A R2R3-MYB, VwMYB1, regulates anthocyanin biosynthesis in pansy petal blotch via transcriptional activation of VwF3’5’H

A R2R3-MYB, VwMYB1, regulates anthocyanin biosynthesis in pansy petal blotch via transcriptional activation of VwF3’5’H | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A R2R3-MYB, VwMYB1, regulates anthocyanin biosynthesis in pansy petal blotch via transcriptional activation of VwF3’5’H Xinyu Ye, Xiaopei Zhu, Yudan Li, Yanke Hu, Jinyan Mu, Xiaohua Du, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8639386/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 May, 2026 Read the published version in Plant Cell Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Anthocyanins play a pivotal role in determining the visual characteristics of ornamental plants. To elucidate the molecular basis of the distinctive “blotch” formation in pansy ( Viola × wittrockiana ), this study employed an integrated approach that combines targeted metabolomics and comparative transcriptomics. Comparative analysis of blotched versus non-blotched petal regions identified VwMYB1 , a differentially expressed R2R3-MYB transcription factor, as a key candidate regulator. Functional validation revealed that the heterologous overexpression of VwMYB1 in tobacco enhanced floral pigmentation, while its transient overexpression in pansy petals induced localized dark blotches, thereby confirming its role in promoting pigmentation. Targeted metabolomic profiling demonstrated that the blotch phenotype is primarily attributed to the substantial and specific accumulation of delphinidin-type anthocyanins. Given that F3’5’H is the pivotal branch-point enzyme for delphinidin biosynthesis, the regulatory interaction between VwMYB1 and VwF3’5’H was further examined. Yeast one-hybrid and dual-luciferase reporter assays confirmed that VwMYB1 directly binds to the promoter of VwF3’5’H and activates its expression. This study not only illustrates a streamlined pipeline from transcriptome-based gene discovery to functional and metabolic validation but also uncovers the core regulatory mechanism by which VwMYB1 governs delphinidin-based blotch formation through the direct transcriptional activation of VwF3’5’H , thereby offering both mechanistic insight and a practical genetic tool for precision breeding in flower color modification. Viola × wittrockiana blotch formation anthocyanin biosynthesis VwMYB1 VwF3’5’H Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Anthocyanins, a class of water-soluble flavonoid pigments, are widely distributed in plants and perform diverse functions (Saito et al., 2013 ). In addition to their well - recognized function of attracting pollinators and increasing the ornamental value of flowers, as demonstrated by the attractive blotches, spots, and stripes in many angiosperms (Moeller et al., 2005; Shang et al., 2011 ), they also act as antioxidants, enhancing the plants' resistance to both biotic and abiotic stresses and being beneficial to human health. (Lu et al., 2024 ). The pansy ( Viola × wittrockiana ) is an excellent ornamental plant, well-known for its unique and stable 'face-like' blotches with anthocyanin enrichment at the flower center, a characteristic that is central to its commercial attractiveness(Endo, 1959 ). Early studies identified delphinidin- and cyanidin-type anthocyanins as the primary pigments responsible for petal blotch in pansy (Endo, 1959 ). Subsequently, this finding was refined by pinpointing floral developmental stages III–V as the critical period for their accumulation (Li et al., 2014 ). The anthocyanin biosynthesis pathway is evolutionarily conserved and well-characterized in higher plants, encompassing six essential structural genes: chalcone synthase ( CHS ), chalcone isomerase ( CHI ), flavonoid 3-hydroxylase ( F3H ), dihydroflavonol 4-reductase ( DFR ), anthocyanin synthase ( ANS ), and UDP-glucose flavonoid-3-O-glucosyltransferase ( UFGT ) (Rausher et al., 1999 ; Cappellini et al, 2021 ). In addition, the cytochrome P450 enzymes flavonoid 3′-hydroxylase ( F3’H ) and flavonoid 3′,5′-hydroxylase ( F3’5’H ) play decisive roles in determining the types of anthocyanin accumulation (Tanaka and Brugliera, 2013 ). For example, F3’H is highly expressed throughout all developmental stages in wild strawberry ( Fragaria vesca ), contributing to elevated cyanidin levels in its fruits (Thill et al., 2013 ). Notably, F3’5’H acts as the key branch-point enzyme that channels metabolic flux toward delphinidin-type anthocyanin synthesis, a function that has been validated in pansy (Huynh et al., 2024 ). These structural genes are commonly classified into early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs) (He et al., 2010 ; Holton and Cornish, 1995 ). LBGs, such as DFR , ANS , and UFGT , encode enzymes essential for the terminal steps of anthocyanin synthesis and thus play a vital role in regulating total anthocyanin accumulation (Winkel-Shirley, 2001 ). Nevertheless, the upstream regulatory mechanism governing the spatially precise expression of F3’5’H during blotch pattern formation remains poorly understood, representing a significant gap in current knowledge. Anthocyanin biosynthesis is predominantly regulated by transcription factors (TFs), including the MYB, bHLH, and WRKY families (Li et al., 2016 ; Hu et al., 2020 ; Ding, 2023 ). The MBW complex, which is formed by MYB, bHLH, and WD40 proteins, serves as a core regulatory module (Zhang et al., 2020 ). Although the regulatory networks governing pigmentation have been clarified in species such as peony and apple, it is still arduous to identify the key regulators from the extensive MYB family in non - model plants like pansy. Previous studies, for example, the transient functional validation of VwMYB8 in heterologous systems, were short of stable genetic evidence in the native plant context (Zeng et al., 2016 ). Moreover, the lack of a reference genome makes traditional screening approaches even more complicated. To directly tackle this mechanistic gap, we devised a strategy of combining targeted metabolomics with, transcriptome for analysis of blotch and non-blotch tissues to identify candidate MYB TFs. Subsequently, the function of the primary candidate was validated via genetic transformation. Ultimately, the molecular mechanism was revealed by clarifying how VwMYB1 regulates the downstream structural genes, such as F3′5′H , for anthocyanin synthesis. This research not only deciphers the core regulatory module governing blotch formation in pansy but also establishes an efficient pipeline for gene discovery in non-model ornamental plants. Materials and methods Plant materials and cultivation environment The plant material utilized in this study was an inbred line DSRFY, which was derived from a pansy cultivar ‘Swiss Giants Finegold’. It displays regular dark-brown blotches on its petals. Seeds of this material were sown on September 30, 2024. Seedlings were transplanted into 12–14 cm pots once they had 3–4 true leaves and were cultivated on the campus of Henan Institute of Science and Technology (35.18°N, 113.52°E) for subsequent care and growth. Petal tissues were collected for total RNA extraction and pigment detection. Determination of anthocyanin content The anthocyanin content was determined in accordance with the methods previously described (Xie et al., 2012 ; Wang et al., 2022 ). For each experimental group, five petal samples were collected, promptly frozen in liquid nitrogen, and stored at − 80°C. The frozen composite samples were pulverized into powder using a cryogenic grinder and weighed for subsequent analysis. Approximately 0.1 g of petal tissue was homogenized in 1 mL of extraction buffer. The homogenate was transferred to a microcentrifuge tube, and the volume was adjusted to 1 mL by adding additional extraction buffer. The tube was firmly sealed and incubated at 60°C for 30 minutes with intermittent shaking. Subsequently, the sample was centrifuged at 12,000 rpm/min for 10 minutes at room temperature, and the supernatant was collected for analysis. The total monomeric anthocyanin content was determined using the pH-differential method (Lee et al., 2005). Briefly, two aliquots of the extract were separately mixed with potassium chloride buffer (pH 1.0) and sodium acetate buffer (pH 4.5). The absorbance of each mixture was measured at 530 nm and 700 nm using a spectrophotometer, and the corrected absorbance difference (ΔA) was calculated according to the standard formula. The total anthocyanin content, expressed as micromoles per gram of fresh weight (µg/g FW), was then calculated from the ΔA value using the established formula which incorporates the molar extinction coefficient and path length. Each sample was analyzed with three independent technical replicates, and the data were subjected to statistical analysis. Transcriptome sequencing and analyses Samples were collected from the petals of floral buds before their blooming according to Li et al ( 2014 ) and dissected into blotch and non-blotch regions, with three biological replicates per group. All samples were immediately flash-frozen in liquid nitrogen and stored at − 80°C. Total RNA was extracted using the Plant Total RNA Extraction Kit (Vazyme, China). RNA integrity was evaluated by agarose gel electrophoresis, and purity and concentration were measured using a NanoDrop 2000 spectrophotometer (OD260/280 between 1.8 and 2.0). High-quality RNA was utilized to construct cDNA libraries, which were sequenced on the Illumina NovaSeq platform (paired-end, 150 bp) by Biomarker Technologies Co., Ltd. (Beijing, China). Raw reads were filtered via Trimmomatic to eliminate adapters and low-quality sequences, and clean reads were assembled using Trinity. Coding sequences (CDS) were predicted with TransDecoder. Unigene functional annotation was carried out using BLASTx (E - value ≤ 1×10⁻⁵) against the NR, Swiss-Prot, Pfam, GO, KEGG, and KOG databases. Transcript abundance was quantified and presented as FPKM using RSEM. Differential expression analysis was executed with the DEGseq R package, with genes meeting |log₂FoldChange| ≥ 1 and FDR < 0.01 regarded as differentially expressed genes (DEGs). Gene Ontology (GO) enrichment analysis of DEGs was performed using Blast2GO. KEGG pathway enrichment analysis was carried out using KOBAS 2.0. Terms and pathways with an adjusted p - value < 0.05 were deemed significantly enriched. Real-time quantitative PCR (qPCR) Total RNA was extracted from various tissues of pansy, including the blotch and non - blotch regions of petals at different developmental stages: S1 (petal length approximately 0.7 cm), S3 (petal length approximately 1.2 cm), S5 (the first day of flowering), and S8 (onset of flower senescence), as well as from roots, stems, and leaves. The extraction was performed using a Plant Total RNA Extraction Kit (Vazyme, China). First-strand cDNA was synthesized from the extracted RNA by utilizing the PrimeScript RT Reagent Kit (Vazyme, China). Subsequently, quantitative real-time PCR (qRT-PCR) was performed with SYBR Premix Ex Taq (Takara, China) on a real-time PCR system. The β-actin gene of pansy was employed as an internal reference for normalization. The quantity of the starting template was adjusted according to the cycle threshold (Ct) values of this endogenous control (usually ranging from 18 to 20 cycles). All the primers utilized in this study are presented in Supplementary Table S1. Isolation of VwMYB1 from pansy Total RNA was extracted from pansy petal tissues using a Plant Total RNA Extraction Kit (Vazyme, China), strictly following the manufacturer's instructions. Subsequently, first-trand cDNA was synthesized from the extracted RNA using the PrimeScript RT Reagent Kit (Vazyme, China). To acquire the full - length cDNA of VwMYB1, PCR amplification was carried out with gene-specific primers (Supplementary Table S1). The resultant PCR product was purified and sequenced. The obtained nucleotide and deduced amino acid sequences were respectively analyzed via the BLASTX and BLASTP programs against the GenBank database to search for homologous sequences and conduct comparative analyses. Subcellular localization assay The full-length cDNA of VwMYB1 was precisely inserted into the pCambia1302 vector to generate the fusion construct pCambia1302:: VwMYB1 . Subsequently, this construct was introduced into Agrobacterium tumefaciens strain GV3101 through electroporation. As a control, the pCambia1302 vector, which contained a CaMV35S::GFP cassette, was employed. Agrobacterium strains carrying the respective vectors were cultured on LB agar plates supplemented with appropriate antibiotics at 28°C for 2–3 days. Bacterial cells were then collected and resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES [pH 5.6], 200 µM acetosyringone) to reach an OD₆₀₀ of 0.4. Before infiltration, the suspensions were left at room temperature for 2 hours without agitation. Approximately 500 µL of the bacterial suspension was infiltrated into the abaxial side of young leaves from 3-to 4-week-old tobacco ( Nicotiana benthamiana ), ensuring at least two infiltration sites per leaf. Subcellular localization analysis was carried out 3 days after infiltration. Confocal images were captured using a Zeiss LSM 710 laser scanning microscope (Zeiss, Germany). VwMYB1 -overexpressing in tobacco The full-length open reading frame (ORF) of VwMYB1 was cloned into the pCambia1302 vector to generate the recombinant construct pCambia1302::VwMYB1. Subsequently, this construct was introduced into the Agrobacterium tumefaciens strain GV3101. Then, tobacco transformation was carried out using the previously described Agrobacterium -mediated method. Transgenic tobacco lines were selected on a medium containing hygromycin. After root induction and acclimatization, the putative transgenic plants were transferred to a greenhouse and grown until they reached the flowering stage. The integration of the VwMYB1 transgene into the genome of the resulting plants was confirmed by PCR analysis. Transient overexpression of VwMYB1 in pansy petals To analyze the function of VwMYB1 , a transient overexpression assay was performed in detached pansy petals. The VwMYB1 overexpression construct (pCambia1302::VwMYB1) and the empty vector control were introduced into Agrobacterium tumefaciens strain GV3101, respectively. The bacterial suspensions were then infiltrated into the petals. After infiltration, the petals were kept in the dark for 24 hours and subsequently transferred to conditions of 25°C with a 16-h-light/8-h-dark photoperiod for three days before phenotypic observation. Detection of anthocyanin composition in petal blotch Freeze-dried petal tissues were thoroughly ground into a fine powder. Approximately 50 mg of the powder was subjected to extraction twice with 0.5 mL of a methanol/water/hydrochloric acid (500:500:1, v/v/v) solution. The extraction process involved vortexing and ultrasonication. After centrifugation, the combined supernatants were carefully collected, filtered through a 0.22 µm membrane, and then used for LC-MS/MS analysis. Anthocyanin separation was conducted on a Waters ACQUITY BEH C18 column (1.7 µm, 2.1 × 100 mm) using a UPLC system (ExionLC™ AD). The mobile phase was composed of water and methanol, each containing 0.5% formic acid, with a flow rate maintained at 0.35 mL/min at a temperature of 40°C. A well-defined gradient elution program was applied. Detection and quantification were accomplished using a triple quadrupole-linear ion trap mass spectrometer (QTRAP® 6500+) operating in positive electrospray ionization (ESI+) mode with scheduled multiple reaction monitoring (MRM). Metabolites were quantified using Multiquant 3.0.3 software (Sciex) with optimized MRM transitions. Dual-luciferase reporter (DLR) assay The perform dual-luciferase activity assays as previously described (Ji et al., 2024). Clone the coding sequence (CDS) of ChMYB1 into the pGreen62-SK vector to serve as the effector construct. Insert the proChGSTF5 into the pGreen0800-LUC vector to generate the reporter construct. Co-infiltrate Agrobacterium tumefaciens (GV3101 pSoup) sus pensions containing the effector and reporter constructs (9:1, OD600 = 0.8) into tobacco leaves for transient expression analysis. Yeast one-hybrid assay The coding sequence (CDS) of VwMYB1 was precisely inserted into the pGADT7 vector to generate the prey construct. The promoter sequence of VwF3’5’H, which contains conserved cis -elements, was carefully cloned into the pAbAi vector to create the bait construct. Subsequently, the paired prey and bait constructs were co-transformed into yeast strain Y1HGold cells. After that, the transformed yeast cells were plated on synthetic dropout (SD) media that lacked tryptophan and uracil and were supplemented with X - gal for blue color development screening. Results Phenotypic and Anthocyanin Content of Blotched and Non-blotched Regions in Pansy To quantitatively clarify the pigment basis of the blotch phenotype, we employed the pansy inbred line DSRFY and separately collected tissue from the blotched and non - blotched regions of petals (Fig. 1a). Spectrophotometric determination of anthocyanin content demonstrated a highly significant disparity between the two regions. Specifically, the anthocyanin content in the blotched region was 8.35 ± 0.09 μg/g fresh weight (mean ± SD), while it was merely 2.28 ± 0.33 μg/g in the non - blotched region. The accumulation level in the blotched region was approximately 3.7 times higher than that in the non-blotched region (Fig. 1b). These results offer direct metabolic evidence indicating that the formation of the blotch is accompanied by highly specific and localized accumulation of anthocyanins. Identification of anthocyanin components in pansy petals To determine the pigment components in the petal blotch area of pansy line DSRFY, a targeted anthocyanin metabolomic analysis was carried out on blotch and non - blotch tissues using LC-MS/MS. The results indicated a highly specific and intense accumulation of anthocyanins in the blotch region. Delphinidin-type anthocyanins were identified as the predominantly dominant pigments, followed by some cyanidin - type anthocyanins (Fig.2a). Among them, delphinidin-3-O-(6''-O-coumaroyl) rutinoside-5-O-glucoside was the most prominent, with an average content reaching as high as 838.7 μg/g in the blotch, which represented a 294-fold increase compared to its level in the non-blotch region, thus constituting the core pigment of the blotch. Moreover, several other delphinidin glycosides, including delphinidin-3-O-glucuronide (mean 42.36 μg/g, ~26-fold blotch/non-blotch ratio) and delphinidin-3-O-rutinoside (mean 19.27 μg/g, ~30-fold ratio), also showed specific and high-level accumulation in the blotch parts (Fig.2b). Certain cyanidin derivatives, such as cyanidin-3,5-O-diglucoside, exhibited accumulation. In contrast, anthocyanins of other types, such as peonidin -, malvidin -, and petunidin-derivatives, were generally present at low concentrations (all below 0.4 μg/g) with no significant difference between the blotch and non-blotch parts of the petals. This extremely distinct metabolite distribution implies that the spatially specific activation of the delphinidin biosynthetic pathway in the cells of petal blotch parts is the key basis for blotch coloration, necessarily involving some functional genes for delphinidin biosynthesis, including F3’5’H and their precise transcriptional regulation by MYB-type transcription factors (Cao et al; 2020). Transcriptome analysis of blotched and non-blotched regions in pansy petals To decipher the transcriptional regulatory mechanism underlying anthocyanin accumulation in the blotched regions of pansy petals, we carried out a comparative transcriptome analysis between the blotched and non-blotched petal regions of DSRFY. A total of 2,554 DEGs were identified, including 1,239 upregulated and 1,315 downregulated genes (Fig.3a). GO enrichment analysis indicated that these DEGs were significantly enriched in terms related to the biological process, cellular component, and molecular function categories (Fig.3b). KEGG pathway analysis demonstrated that the top ten significantly enriched pathways were as follows: Plant-pathogen interaction, Plant hormone signal transduction, MAPK signaling pathway–plant, Carbon metabolism, Biosynthesis of amino acids, Flavonoid biosynthesis, Phenylpropanoid biosynthesis, Starch and sucrose metabolism, Carbon fixation in photosynthetic organisms, and Photosynthesis (Fig.3c). The DEGs enriched in the biosynthesis pathways of flavonoids and anthocyanins revealed that the differences in the expression of genes related to anthocyanin synthesis were the key cause of the differences in anthocyanin accumulation between blotched and non-blotched area. Identification, Expression Pattern, and Subcellular Localization of VwMYB1 We analyzed the expression of eight core structural genes implicated in anthocyanin biosynthesis, namely VwCHI , VwDFR , VwANS , VwUFGT , and the crucial VwF3’5’H . The findings of this analysis indicated that the expression levels of all these genes were elevated in the blotched region when compared to the non-blotched region of the petals (Fig. 4a). This coordinated up-regulation is in line with the metabolic phenotype of increased anthocyanin accumulation in the blotched region. Considering the well-documented regulatory functions of MYB transcription factors in anthocyanin biosynthesis across various plant species, such as peonies and grapevines, our investigation was centered on them. Further screening from previously acquired transcriptome data identified 64 differentially expressed MYB transcription factors (Fig. 4b), suggesting their potential as key regulators of localized anthocyanin synthesis. Among these candidates, the expression pattern of VwiA01G125930 (named VwMYB1 ) exhibited a strong positive correlation with anthocyanin accumulation levels, suggesting that it may serve as a core transcription factor governing the anthocyanin biosynthetic pathway in pansy. Gene expression pattern and protein structural characteristics of VwMYB1 To elucidate the function of the candidate gene VwMYB1 identified from the pansy petal transcriptome, we initially analyzed its expression pattern via quantitative real-time polymerase chain reaction (qRT-PCR). The findings indicated that this gene was specifically and highly expressed in petals, while exhibiting minimal expression in roots, stems, and leaves. Throughout flower development, the transcript abundance of this gene gradually increased, reached its peak at S3 in the blotch regions of petals from the inbred line DSRFY, and then decreased during the senescence stage. Notably, across all the examined developmental stages, its expression in the blotch regions was consistently and significantly higher than that in the non-blotch regions (Fig. 5a). Sequence analysis disclosed that the VwMYB1 protein encompasses canonical R2 and R3 domains at its N-terminus, which validates its categorization as an R2R3 MYB transcription factor (Fig. 5b). Phylogenetic analysis demonstrated that VwMYB1 is most closely associated with PsMYB308 from peony ( Paeonia suffruticosa ) (Fig. 5c), implying potential functional resemblance. Subcellular localization prediction designated VwMYB1 to the nucleus. This prediction was experimentally corroborated by transiently expressing a GFP-VwMYB1 fusion protein in tobacco leaf epidermal cells, wherein the fluorescence signal was solely localized to the nucleus (Fig. 5d). In conclusion, these findings illustrate that VwMYB1 is a nuclear-localized R2R3 MYB transcription factor presumably involved in mediating blotch formation via the regulation of the structure genes involved in anthocyanin biosynthesis. Self-interaction of VwMYB1 in vivo To further analyze the function of VwMYB1, the full-length protein (292 amino acids) was partitioned into two segments according to its domain structure and relevant literature: the N-terminal fragment VwMYB1N (amino acids 1-114) and the C-terminal fragment VwMYB1C (amino acids 115-292). These three fragments, namely the full-length VwMYB1, VwMYB1N, and VwMYB1C, were respectively fused to the GAL4 DNA-binding domain (BD) in the pGBKT7 vector, resulting in the generation of BD-VwMYB1, BD-VwMYB1N, and BD-VwMYB1C, respectively (Fig. 6a). These constructs and the empty BD vector were independently transformed into the yeast strain Y2HGold. All transformants exhibited robust growth on the synthetic dropout (SD) medium lacking tryptophan (SD/-Trp). Nevertheless, on the highly selective medium SD/-Trp-His-Ade supplemented with X-α-gal, only the yeast cells carrying BD-VwMYB1 and BD-VwMYB1C were able to grow and form blue colonies. In contrast, the cells containing BD-VwMYB1N or the empty BD vector were unable to grow (Fig. 6b). These findings suggest that VwMYB1 has transcriptional activation activity, and this activity is confined to its C-terminal region. To investigate the potential of VwMYB1 to form homodimers, the full - length coding sequence of VwMYB1 was cloned into the pGADT7 vector to generate AD-VwMYB1. Various combinations of BD and AD constructs were co-transformed into Y2HGold cells. All co - transformants were able to grow on the SD/-Trp/-Leu medium. On the more selective quadruple dropout medium SD/-Trp/-Leu/-Ade/-His supplemented with X-α-gal, vigorous growth and blue coloration were solely observed for the positive control (BD-p53 × AD-T) and the test combination BD-VwMYB1 × AD-VwMYB1. Other combinations, including the negative controls, failed to grow (Fig. 6c). This finding indicates that VwMYB1 is capable of interacting with itself to form homodimers within yeast cells. Heterologous Stable Expression of VwMYB1 in Tobacco To overcome the limitation of stable genetic transformation in pansy, we heterologously expressed VwMYB1 in tobacco to further verify its conserved function. Ten independent VwMYB1 overexpression transgenic lines (VwMYB1-OE) were generated, all of which exhibited consistent and stable darkening phenotypes. Three T3 lines (OE-1, OE-3, and OE-5) were chosen for in depth analysis (Fig. 7a). In comparison with the wild type (WT), the VwMYB1-OE lines showed significantly darker purplish-red corolla coloration, along with a notable increase in the total anthocyanin content in petals (Fig. 7b). Moreover, qRT-PCR analysis indicated that the expression levels of multiple endogenous key genes in the tobacco anthocyanin biosynthetic pathway (e.g., NtDFR , NtANS ) were significantly up-regulated (Fig. 7c). These findings demonstrate that VwMYB1 can conservatively activate the anthocyanin biosynthetic pathway across species, independently facilitating pigment accumulation and petal coloration. Transient Overexpression of VwMYB1 in Native Pansy Petals To verify the direct regulatory role of VwMYB1 in anthocyanin synthesis, a transient overexpression assay was conducted in living pansy petals. The results indicated that, when compared to the control areas infiltrated with the empty vector (EV), the petal areas infiltrated with the Agrobacterium suspension carrying the VwMYB1 overexpression vector developed well - defined, dark purple spots with intense pigment deposition within 48-72 hours (Fig. 8a). Analysis of the anthocyanin content further confirmed a significant increase in pigmentation within the overexpression zones (Fig. 8b). Additionally, qRT-PCR analysis disclosed that the expression levels of several key genes in the anthocyanin biosynthetic pathway, namely VwF3’5’H, VwDFR, VwUFGT, VwCHI, and VwANS, were significantly up-regulated (Fig. 8c). These findings demonstrate that VwMYB1 directly promotes anthocyanin accumulation by activating the expression of genes in the anthocyanin biosynthetic pathway. VwMYB1 directly targets and activates the promoter of the VwF3'5'H To identify the direct target genes of VwMYB1 in the anthocyanin pathway, a dual-luciferase reporter assay was conducted in transformed tobacco. Considering that delphinidin is the predominant anthocyanin in the pansy blotch region and F3’5’H is a crucial branch-point gene for its biosynthesis, we concentrated on the VwF3’5’H promoter as a potential target for VwMYB1-mediated transcriptional activation. The promoter sequence of VwF3’5’H was cloned from pansy. To verify the interaction between VwMYB1 and the VwF3’5’H promoter, a yeast one-hybrid assay was initially carried out. The full-length VwF3’5’H promoter fragment was cloned into the pAbAi vector to generate the bait construct. The findings confirmed that VwMYB1 specifically binds to this promoter region (Fig. 9a). Subsequently, a transient dual - luciferase reporter assay in tobacco leaves was utilized. A reporter vector was constructed by fusing the VwF3’5’H promoter to the firefly luciferase (LUC) gene, which was then co-transformed with a VwMYB1 overexpression effector vector. The assay indicated that VwMYB1 significantly enhanced the reporter gene expression, resulting in an approximately 2.6-fold increase in luciferase activity compared to the control (Fig. 9b,c). In summary, VwMYB1 directly binds to the promoter of VwF3’5’H and effectively activates its transcription. These findings establish, at the molecular level, the key mechanism through which VwMYB1 precisely controls delphinidin synthesis in the pansy blotch area by directly regulating the VwF3’5’H gene. Discussion Inspired by Zhang et al. ( 2023 ), who adopted an integrated strategy combining metabolomic and comparative transcriptomic analyses to elucidate the transcriptional regulatory mechanisms underlying the fruit coloration of Malus sieversii , and considering the well-established role of MYB transcription factors in anthocyanin biosynthesis (Liu et al., 2021), this study focuses on the MYB transcription factors that exhibited differential expression consistent with the measured anthocyanin accumulation between blotch and non-blotch regions. A key candidate transcription factor, VwMYB1 , was identified. Preliminary characterization indicated that VwMYB1 possesses a canonical R2R3-MYB DNA-binding domain (Fig. 5 b) and is localized to the nucleus (Fig. 5 d) as a transcription factor. The expression pattern of VwMYB1 (Fig. 5 a) was significantly correlated with anthocyanin accumulation during inflorescence development. Its function was further confirmed through both heterologous overexpression in tobacco and transient overexpression in native pansy petals (Fig. 7 a, 8 a), demonstrating that VwMYB1 participates in the regulation of anthocyanin biosynthesis in pansy petals. Targeted metabolomics analysis revealed that the blotch coloration is predominantly caused by a substantial accumulation of delphinidin-type anthocyanins. Furthermore, F3’5’H is a crucial enzyme in the pathway leading to delphinidin production (Huynh et al., 2013), which uncovered a core VwMYB1-VwF3’5’H regulatory module involved in the biosynthesis of anthocyanins in the petal blotch area of pansy. Another notable discovery in our study is the functional complexity of conserved transcription factors among different species. Although VwMYB1 phylogenetically clusters within a subgroup that includes regulators characterized in other plants and exhibits high protein - sequence similarity to the anthocyanin repressor PsMYB308 from peony, and contains a C2 motif and EAR motif typical of repressors (Luan et al., 2024 ), our functional assays clearly demonstrated its robust positive regulatory activity in pansy. Notably, a similar functional divergence was observed in its relative, VwMYB8 , which was classified to SG4 subfamily, in which most members were characterized as anthocyanin repressors, but its transient expression in rose petals resulted in enhanced pigmentation, demonstrating a positive regulatory role (Zeng et al., 2016 ). The overexpression of VwMYB1 significantly enhanced pigmentation both locally (in pansy petal blotches) and systemically (in transgenic tobacco petals), confirming its role as an activator in the anthocyanin pathway. This functional disparity emphasizes the possibility of functional diversification within conserved gene families across horticultural species, highlighting the crucial significance of direct functional validation in the target organism (Wang et al., 2022 ). This finding not only enriches our understanding of the functional diversity of MYB proteins but also offers a novel candidate gene for the precise molecular manipulation of floral color in ornamental plants. While this study elucidates the core biosynthetic module, a fundamental question persists: what delineates the precise spatial boundary of its activity? Current research, including our own, has predominantly concentrated on the synthesis of anthocyanins within the blotch. Future research endeavors must address the question of why this synthesis is spatially constrained. Epigenetic regulation, such as differential DNA methylation or histone modifications, may serve as a key determinant, as indicated by studies on other patterned flowers (Liu et al., 2023 ). Furthermore, the strong association between blotch patterning and petal development suggests the involvement of specific developmental signals. Investigating how signals related to tissue polarity, hormone gradients, or localized conditions are perceived and transduced to differentially regulate the VwMYB1-VwF3’5’H module will be of great significance. Techniques such as spatial transcriptomics and chromatin accessibility mapping will play a vital role in deciphering the upstream signals and regulatory landscape that direct the remarkable precision of floral pattern formation. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8639386","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581443925,"identity":"52dcc8ca-0c19-4c34-9062-acaa9612f458","order_by":0,"name":"Xinyu Ye","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Ye","suffix":""},{"id":581443931,"identity":"adf9a88d-2645-42fc-9a66-81955e2f45ed","order_by":1,"name":"Xiaopei Zhu","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaopei","middleName":"","lastName":"Zhu","suffix":""},{"id":581443933,"identity":"0ee3402f-8a44-42be-922b-fded190b3820","order_by":2,"name":"Yudan Li","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yudan","middleName":"","lastName":"Li","suffix":""},{"id":581443938,"identity":"eafbf15d-5f30-431a-b5a3-f6acbbc98faf","order_by":3,"name":"Yanke Hu","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yanke","middleName":"","lastName":"Hu","suffix":""},{"id":581443941,"identity":"5a17d4d3-a76d-4466-a87a-140783bd9372","order_by":4,"name":"Jinyan Mu","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jinyan","middleName":"","lastName":"Mu","suffix":""},{"id":581443946,"identity":"604267cc-7fe1-42ed-89ec-2fbe97e2f502","order_by":5,"name":"Xiaohua Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYDCCAwxsDB8MJOT4mZkPPyBaC+OMCgtjyXa2NAOitTDznKlI3HCeR0GCKB18N3LMHvC2SRgbH+ZhMGCosYkmqEXyRlq6gWSbhJzZYd4DDxiOpeU2ENJicCP5mIQh0Bazw3wJBowNh4nRktgmAUKbm3kMJIjUArTlwBmJxA3MxGqRPPMsTbKhQsJY4jAwkBOI8Qvf8Rwz6T8GdXL8/YcPP/hQY0NYCypIIE35KBgFo2AUjAJcAACGrT8oGeccvwAAAABJRU5ErkJggg==","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Xiaohua","middleName":"","lastName":"Du","suffix":""},{"id":581443949,"identity":"48b8d189-72f3-43ac-9ad6-4d70b9bcdf6b","order_by":6,"name":"Huichao Liu","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Huichao","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-01-19 12:16:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8639386/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8639386/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-026-03842-5","type":"published","date":"2026-05-02T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101751203,"identity":"98acd61a-8366-4b62-90fb-69c283014477","added_by":"auto","created_at":"2026-02-03 10:18:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62826,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypes of \u003cem\u003eViola\u003c/em\u003e × \u003cem\u003ewittrockiana\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(a) a plant of pansy inbred line DSRFY, scale bar = 1 cm.; (b) Anthocyanin content in petals of blotched and non-blotched area.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/07f8fd17bb245d1d22edf7b6.jpeg"},{"id":101751406,"identity":"0647ea01-984a-40c4-a9a5-0050d72d965a","added_by":"auto","created_at":"2026-02-03 10:20:03","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112915,"visible":true,"origin":"","legend":"\u003cp\u003eTargeted metabolomic analysis of anthocyanins in blotch and non-blotch regions of pansy petals.\u003cbr\u003e\n(a) Composition and distribution of major anthocyanin types.\u003cbr\u003e\n(b) Quantification of key delphinidin derivatives.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/ac37499265fc59d5f60a53f7.jpeg"},{"id":101751354,"identity":"266af7ba-928f-4efd-87ca-faa3fff12ee1","added_by":"auto","created_at":"2026-02-03 10:19:38","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113445,"visible":true,"origin":"","legend":"\u003cp\u003eComparative transcriptome analysis between blotched and non-blotched regions of pansy petals.\u003cbr\u003e\n(a) Heatmap Volcano plot of DEGs. Purple dots represent upregulated genes, and blue dots represent downregulated genes. (b) Gene Ontology (GO) enrichment analysis of DEGs. (c) Top 10 enriched KEGG pathways of DEGs. The bar length indicates the number of enriched genes, and the color represents the primary pathway category.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/567b0d104a8cd45e05177310.jpeg"},{"id":101410510,"identity":"ed05ab30-c0f8-4173-afe6-cb6485cc36a1","added_by":"auto","created_at":"2026-01-29 11:33:50","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132940,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of anthocyanin biosynthetic pathway genes and MYB transcription factors in pansy petals. (a) The anthocyanin biosynthesis pathway diagram, expression levels of key structural genes in blotched (B) and non-blotched (NB) regions, and anthocyanin content. (b) Heatmap showing the expression of 64 differentially expressed MYB transcription factors in blotched (B) and non-blotched (NB) regions.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/9450a8836e237dc9b62abec7.jpeg"},{"id":101410515,"identity":"0b0bbed4-04ae-4df9-b227-65bafb869d53","added_by":"auto","created_at":"2026-01-29 11:33:51","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":102771,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of a R2R3-MYB gene involved in anthocyanin biosynthesis in pansy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eTissue-specific and flower developmental stage-specific expression of VwMYB1 in pansy. Expression levels are presented relative to that in young roots, which was set to 1.\u003cstrong\u003e(b)\u003c/strong\u003e Multiple sequence alignment of \u003cem\u003eVwMYB1\u003c/em\u003e with selected homologs. Alignment was performed using DNAMAN software. Amino acids with 100% identity are shaded in purple.\u003cstrong\u003e (c) \u003c/strong\u003ePhylogenetic analysis of \u003cem\u003eVwMYB1\u003c/em\u003e and other plant R2R3-MYB transcription factors. The phylogenetic tree was constructed using the neighbor-joining method in MEGA software. Numbers next to branch nodes represent bootstrap values from 1000 replicates. The scale bar indicates an evolutionary distance of 0.05 substitutions per site. VwMYB1 is marked in red. Gene accession numbers are as follows: \u003cem\u003eArabidopsis thaliana\u003c/em\u003e(At): AtPAP2 (AAG42002), AtPAP1 (AAG42001), AtMYB113 (NP_176811), \u003cem\u003ePetunia hybrida\u003c/em\u003e (Ph):PhAN2 (AAF66727), \u003cem\u003eNicotiana tabacum\u003c/em\u003e (Nt): NtAN2 (NP_001312447), \u003cem\u003eVitis vinifera\u003c/em\u003e (Vv): VvMYBA2 (BAF06563), \u003cem\u003eMalus domestica\u003c/em\u003e (Md): MdMYB10a (ABB84753), \u003cem\u003ePaeonia suffruticosa\u003c/em\u003e (Ps): PsMYB308 (XP_050686438.1); \u003cstrong\u003e(d) \u003c/strong\u003eSubcellular localization of the GFP:VwMYB1 fusion protein in tobacco leaf epidermal cells. GFP alone served as the control. Nuclei were counterstained with DAPI to confirm localization. Bar = 50 µm.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/e43c2223d5ca84a64462d988.jpeg"},{"id":101751242,"identity":"44c4bb9a-52ed-4bd7-948c-6d758742ce2d","added_by":"auto","created_at":"2026-02-03 10:18:40","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":89035,"visible":true,"origin":"","legend":"\u003cp\u003eSelf-activation assay and verification of homodimerization for VwMYB1.\u003cbr\u003e\n(a) Schematic diagram of the full-length and truncated fragments (VwMYB1N and VwMYB1C) of VwMYB1 used for vector construction.(b) Yeast two-hybrid assay showing the self-activation of VwMYB1 in yeast cells.(c) In vivo evidence for VwMYB1 forming homodimers. BD-p53 × AD-T-antigen served as the positive control, and BD-Lamin × AD-T-antigen served as the negative control.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/adcd620e78577d6a7f80bbff.jpeg"},{"id":101410517,"identity":"5471c9d5-b021-4b0b-963c-542133713dba","added_by":"auto","created_at":"2026-01-29 11:33:51","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":66106,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of heterologous stable expression of VwMYB1 in tobacco. (a) Phenotypic comparison of corollas between wild-type (WT) and three representative VwMYB1-overexpression lines (OE-1, OE-3, OE-5). (b) Determination of total anthocyanin content in petals of the corresponding lines. (c) Effect of VwMYB1 overexpression on the transcript levels of endogenous key genes in the tobacco anthocyanin biosynthetic pathway.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/7335e8bf7227b6817e843c32.jpeg"},{"id":101410512,"identity":"fc3908c4-de32-436c-a244-e0423111b754","added_by":"auto","created_at":"2026-01-29 11:33:51","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":56306,"visible":true,"origin":"","legend":"\u003cp\u003eHomologous expression of VwMYB1 in pansy petals.\u003c/p\u003e\n\u003cp\u003e(a) Phenotype of pansy petals after transient\u003cstrong\u003e \u003c/strong\u003eoverexpressing VwMYB1. Scale bar = 1 cm. (b) Anthocyanin content in corollas of empty vector (EV) and VwMYB1-overexpressing (OE) pansy petlas. (c) Effect of VwMYB1 overexpression on the transcript levels of endogenous key genes in the anthocyanin biosynthetic pathway.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/6ba46a9ea70d82e73ae006f7.jpeg"},{"id":101410514,"identity":"0ee8215a-905f-4be0-884b-28c86928f882","added_by":"auto","created_at":"2026-01-29 11:33:51","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":98549,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional activation analysis of the anthocyanin-related gene promoter by VwMYB1.\u003cbr\u003e\n(a) Representative images of tobacco leaves co-infiltrated with agrobacterium harboring the VwF3’5’H promoter-LUC reporter construct and the VwMYB1 effector construct, or corresponding controls, 3 days post-infiltration. (b) Quantitative results of the dual-luciferase reporter assay. Relative LUC/REN ratios show that VwMYB1 significantly activates the \u003cem\u003eVwF3’5’H\u003c/em\u003e promoter approximately 2.6-fold. Yeast one-hybrid assay confirming the direct interaction between VwMYB1 and the \u003cem\u003eVwF3’5’H\u003c/em\u003e promoter. Data in panel C are presented as mean ± SD (n=4).1：0800-luc-VwF3’5’H+62-sk-VwMYB1；2：0800-1uc-VwF3’5’H+62-sk；3：0800-luc+62-sk-VwMYB1；4：0800-1uc+62-sk.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/6ed5c01c2d0b9ea189c82358.jpeg"},{"id":108804877,"identity":"e94bed7e-fb32-43a1-b85b-1affc31eb144","added_by":"auto","created_at":"2026-05-08 15:24:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1132030,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8639386/v1/47a0fe0a-a0d9-45f0-8277-690bcb75b3dd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A R2R3-MYB, VwMYB1, regulates anthocyanin biosynthesis in pansy petal blotch via transcriptional activation of VwF3’5’H","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnthocyanins, a class of water-soluble flavonoid pigments, are widely distributed in plants and perform diverse functions (Saito et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In addition to their well - recognized function of attracting pollinators and increasing the ornamental value of flowers, as demonstrated by the attractive blotches, spots, and stripes in many angiosperms (Moeller et al., 2005; Shang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), they also act as antioxidants, enhancing the plants' resistance to both biotic and abiotic stresses and being beneficial to human health. (Lu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The pansy (\u003cem\u003eViola \u0026times; wittrockiana\u003c/em\u003e) is an excellent ornamental plant, well-known for its unique and stable 'face-like' blotches with anthocyanin enrichment at the flower center, a characteristic that is central to its commercial attractiveness(Endo, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1959\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEarly studies identified delphinidin- and cyanidin-type anthocyanins as the primary pigments responsible for petal blotch in pansy (Endo, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1959\u003c/span\u003e). Subsequently, this finding was refined by pinpointing floral developmental stages III\u0026ndash;V as the critical period for their accumulation (Li et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The anthocyanin biosynthesis pathway is evolutionarily conserved and well-characterized in higher plants, encompassing six essential structural genes: chalcone synthase (\u003cem\u003eCHS\u003c/em\u003e), chalcone isomerase (\u003cem\u003eCHI\u003c/em\u003e), flavonoid 3-hydroxylase (\u003cem\u003eF3H\u003c/em\u003e), dihydroflavonol 4-reductase (\u003cem\u003eDFR\u003c/em\u003e), anthocyanin synthase (\u003cem\u003eANS\u003c/em\u003e), and UDP-glucose flavonoid-3-O-glucosyltransferase (\u003cem\u003eUFGT\u003c/em\u003e) (Rausher et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Cappellini et al, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, the cytochrome P450 enzymes flavonoid 3\u0026prime;-hydroxylase (\u003cem\u003eF3\u0026rsquo;H\u003c/em\u003e) and flavonoid 3\u0026prime;,5\u0026prime;-hydroxylase (\u003cem\u003eF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e) play decisive roles in determining the types of anthocyanin accumulation (Tanaka and Brugliera, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For example, \u003cem\u003eF3\u0026rsquo;H\u003c/em\u003e is highly expressed throughout all developmental stages in wild strawberry (\u003cem\u003eFragaria vesca\u003c/em\u003e), contributing to elevated cyanidin levels in its fruits (Thill et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Notably, \u003cem\u003eF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e acts as the key branch-point enzyme that channels metabolic flux toward delphinidin-type anthocyanin synthesis, a function that has been validated in pansy (Huynh et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These structural genes are commonly classified into early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs) (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Holton and Cornish, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). LBGs, such as \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e, and \u003cem\u003eUFGT\u003c/em\u003e, encode enzymes essential for the terminal steps of anthocyanin synthesis and thus play a vital role in regulating total anthocyanin accumulation (Winkel-Shirley, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Nevertheless, the upstream regulatory mechanism governing the spatially precise expression of \u003cem\u003eF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e during blotch pattern formation remains poorly understood, representing a significant gap in current knowledge.\u003c/p\u003e \u003cp\u003eAnthocyanin biosynthesis is predominantly regulated by transcription factors (TFs), including the MYB, bHLH, and WRKY families (Li et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ding, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The MBW complex, which is formed by MYB, bHLH, and WD40 proteins, serves as a core regulatory module (Zhang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although the regulatory networks governing pigmentation have been clarified in species such as peony and apple, it is still arduous to identify the key regulators from the extensive MYB family in non - model plants like pansy. Previous studies, for example, the transient functional validation of \u003cem\u003eVwMYB8\u003c/em\u003e in heterologous systems, were short of stable genetic evidence in the native plant context (Zeng et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, the lack of a reference genome makes traditional screening approaches even more complicated.\u003c/p\u003e \u003cp\u003eTo directly tackle this mechanistic gap, we devised a strategy of combining targeted metabolomics with, transcriptome for analysis of blotch and non-blotch tissues to identify candidate MYB TFs. Subsequently, the function of the primary candidate was validated via genetic transformation. Ultimately, the molecular mechanism was revealed by clarifying how \u003cem\u003eVwMYB1\u003c/em\u003e regulates the downstream structural genes, such as \u003cem\u003eF3\u0026prime;5\u0026prime;H\u003c/em\u003e, for anthocyanin synthesis. This research not only deciphers the core regulatory module governing blotch formation in pansy but also establishes an efficient pipeline for gene discovery in non-model ornamental plants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and cultivation environment\u003c/h2\u003e \u003cp\u003eThe plant material utilized in this study was an inbred line DSRFY, which was derived from a pansy cultivar \u0026lsquo;Swiss Giants Finegold\u0026rsquo;. It displays regular dark-brown blotches on its petals. Seeds of this material were sown on September 30, 2024. Seedlings were transplanted into 12\u0026ndash;14 cm pots once they had 3\u0026ndash;4 true leaves and were cultivated on the campus of Henan Institute of Science and Technology (35.18\u0026deg;N, 113.52\u0026deg;E) for subsequent care and growth. Petal tissues were collected for total RNA extraction and pigment detection.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of anthocyanin content\u003c/h3\u003e\n\u003cp\u003eThe anthocyanin content was determined in accordance with the methods previously described (Xie et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For each experimental group, five petal samples were collected, promptly frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. The frozen composite samples were pulverized into powder using a cryogenic grinder and weighed for subsequent analysis. Approximately 0.1 g of petal tissue was homogenized in 1 mL of extraction buffer. The homogenate was transferred to a microcentrifuge tube, and the volume was adjusted to 1 mL by adding additional extraction buffer. The tube was firmly sealed and incubated at 60\u0026deg;C for 30 minutes with intermittent shaking. Subsequently, the sample was centrifuged at 12,000 rpm/min for 10 minutes at room temperature, and the supernatant was collected for analysis.\u003c/p\u003e \u003cp\u003eThe total monomeric anthocyanin content was determined using the pH-differential method (Lee et al., 2005). Briefly, two aliquots of the extract were separately mixed with potassium chloride buffer (pH 1.0) and sodium acetate buffer (pH 4.5). The absorbance of each mixture was measured at 530 nm and 700 nm using a spectrophotometer, and the corrected absorbance difference (ΔA) was calculated according to the standard formula. The total anthocyanin content, expressed as micromoles per gram of fresh weight (\u0026micro;g/g FW), was then calculated from the ΔA value using the established formula which incorporates the molar extinction coefficient and path length. Each sample was analyzed with three independent technical replicates, and the data were subjected to statistical analysis.\u003c/p\u003e\n\u003ch3\u003eTranscriptome sequencing and analyses\u003c/h3\u003e\n\u003cp\u003eSamples were collected from the petals of floral buds before their blooming according to Li et al (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and dissected into blotch and non-blotch regions, with three biological replicates per group. All samples were immediately flash-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Total RNA was extracted using the Plant Total RNA Extraction Kit (Vazyme, China). RNA integrity was evaluated by agarose gel electrophoresis, and purity and concentration were measured using a NanoDrop 2000 spectrophotometer (OD260/280 between 1.8 and 2.0). High-quality RNA was utilized to construct cDNA libraries, which were sequenced on the Illumina NovaSeq platform (paired-end, 150 bp) by Biomarker Technologies Co., Ltd. (Beijing, China). Raw reads were filtered via Trimmomatic to eliminate adapters and low-quality sequences, and clean reads were assembled using Trinity. Coding sequences (CDS) were predicted with TransDecoder. Unigene functional annotation was carried out using BLASTx (E - value\u0026thinsp;\u0026le;\u0026thinsp;1\u0026times;10⁻⁵) against the NR, Swiss-Prot, Pfam, GO, KEGG, and KOG databases. Transcript abundance was quantified and presented as FPKM using RSEM. Differential expression analysis was executed with the DEGseq R package, with genes meeting |log₂FoldChange| \u0026ge; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01 regarded as differentially expressed genes (DEGs). Gene Ontology (GO) enrichment analysis of DEGs was performed using Blast2GO. KEGG pathway enrichment analysis was carried out using KOBAS 2.0. Terms and pathways with an adjusted \u003cem\u003ep\u003c/em\u003e - value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were deemed significantly enriched.\u003c/p\u003e\n\u003ch3\u003eReal-time quantitative PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from various tissues of pansy, including the blotch and non - blotch regions of petals at different developmental stages: S1 (petal length approximately 0.7 cm), S3 (petal length approximately 1.2 cm), S5 (the first day of flowering), and S8 (onset of flower senescence), as well as from roots, stems, and leaves. The extraction was performed using a Plant Total RNA Extraction Kit (Vazyme, China).\u003c/p\u003e \u003cp\u003eFirst-strand cDNA was synthesized from the extracted RNA by utilizing the PrimeScript RT Reagent Kit (Vazyme, China). Subsequently, quantitative real-time PCR (qRT-PCR) was performed with SYBR Premix Ex Taq (Takara, China) on a real-time PCR system. The β-actin gene of pansy was employed as an internal reference for normalization. The quantity of the starting template was adjusted according to the cycle threshold (Ct) values of this endogenous control (usually ranging from 18 to 20 cycles). All the primers utilized in this study are presented in Supplementary Table S1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation of\u003c/b\u003e \u003cb\u003eVwMYB1\u003c/b\u003e \u003cb\u003efrom pansy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from pansy petal tissues using a Plant Total RNA Extraction Kit (Vazyme, China), strictly following the manufacturer's instructions. Subsequently, first-trand cDNA was synthesized from the extracted RNA using the PrimeScript RT Reagent Kit (Vazyme, China). To acquire the full - length cDNA of VwMYB1, PCR amplification was carried out with gene-specific primers (Supplementary Table S1). The resultant PCR product was purified and sequenced. The obtained nucleotide and deduced amino acid sequences were respectively analyzed via the BLASTX and BLASTP programs against the GenBank database to search for homologous sequences and conduct comparative analyses.\u003c/p\u003e\n\u003ch3\u003eSubcellular localization assay\u003c/h3\u003e\n\u003cp\u003eThe full-length cDNA of \u003cem\u003eVwMYB1\u003c/em\u003e was precisely inserted into the pCambia1302 vector to generate the fusion construct pCambia1302::\u003cem\u003eVwMYB1\u003c/em\u003e. Subsequently, this construct was introduced into \u003cem\u003eAgrobacterium\u003c/em\u003e tumefaciens strain GV3101 through electroporation. As a control, the pCambia1302 vector, which contained a CaMV35S::GFP cassette, was employed. Agrobacterium strains carrying the respective vectors were cultured on LB agar plates supplemented with appropriate antibiotics at 28\u0026deg;C for 2\u0026ndash;3 days. Bacterial cells were then collected and resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES [pH 5.6], 200 \u0026micro;M acetosyringone) to reach an OD₆₀₀ of 0.4. Before infiltration, the suspensions were left at room temperature for 2 hours without agitation. Approximately 500 \u0026micro;L of the bacterial suspension was infiltrated into the abaxial side of young leaves from 3-to 4-week-old tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e), ensuring at least two infiltration sites per leaf. Subcellular localization analysis was carried out 3 days after infiltration. Confocal images were captured using a Zeiss LSM 710 laser scanning microscope (Zeiss, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eVwMYB1\u003c/b\u003e \u003cb\u003e-overexpressing in tobacco\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe full-length open reading frame (ORF) of \u003cem\u003eVwMYB1\u003c/em\u003e was cloned into the pCambia1302 vector to generate the recombinant construct pCambia1302::VwMYB1. Subsequently, this construct was introduced into the Agrobacterium tumefaciens strain GV3101. Then, tobacco transformation was carried out using the previously described \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated method. Transgenic tobacco lines were selected on a medium containing hygromycin. After root induction and acclimatization, the putative transgenic plants were transferred to a greenhouse and grown until they reached the flowering stage. The integration of the \u003cem\u003eVwMYB1\u003c/em\u003e transgene into the genome of the resulting plants was confirmed by PCR analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransient overexpression of\u003c/b\u003e \u003cb\u003eVwMYB1\u003c/b\u003e \u003cb\u003ein pansy petals\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo analyze the function of \u003cem\u003eVwMYB1\u003c/em\u003e, a transient overexpression assay was performed in detached pansy petals. The \u003cem\u003eVwMYB1\u003c/em\u003e overexpression construct (pCambia1302::VwMYB1) and the empty vector control were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101, respectively. The bacterial suspensions were then infiltrated into the petals. After infiltration, the petals were kept in the dark for 24 hours and subsequently transferred to conditions of 25\u0026deg;C with a 16-h-light/8-h-dark photoperiod for three days before phenotypic observation.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetection of anthocyanin composition in petal blotch\u003c/h2\u003e \u003cp\u003eFreeze-dried petal tissues were thoroughly ground into a fine powder. Approximately 50 mg of the powder was subjected to extraction twice with 0.5 mL of a methanol/water/hydrochloric acid (500:500:1, v/v/v) solution. The extraction process involved vortexing and ultrasonication. After centrifugation, the combined supernatants were carefully collected, filtered through a 0.22 \u0026micro;m membrane, and then used for LC-MS/MS analysis. Anthocyanin separation was conducted on a Waters ACQUITY BEH C18 column (1.7 \u0026micro;m, 2.1 \u0026times; 100 mm) using a UPLC system (ExionLC\u0026trade; AD). The mobile phase was composed of water and methanol, each containing 0.5% formic acid, with a flow rate maintained at 0.35 mL/min at a temperature of 40\u0026deg;C. A well-defined gradient elution program was applied. Detection and quantification were accomplished using a triple quadrupole-linear ion trap mass spectrometer (QTRAP\u0026reg; 6500+) operating in positive electrospray ionization (ESI+) mode with scheduled multiple reaction monitoring (MRM). Metabolites were quantified using Multiquant 3.0.3 software (Sciex) with optimized MRM transitions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDual-luciferase reporter (DLR) assay\u003c/h3\u003e\n\u003cp\u003eThe perform dual-luciferase activity assays as previously described (Ji et al., 2024). Clone the coding sequence (CDS) of ChMYB1 into the pGreen62-SK vector to serve as the effector construct. Insert the proChGSTF5 into the pGreen0800-LUC vector to generate the reporter construct. Co-infiltrate Agrobacterium tumefaciens (GV3101 pSoup) sus pensions containing the effector and reporter constructs (9:1, OD600\u0026thinsp;=\u0026thinsp;0.8) into tobacco leaves for transient expression analysis.\u003c/p\u003e\n\u003ch3\u003eYeast one-hybrid assay\u003c/h3\u003e\n\u003cp\u003eThe coding sequence (CDS) of \u003cem\u003eVwMYB1\u003c/em\u003e was precisely inserted into the pGADT7 vector to generate the prey construct. The promoter sequence of VwF3\u0026rsquo;5\u0026rsquo;H, which contains conserved \u003cem\u003ecis\u003c/em\u003e-elements, was carefully cloned into the pAbAi vector to create the bait construct. Subsequently, the paired prey and bait constructs were co-transformed into yeast strain Y1HGold cells. After that, the transformed yeast cells were plated on synthetic dropout (SD) media that lacked tryptophan and uracil and were supplemented with X - gal for blue color development screening.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhenotypic and Anthocyanin Content of Blotched and Non-blotched Regions in Pansy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantitatively clarify the pigment basis of the blotch phenotype, we employed the pansy inbred line DSRFY and separately collected tissue from the blotched and non - blotched regions of petals (Fig. 1a). Spectrophotometric determination of anthocyanin content demonstrated a highly significant disparity between the two regions. Specifically, the anthocyanin content in the blotched region was 8.35 \u0026plusmn; 0.09 \u0026mu;g/g fresh weight (mean \u0026plusmn; SD), while it was merely 2.28 \u0026plusmn; 0.33 \u0026mu;g/g in the non - blotched region. The accumulation level in the blotched region was approximately 3.7 times higher than that in the non-blotched region (Fig. 1b). These results offer direct metabolic evidence indicating that the formation of the blotch is accompanied by highly specific and localized accumulation of anthocyanins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of anthocyanin components in pansy petals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the pigment components in the petal blotch area of pansy line DSRFY, a targeted anthocyanin metabolomic analysis was carried out on blotch and non - blotch tissues using LC-MS/MS. The results indicated a highly specific and intense accumulation of anthocyanins in the blotch region. Delphinidin-type anthocyanins were identified as the predominantly dominant pigments, followed by some cyanidin - type anthocyanins (Fig.2a). Among them, delphinidin-3-O-(6\u0026apos;\u0026apos;-O-coumaroyl) rutinoside-5-O-glucoside was the most prominent, with an average content reaching as high as 838.7 \u0026mu;g/g in the blotch, which represented a 294-fold increase compared to its level in the non-blotch region, thus constituting the core pigment of the blotch. Moreover, several other delphinidin glycosides, including delphinidin-3-O-glucuronide (mean 42.36 \u0026mu;g/g, ~26-fold blotch/non-blotch ratio) and delphinidin-3-O-rutinoside (mean 19.27 \u0026mu;g/g, ~30-fold ratio), also showed specific and high-level accumulation in the blotch parts (Fig.2b). Certain cyanidin derivatives, such as cyanidin-3,5-O-diglucoside, exhibited accumulation. In contrast, anthocyanins of other types, such as peonidin -, malvidin -, and petunidin-derivatives, were generally present at low concentrations (all below 0.4 \u0026mu;g/g) with no significant difference between the blotch and non-blotch parts of the petals. This extremely distinct metabolite distribution implies that the spatially specific activation of the delphinidin biosynthetic pathway in the cells of petal blotch parts is the key basis for blotch coloration, necessarily involving some functional genes for delphinidin biosynthesis, including F3\u0026rsquo;5\u0026rsquo;H and their precise transcriptional regulation by MYB-type transcription factors (Cao et al; 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of blotched and non-blotched regions in pansy petals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo decipher the transcriptional regulatory mechanism underlying anthocyanin accumulation in the blotched regions of pansy petals, we carried out a comparative transcriptome analysis between the blotched and non-blotched petal regions of DSRFY. A total of 2,554 DEGs were identified, including 1,239 upregulated and 1,315 downregulated genes (Fig.3a). GO enrichment analysis indicated that these DEGs were significantly enriched in terms related to the biological process, cellular component, and molecular function categories (Fig.3b). KEGG pathway analysis demonstrated that the top ten significantly enriched pathways were as follows: Plant-pathogen interaction, Plant hormone signal transduction, MAPK signaling pathway\u0026ndash;plant, Carbon metabolism, Biosynthesis of amino acids, Flavonoid biosynthesis, Phenylpropanoid biosynthesis, Starch and sucrose metabolism, Carbon fixation in photosynthetic organisms, and Photosynthesis (Fig.3c). The DEGs enriched in the biosynthesis pathways of flavonoids and anthocyanins revealed that the differences in the expression of genes related to anthocyanin synthesis were the key cause of the differences in anthocyanin accumulation between blotched and non-blotched area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification, Expression Pattern, and Subcellular Localization of \u003cem\u003eVwMYB1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed the expression of eight core structural genes implicated in anthocyanin biosynthesis, namely \u003cem\u003eVwCHI\u003c/em\u003e, \u003cem\u003eVwDFR\u003c/em\u003e, \u003cem\u003eVwANS\u003c/em\u003e, \u003cem\u003eVwUFGT\u003c/em\u003e, and the crucial \u003cem\u003eVwF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e. The findings of this analysis indicated that the expression levels of all these genes were elevated in the blotched region when compared to the non-blotched region of the petals (Fig. 4a). This coordinated up-regulation is in line with the metabolic phenotype of increased anthocyanin accumulation in the blotched region. Considering the well-documented regulatory functions of MYB transcription factors in anthocyanin biosynthesis across various plant species, such as peonies and grapevines, our investigation was centered on them. Further screening from previously acquired transcriptome data identified 64 differentially expressed MYB transcription factors (Fig. 4b), suggesting their potential as key regulators of localized anthocyanin synthesis. Among these candidates, the expression pattern of \u003cem\u003eVwiA01G125930\u003c/em\u003e (named \u003cem\u003eVwMYB1\u003c/em\u003e) exhibited a strong positive correlation with anthocyanin accumulation levels, suggesting that it may serve as a core transcription factor governing the anthocyanin biosynthetic pathway in pansy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression pattern and protein structural characteristics of VwMYB1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the function of the candidate gene\u0026nbsp;\u003cem\u003eVwMYB1\u003c/em\u003e identified from the pansy petal transcriptome, we initially analyzed its expression pattern via quantitative real-time polymerase chain reaction (qRT-PCR). The findings indicated that this gene was specifically and highly expressed in petals, while exhibiting minimal expression in roots, stems, and leaves. Throughout flower development, the transcript abundance of this gene gradually increased, reached its peak at S3 in the blotch regions of petals from the inbred line DSRFY, and then decreased during the senescence stage. Notably, across all the examined developmental stages, its expression in the blotch regions was consistently and significantly higher than that in the non-blotch regions (Fig. 5a).\u003cbr\u003eSequence analysis disclosed that the VwMYB1 protein encompasses canonical R2 and R3 domains at its N-terminus, which validates its categorization as an R2R3 MYB transcription factor (Fig. 5b). Phylogenetic analysis demonstrated that VwMYB1 is most closely associated with PsMYB308 from peony (\u003cem\u003ePaeonia suffruticosa\u003c/em\u003e) (Fig. 5c), implying potential functional resemblance. Subcellular localization prediction designated VwMYB1 to the nucleus. This prediction was experimentally corroborated by transiently expressing a GFP-VwMYB1 fusion protein in tobacco leaf epidermal cells, wherein the fluorescence signal was solely localized to the nucleus (Fig. 5d). In conclusion, these findings illustrate that VwMYB1 is a nuclear-localized R2R3 MYB transcription factor presumably involved in mediating blotch formation via the regulation of the structure genes involved in anthocyanin biosynthesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelf-interaction of VwMYB1 in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further analyze the function of VwMYB1, the full-length protein (292 amino acids) was partitioned into two segments according to its domain structure and relevant literature: the N-terminal fragment VwMYB1N (amino acids 1-114) and the C-terminal fragment VwMYB1C (amino acids 115-292). These three fragments, namely the full-length VwMYB1, VwMYB1N, and VwMYB1C, were respectively fused to the GAL4 DNA-binding domain (BD) in the pGBKT7 vector, resulting in the generation of BD-VwMYB1, BD-VwMYB1N, and BD-VwMYB1C, respectively (Fig. 6a). These constructs and the empty BD vector were independently transformed into the yeast strain Y2HGold.\u003c/p\u003e\n\u003cp\u003eAll transformants exhibited robust growth on the synthetic dropout (SD) medium lacking tryptophan (SD/-Trp). Nevertheless, on the highly selective medium SD/-Trp-His-Ade supplemented with X-\u0026alpha;-gal, only the yeast cells carrying BD-VwMYB1 and BD-VwMYB1C were able to grow and form blue colonies. In contrast, the cells containing BD-VwMYB1N or the empty BD vector were unable to grow (Fig. 6b). These findings suggest that VwMYB1 has transcriptional activation activity, and this activity is confined to its C-terminal region.\u003c/p\u003e\n\u003cp\u003eTo investigate the potential of VwMYB1 to form homodimers, the full - length coding sequence of VwMYB1 was cloned into the pGADT7 vector to generate AD-VwMYB1. Various combinations of BD and AD constructs were co-transformed into Y2HGold cells. All co - transformants were able to grow on the SD/-Trp/-Leu medium. On the more selective quadruple dropout medium SD/-Trp/-Leu/-Ade/-His supplemented with X-\u0026alpha;-gal, vigorous growth and blue coloration were solely observed for the positive control (BD-p53 \u0026times; AD-T) and the test combination BD-VwMYB1 \u0026times; AD-VwMYB1. Other combinations, including the negative controls, failed to grow (Fig. 6c). This finding indicates that VwMYB1 is capable of interacting with itself to form homodimers within yeast cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHeterologous Stable Expression of\u0026nbsp;\u003cem\u003eVwMYB1\u003c/em\u003e in Tobacco\u003cbr\u003e\u003c/strong\u003eTo overcome the limitation of stable genetic transformation in pansy, we heterologously expressed VwMYB1 in tobacco to further verify its conserved function. Ten independent VwMYB1 overexpression transgenic lines (VwMYB1-OE) were generated, all of which exhibited consistent and stable darkening phenotypes. Three T3 lines (OE-1, OE-3, and OE-5) were chosen for in depth analysis (Fig. 7a). In comparison with the wild type (WT), the VwMYB1-OE lines showed significantly darker purplish-red corolla coloration, along with a notable increase in the total anthocyanin content in petals (Fig. 7b). Moreover, qRT-PCR analysis indicated that the expression levels of multiple endogenous key genes in the tobacco anthocyanin biosynthetic pathway (e.g., \u003cem\u003eNtDFR\u003c/em\u003e, \u003cem\u003eNtANS\u003c/em\u003e) were significantly up-regulated (Fig. 7c). These findings demonstrate that VwMYB1 can conservatively activate the anthocyanin biosynthetic pathway across species, independently facilitating pigment accumulation and petal coloration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransient Overexpression of VwMYB1 in Native Pansy Petals\u003cbr\u003e\u003c/strong\u003eTo verify the direct regulatory role of VwMYB1 in anthocyanin synthesis, a transient overexpression assay was conducted in living pansy petals. The results indicated that, when compared to the control areas infiltrated with the empty vector (EV), the petal areas infiltrated with the Agrobacterium suspension carrying the VwMYB1 overexpression vector developed well - defined, dark purple spots with intense pigment deposition within 48-72 hours (Fig. 8a). Analysis of the anthocyanin content further confirmed a significant increase in pigmentation within the overexpression zones (Fig. 8b). Additionally, qRT-PCR analysis disclosed that the expression levels of several key genes in the anthocyanin biosynthetic pathway, namely VwF3\u0026rsquo;5\u0026rsquo;H, VwDFR, VwUFGT, VwCHI, and VwANS, were significantly up-regulated (Fig. 8c). These findings demonstrate that VwMYB1 directly promotes anthocyanin accumulation by activating the expression of genes in the anthocyanin biosynthetic pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVwMYB1 directly targets and activates the promoter of the \u003cem\u003eVwF3\u0026apos;5\u0026apos;H\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the direct target genes of VwMYB1 in the anthocyanin pathway, a dual-luciferase reporter assay was conducted in transformed tobacco. Considering that delphinidin is the predominant anthocyanin in the pansy blotch region and \u003cem\u003eF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e is a crucial branch-point gene for its biosynthesis, we concentrated on the VwF3\u0026rsquo;5\u0026rsquo;H promoter as a potential target for VwMYB1-mediated transcriptional activation. The promoter sequence of VwF3\u0026rsquo;5\u0026rsquo;H was cloned from pansy. To verify the interaction between VwMYB1 and the VwF3\u0026rsquo;5\u0026rsquo;H promoter, a yeast one-hybrid assay was initially carried out. The full-length VwF3\u0026rsquo;5\u0026rsquo;H promoter fragment was cloned into the pAbAi vector to generate the bait construct. The findings confirmed that VwMYB1 specifically binds to this promoter region (Fig. 9a). Subsequently, a transient dual - luciferase reporter assay in tobacco leaves was utilized. A reporter vector was constructed by fusing the VwF3\u0026rsquo;5\u0026rsquo;H promoter to the firefly luciferase (LUC) gene, which was then co-transformed with a VwMYB1 overexpression effector vector. The assay indicated that VwMYB1 significantly enhanced the reporter gene expression, resulting in an approximately 2.6-fold increase in luciferase activity compared to the control (Fig. 9b,c). In summary, VwMYB1 directly binds to the promoter of \u003cem\u003eVwF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e and effectively activates its transcription. These findings establish, at the molecular level, the key mechanism through which VwMYB1 precisely controls delphinidin synthesis in the pansy blotch area by directly regulating the \u003cem\u003eVwF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e gene.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInspired by Zhang et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who adopted an integrated strategy combining metabolomic and comparative transcriptomic analyses to elucidate the transcriptional regulatory mechanisms underlying the fruit coloration of \u003cem\u003eMalus sieversii\u003c/em\u003e, and considering the well-established role of MYB transcription factors in anthocyanin biosynthesis (Liu et al., 2021), this study focuses on the MYB transcription factors that exhibited differential expression consistent with the measured anthocyanin accumulation between blotch and non-blotch regions. A key candidate transcription factor, \u003cem\u003eVwMYB1\u003c/em\u003e, was identified. Preliminary characterization indicated that \u003cem\u003eVwMYB1\u003c/em\u003e possesses a canonical R2R3-MYB DNA-binding domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and is localized to the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) as a transcription factor. The expression pattern of \u003cem\u003eVwMYB1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) was significantly correlated with anthocyanin accumulation during inflorescence development. Its function was further confirmed through both heterologous overexpression in tobacco and transient overexpression in native pansy petals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea), demonstrating that VwMYB1 participates in the regulation of anthocyanin biosynthesis in pansy petals. Targeted metabolomics analysis revealed that the blotch coloration is predominantly caused by a substantial accumulation of delphinidin-type anthocyanins. Furthermore, \u003cem\u003eF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e is a crucial enzyme in the pathway leading to delphinidin production (Huynh et al., 2013), which uncovered a core VwMYB1-VwF3\u0026rsquo;5\u0026rsquo;H regulatory module involved in the biosynthesis of anthocyanins in the petal blotch area of pansy.\u003c/p\u003e \u003cp\u003eAnother notable discovery in our study is the functional complexity of conserved transcription factors among different species. Although \u003cem\u003eVwMYB1\u003c/em\u003e phylogenetically clusters within a subgroup that includes regulators characterized in other plants and exhibits high protein - sequence similarity to the anthocyanin repressor \u003cem\u003ePsMYB308\u003c/em\u003e from peony, and contains a C2 motif and EAR motif typical of repressors (Luan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), our functional assays clearly demonstrated its robust positive regulatory activity in pansy. Notably, a similar functional divergence was observed in its relative, \u003cem\u003eVwMYB8\u003c/em\u003e, which was classified to SG4 subfamily, in which most members were characterized as anthocyanin repressors, but its transient expression in rose petals resulted in enhanced pigmentation, demonstrating a positive regulatory role (Zeng et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The overexpression of \u003cem\u003eVwMYB1\u003c/em\u003e significantly enhanced pigmentation both locally (in pansy petal blotches) and systemically (in transgenic tobacco petals), confirming its role as an activator in the anthocyanin pathway. This functional disparity emphasizes the possibility of functional diversification within conserved gene families across horticultural species, highlighting the crucial significance of direct functional validation in the target organism (Wang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This finding not only enriches our understanding of the functional diversity of MYB proteins but also offers a novel candidate gene for the precise molecular manipulation of floral color in ornamental plants.\u003c/p\u003e \u003cp\u003eWhile this study elucidates the core biosynthetic module, a fundamental question persists: what delineates the precise spatial boundary of its activity? Current research, including our own, has predominantly concentrated on the synthesis of anthocyanins within the blotch. Future research endeavors must address the question of why this synthesis is spatially constrained. Epigenetic regulation, such as differential DNA methylation or histone modifications, may serve as a key determinant, as indicated by studies on other patterned flowers (Liu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, the strong association between blotch patterning and petal development suggests the involvement of specific developmental signals. Investigating how signals related to tissue polarity, hormone gradients, or localized conditions are perceived and transduced to differentially regulate the VwMYB1-VwF3\u0026rsquo;5\u0026rsquo;H module will be of great significance. Techniques such as spatial transcriptomics and chromatin accessibility mapping will play a vital role in deciphering the upstream signals and regulatory landscape that direct the remarkable precision of floral pattern formation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the key research and development program of Henan province (241111113100) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplemental data associated with this article can be found online at China National Center for Bioinformation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJungmin Lee, Robert W Durst, Ronald E Wrolstad, Collaborators (2005) Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the pH Differential Method: Collaborative Study. 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DOI:10.17660/ActaHortic.2024.1404.117\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Viola × wittrockiana, blotch formation, anthocyanin biosynthesis, VwMYB1, VwF3’5’H","lastPublishedDoi":"10.21203/rs.3.rs-8639386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8639386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnthocyanins play a pivotal role in determining the visual characteristics of ornamental plants. To elucidate the molecular basis of the distinctive \u0026ldquo;blotch\u0026rdquo; formation in pansy (\u003cem\u003eViola \u0026times; wittrockiana\u003c/em\u003e), this study employed an integrated approach that combines targeted metabolomics and comparative transcriptomics. Comparative analysis of blotched versus non-blotched petal regions identified \u003cem\u003eVwMYB1\u003c/em\u003e, a differentially expressed R2R3-MYB transcription factor, as a key candidate regulator. Functional validation revealed that the heterologous overexpression of \u003cem\u003eVwMYB1\u003c/em\u003e in tobacco enhanced floral pigmentation, while its transient overexpression in pansy petals induced localized dark blotches, thereby confirming its role in promoting pigmentation. Targeted metabolomic profiling demonstrated that the blotch phenotype is primarily attributed to the substantial and specific accumulation of delphinidin-type anthocyanins. Given that \u003cem\u003eF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e is the pivotal branch-point enzyme for delphinidin biosynthesis, the regulatory interaction between \u003cem\u003eVwMYB1\u003c/em\u003e and \u003cem\u003eVwF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e was further examined. Yeast one-hybrid and dual-luciferase reporter assays confirmed that \u003cem\u003eVwMYB1\u003c/em\u003e directly binds to the promoter of \u003cem\u003eVwF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e and activates its expression. This study not only illustrates a streamlined pipeline from transcriptome-based gene discovery to functional and metabolic validation but also uncovers the core regulatory mechanism by which \u003cem\u003eVwMYB1\u003c/em\u003e governs delphinidin-based blotch formation through the direct transcriptional activation of \u003cem\u003eVwF3\u0026rsquo;5\u0026rsquo;H\u003c/em\u003e, thereby offering both mechanistic insight and a practical genetic tool for precision breeding in flower color modification.\u003c/p\u003e","manuscriptTitle":"A R2R3-MYB, VwMYB1, regulates anthocyanin biosynthesis in pansy petal blotch via transcriptional activation of VwF3’5’H","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 11:33:42","doi":"10.21203/rs.3.rs-8639386/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-05T14:07:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-05T04:48:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T08:01:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159881450047926864288725766694583497537","date":"2026-02-01T23:25:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182136406093867227957169246935649851129","date":"2026-02-01T21:23:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328515927337189122948909379015495461330","date":"2026-01-28T00:54:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T10:25:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T07:14:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-21T07:12:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2026-01-19T12:05:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fb4474cc-3e4e-48ce-9c58-c269f49a92d8","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T10:02:50+00:00","versionOfRecord":{"articleIdentity":"rs-8639386","link":"https://doi.org/10.1007/s00299-026-03842-5","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2026-05-02 15:57:58","publishedOnDateReadable":"May 2nd, 2026"},"versionCreatedAt":"2026-01-29 11:33:42","video":"","vorDoi":"10.1007/s00299-026-03842-5","vorDoiUrl":"https://doi.org/10.1007/s00299-026-03842-5","workflowStages":[]},"version":"v1","identity":"rs-8639386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8639386","identity":"rs-8639386","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}