The experiments were carried out at the College of Horticulture Sciences, Shandong Agricultural University (Tai’an, Shandong, China) and Ministry of Agriculture Key Laboratory for utilization of horticultural crop germplasm resources, Fruit Tree Research Institute, Chinese Academy of Agricultural Sciences (Xingcheng, Liaoning, China) from April 2018 to January 2021. The experimental material were 9-year-old blueberry (V. corymbosum “Duke”) seedlings that were collected from the small berry garden in the Fruit Tree Research Institute, Chinese Academy of Agricultural Sciences. Fruits were collected 40 days (green fruits), 65 days (pink fruits), or 80 days (blue fruits) after flowering. To ensure consistency across the materials, fruits were collected from the tips of inflorescences (first to mature), snap-frozen in liquid nitrogen, and stored at –80°C until analysis.

Plant total RNA extraction kits (TaKaRa, Beijing, China, No. 9769S) were used for extraction of total RNA from fruits of each of the three stages. Agarose gel electrophoresis (Agilent 2100 bioanalyzer, Agilent),¹ and spectrophotometry (NanoDrop spectrophotometer, Thermo Fisher Scientific)² were used to examine the integrity, purity, and quality of total RNA. Oligo (dT) magnetic beads were used for mRNA enrichment and the SMART PCR cDNA Synthesis Kit was used to synthesize full-length cDNA from mRNA. BluePippin (Sage Science)³ was used to screen full-length cDNA fragments and three types of insert libraries were constructed (1–2, 2–3, and 3–6 kb). PCR amplification was carried out on full-length, screened cDNA. End-terminal repair was carried out on full-length cDNA and the cDNAs were ligated to SMRT bell adapters before digestion using exonucleases. Secondary screening of the libraries was carried out using BluePippin to obtain the sequencing libraries. After library construction, Qubit 2.0 (Thermo Fisher Scientific) was used for quantitative analysis. The Agilent 2100 bioanalyzer (Agilent 2100 bioanalyzer) was used for examination of the insert fragments in the libraries. After the libraries passed the quality check, the PacBio RS II platform was used for full-length transcriptome sequencing. Two SMRT cells were used for sequencing the 1–2 and 3–6 kb fragment libraries, and three SMRT cells were used for sequencing the 2–3 kb fragment libraries. The LoRDEC software was used to correct the sequencing errors in the consensus transcripts using Illumina reads as the reference (Salmela and Rivals, 2014).

The methods for total RNA extraction from fruits and examination of total RNA integrity, purity, and quality were carried out as described above. After DNAse I digestion of total RNA from fruit samples, oligo (dT) magnetic beads were used for mRNA enrichment. After mRNAs were made into short fragments, first-strand cDNA and second-strand cDNA were synthesized, purified and recovered, and the sticky ends were repaired. An “A” adapter was added to the 3′ ends of the cDNA before selection of fragment sizes. Finally, PCR amplification was used to construct the libraries. Qubit 2.0 (Thermo Fisher Scientific; see text footnote 2) and Agilent 2100 bioanalyzers were used for examination of library concentration and the fragment sizes of the inserts in the constructed libraries. We used Quantitative PCR to verify quantitation of the effective concentrations of the libraries. After the libraries passed the quality check, the Illumina HiSeq 2500 sequencer was used for sequencing and the sequencing read length was 125 bp paired-ends. RNA samples from green, pink, and blue fruits were sequenced in duplicates.

The IsoSeq^TM protocol (Pacific Biosciences, United States) was used to process the sequenced reads using circular consensus sequencing RNA BIOLOGY 5. Effective subreads were obtained using the P_Fetch and P_Filter function (parameters: miniLength = 50, read Score = 0.75, artifact = –1,000) in the SMRT Analysis software suite.⁴ CCS was obtained from the P_CCS module using the parameter Min Complete Passes = 2 and Min Predicted Accuracy = 0. After examining for poly (A) signal and 5′ and 3′ adaptors, only the CCS with all three signals was considered as a FLNC read (Du et al., 2021). Unmerged subreads were also examined for the three signals, and those with three signals were incorporated into the final FLNC read set.

Blueberry transcriptome data from Illumina and SMRT sequencing were used for alignment with COG, GO, and NR databases to obtain VcMYB genes. Phylogenetic analysis of all the excavated VcMYB protein sequences and Arabidopsis MYB protein sequences showed that only one VcMYB sequence has extremely high homology with the Arabidopsis sg6 R2R3 MYB protein, and it was named VcMYB1.

Three RNA samples were isolated from fruits of each of the three fruit development stages of blueberries. RNA samples were isolated from leaves of Arabidopsis. Single-stranded cDNA was obtained using a reverse transcription kit (TaKaRa, Shiga, Japan). All qRT-PCR analyses were performed with three biological repeats and three techincal repeats. Arabidopsis polyubiquitin 10 (AtUBQ10) was used as housekeeping gene. Supplementary Table 4 shows the primers used.

The extraction methods for anthocyanins, flavonols, and proanthocyanidins were referenced from Prior et al. (2001); Lätti et al. (2010), and Pertuzatti et al. (2016), respectively. High performance liquid chromatography (HPLC) separation, identification and quantitation of anthocyanins, proanthocyanidins, and flavonols were performed on an Agilent 1100 Series system (Agilent, Germany), equipped with DAD (G1315B) and a LC/MSD Trap VL (G2445C VL) electrospray ionization mass spectrometry (ESIMSn) system that was coupled to an Agilent ChemStation (version B.01.03) data-processing station. The mass spectra data were processed with the Agilent LC/MS Trap software (Version 5.3).

ESI-MSn was used to identify the anthocyanins, proanthocyanidins, and flavonols profile and the following parameters were employed: positive ionization mode; dry gas, N2, 11 mL/min; drying temperature, 350°C; nebulizer, 65 psi; capillary, –2500 V; capillary exit offset, 70 V; skimmer1, 20 V; skimmer 2, 6 V; compound stability, 100%; scan range, 50–1,200 m/z. For the purposes of quantitation, For quantification, DAD chromatograms were extracted at 520 nm for anthocyanins, proanthocyanidins at 280 nm, and flavonols at 360 nm.

The Protein BLAST program⁵ was used to obtain homologs of Arabidopsis, Vitis vinifera and Malus domestica MYB. The amino-acid secondary structure of VcMYB was predicted using the Simple Modular Architecture Research Tool (SMART) software program.⁶

The ORF (Open Reading Frame) was cloned and inserted into the pRI101-AN vector, and used to transform Agrobacterium tumefaciens GV3101. Arabidopsis plants were transformed with Agrobacterium by the floral dipping method. T1 VcMYB1 transgenic plants were selected on Murashige and Skoog (MS) media containing 50 mg L^–1 kanamycin. Kanamycin-resistant T1 seedlings were transferred to soil and grown at 22°C in a growth chamber with a 16 h day length. T2 seedlings were selected on MS media containing 50 mg L^–1 kanamycin.

The Agrobacterium GV3101 strains containing the pRI101-VcMYB1 were infiltrated into the abaxial leaf surface of tobacco according to Espley et al. (2007). Each infiltration was performed using three leaves from the same plants. Photographs were taken 4–7 days after infiltration. To control for leaf-to-leaf variability, at least two plants were used for infiltration, and each leaf included negative controls (Agrobacterium with pRI101 empty vector).

Fruit injection assays were carried out according to Li X. Y. et al. (2012), Li Y. Y. et al. (2012), and An et al. (2017b). The VcMYB1-pGreenII62-SK vector was generated by cloning the ORF of VcMYB1 into the pGreenII62-SK vector. The mixed vectors and the A. tumefaciens solutions were injected into the fruit peels.

Chromosome walking techniques were used to isolate the promoter sequence for VcDFR and the method was referenced from the Genome Walking Kit (TaKaRa).⁷ Specific primers (SP1, SP2, and SP3) were designed according to the open reading frame of VcDFR. Specific primers and four degenerate primers in the reagent kit were used for three rounds of thermal asymmetric PCR. Finally, a 834 bp fragment was obtained through three rounds of PCR.

Y1H assays were performed with the yeast strain Y1HGold (Clontech).⁸ The full-length cDNA of VcMYB1 was cloned and inserted into the pGADT7 vector. The promoter fragments of VcDFR were cloned into the pAbAi vector to generated the construct pAbAi-VcDFR_pro, and then transformed into Y1HGold using a PEG/LiAc method. Yeast cells were cultured on the medium lacking Ura (SD/-Ura). Yeast colony PCR for confirming that positive plasmid has integrated into the Y1HGold genome, and then transformed pGADT7-VcMYB1 into Y1HGold-pAbAi-VcDFR_pro. Yeast cells were cultured on media lacking Leu (SD/-Leu) with Aureobasidin A to identify possible interactions.

The EMSA was performed using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, Waltham, MA, United States). Briefly, biotin-labeled probes were incubated in a 1 × binding buffer containing 5 mM MgCl₂, 50 mM KCl and 2.5% glycerol with or without VcMYB1-His fusion proteins at room temperature for 20 min. An unlabeled probe was added to the reactions for unlabeled probe competition.

Transient expression assay in tobacco leaves were performed according to Shang et al. (2010). The VcDFR promoter was amplified and cloned into pGreenII0800-LUC vector, which generated the reporter constructs VcDFR_pro:Luc. The effector (35S_pro:VcMYB1) was constructed by cloning the ORF of VcMYB1 into the pGreenII62-SK vector. Transformed leaves were sprayed with 100 mM luciferin, after which they were placed in darkness for 5 min before examining for luminescence. A charge-coupled device-imaging apparatus (NightOWL LB983 in conjunction with Indigo software) was used to collect the LUC images.

The Agrobacterium GV3101 strains containing the pRI101-VcMYB1 generated effector constructs. Reporter constructs were generated using the promoter sequences of VcDFR cloned upstream of the β-glucuronidase (GUS) reporter gene in the pCAMBIA1301 vector, and used to transform Agrobacterium GV3101. For the transient expression assay, Agrobacterium GV3101 strains containing the pRI101-VcMYB1 were co-infiltrated into the abaxial leaf surface of tobacco. Each infection was performed in three biological replicates. After grow in a growth chamber for 3–4 days, the infected leaves were used to analyze GUS activity. Proteins were extracted from infected leaves and fluorescence was measured using a fluorometer (VersaFluor Fluorometer, Bio-Rad)⁹ as performed according to Jefferson et al. (1987). The values of GUS activity were calculated from three biological replicates.

The Illumina and SMRT sequencing platforms were used to analyze transcriptome data of blueberries. First, the Hiseq 2500 platform was used for sequencing of three samples from blueberry fruits of each of three developmental stage, green (diameter 8–10 mm), pink (12–14 mm), and blue (12–14 mm) (Figure 1A). Samples were sequenced in duplicates. After removal of sequencing adapters and primer sequences the high-quality reads (clean data) totaled 112,625,479 (Table 1) and the total number of bases was 28,376,811,360. After Trinity assembly, a total of 104,068 Unigenes were obtained (Supplementary Table 1).

Next, SMRT sequencing was carried out on the same samples. Equal amounts of six types of mRNAs from the three fruit stages were mixed to construct a sample pool. Three insert libraries were constructed and seven SMRT cell (two cells for 1–2 kb fragments, three cells for 2–3 kb fragments, and two cells for 3–6 kb fragments) sequencing microarrays were used. After removal of adapters and low-quality reads, 572,495 subreads (1.9 billion) were obtained. These subreads were screened to obtain 458,964 reads of insert (RoIs), of which there were 209,715 full-length non-chimeric reads (FLNCs) with 5′ and 3′ primer sequences and poly (A) sequences (Table 1). The average length of RoIs and FLNC reads were 2,938 and 2,785 bp, respectively. The iterative isoform-clustering (ICE) algorithm and Quiver software were used for sequence calibration and sequences with accuracy greater than 0.99 were retained to obtain 117,784 consensus reads. Then, CD-HIT EST software was used to remove redundant sequences from the above high-quality transcripts. We obtained 42,740 non-redundant sequences with an average length of 2,452 bases (Supplementary Table 1).

Although Illumina sequencing has extremely high coverage, the transcripts assembled by Trinity cannot represent full-length cDNA. In this study, the Illumina sequencing results of blueberry messenger RNA (mRNA) samples showed that approximately 37.81% of Unigenes were shorter than 300 bases. The number of Unigenes that were greater than 1, 2, and 3 kb were 18,849, 7,042, and 2,476, respectively, with an average length of 677 bases. In the SMRT sequencing data, the percentage of RoIs that were smaller than 300 bases was only 5.6%. The number of FLNCs that were greater than 1, 2, and 3 kb were 42,646, 24,312, and 9,896, respectively. The average length of FLNC reads in the three insert libraries (1–2, 2–3, and 3–6 kb) were 1,757, 2,823, and 3,775 bases. Overall, sequences obtained through SMRT sequencing were longer and the transcripts were more intact.

Blast alignment analysis of the corresponding relationship between 104,068 Unigene sequences obtained from Illumina sequencing and 42,740 non-redundant sequences from SMRT sequencing showed that the sequences do not show one-to-one correspondence as a single SMRT transcript corresponds to a single or multiple Illumina transcripts. There were 15,596 transcripts present in both Illumina and SMRT sequencing; 27,144 transcripts were only found in SMRT sequencing, and 80,624 transcripts were only found in Illumina sequencing (Figure 1B). The Unigene sequences obtained from Illumina sequencing and the non-redundant sequences obtained from SMRT sequencing were used for data alignment in Clusters of Orthologous Groups (COG), Gene Ontology (GO), and non-redundant (NR) databases. The number of annotated genes obtained through Illumina sequencing was lower than that obtained through SMRT sequencing. There were 32,916 annotated genes that were obtained through Illumina sequencing, which accounted for 31.6% of Unigenes and there were 40,828 annotated genes that were obtained through SMRT sequencing, which accounted for 95.5% of non-redundant sequences. Of the obtained annotated genes, 14,576 genes were common to both Illumina and SMRT sequencing; 18,340 genes were only found in Illumina sequencing, and 26,252, genes were only found in SMRT sequencing (Figure 1C).

We used three methods [coding-non-coding index (CNCI), coding potential calculator (CPC), and Pfamscan (PFAM)] to predict long non-coding RNAs (lncRNAs) longer than 200 bases. We obtained 6,251, 3,322, and 7,610 potential lncRNA sequences, respectively, using these three methods. Among these sequences, 2,171 potential lncRNA sequences simultaneously appeared in all three methods and these sequences were regarded as candidate lncRNAs in the target dataset (Supplementary Figure 1).

Plants employ the phenylalanine metabolic pathway to synthesize flavonoids such as anthocyanins, proanthocyanidins, and flavonols. Here, blueberry transcriptome data from Illumina and SMRT sequencing were used for alignment with COG, GO, and NR databases to obtain genes that regulate flavonoid synthesis.

To study the correlation between expression patterns of flavonoid synthesis genes and accumulated flavonoids in fruits, this study employed HPLC-DAD-ESI-MS to quantitate anthocyanin, flavonol, and proanthocyanidin content in fruits from three developmental stages. Total anthocyanin content showed an increasing trend by development stage, while the total flavonol and proanthocyanidin content decreased (Figure 2A).

The anthocyanin synthesis pathway is an important branch of the flavonoid synthesis pathway, which is relatively conserved in higher plants, and its process involves multiple complex enzymatic reactions (Supplementary Figure 2). Most studies on flavonoid synthesis have been in model plants such as apples and peaches, whereas fewer studies have used blueberries (Zhou et al., 2015; An et al., 2017a, An et al., 2020b). To validate the bioinformatics analysis results of annotated genes in the sequencing libraries and study the expression of genes in the phenylalanine metabolic pathways in blueberries, we used homology alignment and phylogenetic analysis to analyze structural genes of 15 main enzymes involved in anthocyanin synthesis pathway: PAL, C4H, 4CL, CHS, CHI (chalcone isomerase), F3H (flavanone 3′-hydroxylase), F3′H (flavonoid 3′ -hydroxylase), F3′5′H (flavonoid 3′, 5′-hydroxylase), DFR, ANS/LDOX (leucoanthocyanidin dioxygenase), UFGT (UDP-glucose: flavonoid 3-O-glucosyltransferase), CCoAOMT (caffeoyl-CoA O-methyltransferase), FLS (flavonol synthase), ANR (anthocyanidin reductase) and LAR (leucoanthocyanidin reductase). These genes have the largest FPKM (Fragments Per Kilobase of transcript per Million mapped reads).

Fluorescence quantitative PCR was used to quantitate the expression status of the structural genes and regulatory genes in flavonoid synthesis during the three developmental stages of blueberries. In this study, we referenced the results from the literature (Zhou et al., 2015) and selected VcGADPH and VcSAND as internal reference genes. The geometric means of the Ct values of these two genes were used as calibration values to analyze the relative expression of other genes. Supplementary Table 2 shows the annotated functions and FPKM (fragments per kb per million) values obtained from transcriptome sequencing of these genes. Supplementary Table 3 shows the primer sequences for qPCR.

Fluorescence qPCR results showed that the genes for flavonoid synthesis exhibited varied trends in expression during the three developmental stages of fruits. We first analyzed the expression of VcC4H, VcPAL, Vc4CL, VcCHS, VcCHI, and VcF3H (Figure 2B). These genes are located upstream of the phenylalanine metabolic pathway and the enzymes encoded by them can catalyze the synthesis of anthocyanins, proanthocyanidins, and flavonols. The results of qPCR showed that with the exception of VcPAL and Vc4CL, the expression of the remaining four genes was highest in the most mature fruits (blue fruits). VcPAL had the highest expression in the least mature (green) fruits and extremely low expression in pink and blue fruits. The expression of Vc4CL was the highest in green fruits, and lowest in pink fruits (Figure 2B).

Next, we analyzed six genes that encode for anthocyanin biosynthesis (VcF3′H, VcF3′5′H, VcDFR, VcANS, VcUFGT, and VcCCoAOMT). In the middle of the anthocyanin synthetic pathway, VcF3′5′H and VcF3′H are branch points as VcF3′5′H can regulate the biosynthesis of delphinidin, petunidin, and malvidin in blueberries whereas VcF3′H can regulate the synthesis of cyanidin and peonidin. In all three fruit developmental stages, VcF3′5′H and VcANS had persistently elevated levels whereas VcF3′H, VcDFR, VcUFGT, and VcCCoAOMT varied; levels were low in immature (green) fruit and high in mature (blue) fruit, then even higher in intermediate (pink) fruits (Figure 2C). Interestingly, in pink and blue fruits, the expression of VcF3′5′H was 31- and 40-fold that of VcF3′H (Figure 2C). This was also verified in quantitation of anthocyanin contents. In blue fruits, the total content of delphinidin, petunidin, and malvidin was 8.1 times that of the total content of cyanidin and peonidin (Table 2). These results suggest that anthocyanin biosynthesis mainly occurs during the fruit maturation phase in “Duke” blueberries, particularly the synthesis of delphinidin, petunidin, and malvidin, and also express the VcF3′5′H gene was upregulated in colored fruits, which changed the ratio of different anthocyanins.

Then, we analyzed the VcFLS gene that regulates flavonol synthesis. VcFLS had the highest expression in green fruits and extremely low expression in pink and blue fruits (Figure 2D). This result was consistent with quantitation of flavonol content, which showed that flavonol contents in green fruits were approximately 1.69 times greater than in pink or blue fruits (Figure 2A). Subsequently, we analyzed two genes that regulate proanthocyanidin biosynthesis, VcANR and VcLAR, which were mainly expressed in green fruits and the expression of VcLAR was approximately 60 times that of VcANR (Figure 2D). The quantitation results of proanthocyanidins in fruits showed that catechin content was 2.19 times that of epicatechin content in green fruits (Figure 2A). The results suggest that the differential expression of VcLAR and VcANR may be responsible for differences in catechin and epicatechin contents. These results suggest that the biosynthetic characteristics of flavonols and proanthocyanidins are relatively similar as they are mainly carried out in immature fruits and there is almost little or no biosynthesis in mature fruits. Of these enzymes, VcLAR may play a dominant role in proanthocyanidin synthesis in green fruits compared with VcANR.

In the blueberry transcriptome data, we obtained a total of 45 blueberry MYB gene sequences named VcMYB1-45, respectively. A heat map analysis of these gene sequences revealed that 14 MYB genes were up-regulated in the stages of green fruit to pink fruit or from green fruit to blue fruit, and 31 MYB genes were down-regulated at the above stage (Figure 3A). The amino acid sequences of R2R3 MYB transcription factors in other plants that regulate anthocyanin synthesis, such as grapes (VvMYBA1), apples (MdMYB10), and Arabidopsis (PAP1) were used as probes for alignment and screening of the MYB amino acid sequence of up-regulated gene expression in blueberries. It was found that only VcMYB1 contained the sg6-motif among the genes up-regulated in blueberries (Figure 3B). Studies have shown that the sg6 motif can promote the synthesis of plant anthocyanins (Stracke et al., 2001; Muñoz et al., 2021). For further verification, Phylogenetic analysis of all the excavated VcMYB protein sequences and homologous sequences of other 138 species of plants showed that only the VcMYB1 gene has extremely high homology with the Arabidopsis SG-6 R2R3 MYB protein (Figure 3C).

To further verify the reliability of the blueberry transcriptome data. Fluorescence qPCR results using 9 blueberry VcMYB transcription factors with larger FPKM showed that the expression trends of VcMYB1 were similar to the expression trends of the six genes that regulate anthocyanin synthesis, such as VcDFR and VcF3′5′ H, which are mainly expressed in colored fruits. VcMYB23, VcMYB26, and VcMYB28 are almost only expressed in green fruits. VcMYB25, VcMYB27, and VcMYB29 exhibited a continuously declining trend. VcMYB24 levels varied; in green fruit VcMYB24 was high; in mature fruits it was lower. VcMYB22 expression varied; it was highest in immature (green) fruits, lower in pink fruits, and higher in the mature (blue) fruits (Supplementary Figure 3). The results suggest that the upregulated expression of VcMYB1 in pink and blue fruits may promote the synthesis of anthocyanins.

Given that the expression patterns of VcMYB1 and anthocyanin-synthesis genes are similar, i.e., relatively high expression in colored fruits and VcMYB1 has relatively high homology and similarity to MYB transcription factors in other plants that regulate anthocyanin synthesis (Figures 3B,C), we hypothesized that VcMYB1 may positively regulate anthocyanin synthesis. To investigate whether VcMYB1 can promote anthocyanin synthesis, we used a heterologous expression system and validated the function of VcMYB1 in Arabidopsis (ecotype Columbia) under the control of CaMV-35S. Results showed that under the same conditions, red pigmentation was present in Arabidopsis seeds (8–10 days after flowering), cotyledons, and hypocotyls overexpressed 35S_pro:VcMYB1 whereas red pigmentation was absent in wild-type (WT) Arabidopsis in these three tissues (Figures 4A,C). Anthocyanin contents in transgenic plants and control plants were 311.61 mg/kg fresh weight and 2.82 mg/kg fresh weight, respectively. Thus, anthocyanin contents in plants overexpressing VcMYB1 was approximately 110 times the expression levels in control plants (Figure 4B). To further understand the molecular mechanisms by which VcMYB1 regulates anthocyanin synthesis in Arabidopsis, we extracted RNA from 35S_pro:VcMYB1 Arabidopsis and WT Arabidopsis. Fluorescence qPCR was used to measure the expression of structural genes for anthocyanin synthesis in Arabidopsis such as AtPAL, AtCHS and AtDFR, and VcMYB1. Except for At4CL, expression of all other genes in the transgenic plants was significantly higher than in WT plants (Figure 4D). This suggests that VcMYB1 affects anthocyanin synthesis in Arabidopsis by regulating structural genes that are related to anthocyanin synthesis.

To confirm the function of VcMYB1, we conducted transient expression experiments and validated the function of VcMYB1 in tobacco (Nicotiana benthamiana). 35S_pro:VcMYB1 was transformed into Agrobacterium before injecting into tobacco leaves. Agrobacterium that were transformed with empty vectors were used as controls. Results showed that 4 days after injection, tobacco leaves overexpressing VcMYB1 were colored whereas no color changes were observed in the control (Figure 4E). Anthocyanin content measurement results showed that the total anthocyanin content in tobacco leaves overexpressing VcMYB1 was 6.8-fold higher than in the control leaves (Figure 4F). The above data shows that VcMYB1 can positively regulate anthocyanin synthesis.

Moreover, vector-mediated overexpression was conducted using 45 days blueberries. VcMYB1-pGreenII62-SK vectors (VcMYB1) were generated, and pGreenII62-SK were used as a control. Compared with the empty vector control (pGreenII62-SK), overexpression of VcMYB1 promoted anthocyanin accumulation in the blueberry shin around the injection sites (Figures 5A,B), The total content of delphinidin, petunidin, and malvidin was higher than the total content of cyanidin and peonidin, consistent with Supplementary Table 2. The expression levels of VcMYB1, VcDFR3, VcANS1 and VcUFGT1 were elevated in the VcMYB1-pGreenII62-SK vectors injection areas compared with the controls (Figure 5C). Therefore, these results demonstrated that VcMYB1 played a positive role in anthocyanin synthesis and fruit coloration.

Because VcMYB1 can promote anthocyanin synthesis, we predicted that VcMYB1 may directly regulate the expression of structural genes in anthocyanin synthesis to regulate anthocyanin synthesis. Previous studies have reported that MYB function by binding to the promoters of their target genes to modulate their expressions (Takos et al., 2006; Ban et al., 2007; Espley et al., 2007; An et al., 2020a). We employed chromosome walking techniques to clone the promoter sequence of VcDFR, VcF3′5′ H and VcUFGT, which was found to have a length of 834, 1,828, and 2,362 bp respectively. Unfortunately, the promoter sequence of VcANS is not available. This promoter was inserted into a pAbAi vector before transformation into a Y1HGoldstrain. The open reading frame of VcMYB1 was inserted into a pGADT7 vector. The Y1H assay was used to analyze whether VcMYB1 can interact with these promoters. Results showed that VcMYB1 can bind the MYB binding site (MBS) motifs in the VcDFR promoter (Figure 6A). For further research, we used the promoter prediction website¹⁰ to predict and analyze the VcDFR, VcF3′5′H and VcUFGT promoter sequences. It was found that only the VcDFR promoter sequence contained the MBS motif. In the anthocyanin synthesis pathway, the action site of VcDRF is adjacent to the action sites of the other three structural genes. Therefore, it is speculated that the expression of VcDRF will promote their expression. We also used a prokaryotic expression system to induce and purify the VcMYB1 protein and used electrophoretic mobility shift assay (EMSA) to validate the results of the Y1H assay. Results showed that VcMYB1 can bind to the biotinylated MBS motif on the VcDFR promoter (Figure 6B).

To investigate whether VcMYB1 can activate the VcDFR promoter, we carried out firefly luciferase (Luc) complementation experiments for validation. We inserted the VcDFR promoter upstream of the Luc gene to generate VcDFR_pro:Luc. VcMYB1 was inserted downstream of the 35S promoter to generate 35S_pro:VcMYB1. Both vectors were transformed into Agrobacterium before co-injection into tobacco leaves. Results showed that luminescence signals could be detected in tobacco plants that were co-injected with VcDFR_pro:Luc and 35S_pro:VcMYB1 whereas no luminescence signals were detected in the control (VcDFR_pro:Luc + 35S_pro:empty) (Figures 6C,D). Following that, we validated the regulation of the VcDFR promoter by VcMYB1 in tobacco leaves by analyzing the activity of the β-glucuronidase (GUS) reporter gene. The results showed that co-transforming VcDFR_pro:Luc and 35S_pro:VcMYB1 into tobacco leaves elevated VcDFR promoter activity (Figure 6E). Therefore, we believe that VcMYB1 directly binds to the promoter of VcDFR to activate the expression of this gene, thereby promoting anthocyanin synthesis.