,72.

To date, research on the MYB gene family, particularly concerning its functional characterization, has predominantly focused on select members, especially within the model organism Arabidopsis thaliana. Studies have illustrated the significant roles of MYB transcription factors in regulating anthocyanin biosynthesis across various plant species, providing insights applicable to non-model flowering plants such as H macrophylla. For instance, R2R3-MYB transcription factors are highlighted as crucial regulators of anthocyanin synthesis, playing pivotal roles in the transcriptional regulation of biosynthetic genes69. In the Rosaceae family, MYB genes, particularly MYB10 and MYBA, have been shown to control anthocyanin levels in apple fruits, with functional studies indicating that these transcription factors can induce anthocyanin pathways in heterologous systems, such as tobacco and strawberries73. Similarly, other investigations revealed that MYB112 in Arabidopsis enhances anthocyanin accumulation under stress conditions, further emphasizing the complex regulatory networks involving MYB factors74. Moreover, the investigation into petunia has revealed that members like DEEP PURPLE and PURPLE HAZE are instrumental in both floral and vegetative pigmentation, demonstrating the adaptability of MYB regulators in different contexts75. These findings underscore the diverse functions and evolutionary significance of MYB transcription factors across flowering plants. Functional analyses conducted in various species suggest that the modulation of anthocyanin biosynthesis by MYBs can be leveraged for enhancing color quality in ornamental and food crops, as seen in the manipulation of MYB119 in poplar to boost anthocyanin levels without detriment to plant growth76. The ongoing exploration of these transcription factors not only sheds light on the genetic basis of pigmentation but also paves the way for future research into the regulatory mechanisms governing anthocyanin synthesis in non-model plants like H. macrophylla, which remains underexplored in the context of MYB functionality.

Conclusion

The findings from this study significantly advance the understanding of the genetic mechanisms underlying flower color variation in H macrophylla. The identification of 72 MYB genes, particularly the roles of R2R3-MYB transcription factors such as HmMYB15 and HmMYB54, provides a robust framework for further exploration of anthocyanin biosynthesis pathways. These transcription factors not only correlate with anthocyanin accumulation during flowering but also exhibit responsiveness to environmental stressors, suggesting that they play a dual role in both ornamental traits and adaptive strategies. The correlation between MYB gene expression and anthocyanin levels points to a complex regulatory network that governs flower coloration, integrating genetic, environmental, and developmental factors. By situating these results within the context of existing literature, it becomes evident that MYB transcription factors are critical not just for H. macrophylla, but potentially for a wide range of plant species, as they contribute to phenotypic plasticity in response to changing environments. This adaptive capacity is essential for survival and reproduction, particularly in the face of climate change and habitat alterations. Additionally, the proposed regulatory model highlights the intricate interactions between MYB genes and structural genes involved in anthocyanin biosynthesis, thereby offering potential pathways for targeted genetic manipulation to enhance desirable traits in ornamental horticulture. Future research should aim to further dissect these interactions, perhaps exploring how external factors modulate MYB gene expression and flower color dynamics. In summary, this study not only enhances our comprehension of flower color genetics in H. macrophylla but also underscores the broader ecological significance of MYB transcription factors in plant adaptation and resilience.