The PpMYB75-PpDFR module reveals the difference between ‘SR’ and its Bud Variant ‘RMHC’ in Peach Red Flesh

The PpMYB75-PpDFR module reveals the difference between ‘SR’ and its Bud Variant ‘RMHC’ in Peach Red Flesh | 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 The PpMYB75-PpDFR module reveals the difference between ‘SR’ and its Bud Variant ‘RMHC’ in Peach Red Flesh Chao Xu, Xiaomin Xue, Zhixing Li, Mingguang Chen, Siyu Wang, Yating Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3378595/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jan, 2024 Read the published version in Journal of Plant Research → Version 1 posted 4 You are reading this latest preprint version Abstract ‘Red Meat Honey Crisp (RMHC)’ has been widely cultivated by growers in recent years due to its early maturity, and red meat type characteristics. As a bud variant of ‘Super Red (SR)’ peach, red flesh is the most distinctive characteristic of ‘Red Meat Honey Crisp (RMHC)’. However, the mechanism of red flesh formation in ‘RMHC’ remains unclear. In this study, 79 differentially produced metabolites were identified by metabolomics analysis. The anthocyanin content in ‘RMHC’ was significantly higher than that in ‘SR’ during the same period, such as cyanidin O-syringic acid and cyanidin 3-O-glucoside. Other flavonoids also increased during the formation of red flesh, including flavonols (6-hydroxykaempferol-7-O-glucoside, hyperin), flavanols (protocatechuic acid, (+)-gallocatechin), and flavonoids (chrysoeriol 5-O-hexoside, tricetin). In addition, transcriptomic analysis and RT-qPCR showed that the expression levels of the flavonoid synthesis pathway transcription factor MYB75 and some structural genes, such as PpDFR , PpCHS , PpC4H , and PpLDOX increased significantly in ‘RMHC’. Subcellular localization analysis revealed that MYB75 was localized to the nucleus. Yeast single hybridization assays showed that MYB75 bound to the cis-acting element CCGTTG of the PpDFR promoter region. The MYB75- PpDFR regulatory network was identified to be a key pathway in the reddening of ‘RMHC’ flesh. Moreover, this is the first study to describe the cause for red meat reddening in ‘RMHC’ compared to ‘SR’ peaches using transcriptomics, metabolomics and molecular methods. Our study identified a key transcription factor involved in the regulation of the flavonoid synthetic pathway and contributes to peach breeding-related efforts as well as the identification of genes involved in color formation in other species. SR RMHC Flavonoids Flesh reddening Anthocyanins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Peach is a fruit with high commercial and nutritional value that has a wide market demand worldwide (Zhao et al. 2015 ). Consumers and cultivators have a very intuitive feeling about the coloring of peaches when choosing fruit. Peach varieties have been divided into white, yellow, and red flesh color categories. The type and content of flavonoids, carotenoids, and chlorophyll in the plants are the main reasons for the different colored fruits and leaves (Drogoudi et al. 2017 ). Many studies have confirmed that yellow pulp is due to the accumulation of carotenoids, while white pulp has a lower pigment content (Falchi et al. 2013 ). The red-fleshed peach, which is mainly determined by the accumulation of flavonoids, such as anthocyanins, flavonoids, and flavonols, is a rare peach germplasm resource with important potential value in production and breeding (Cao et al. 2018 ). Thus, studies on the mechanism for the formation of red peach pulp are significant. The flavonoid biosynthetic pathway in peaches as well as other plants is highly conserved (Araguirang and Richter 2022 ). The enzymes and related structural genes associated with the flavonoid biosynthetic pathway have been well studied. First, phenylalanine serves as a precursor for the biosynthesis of many plant secondary metabolites, such as flavonols, anthocyanins, and proanthocyanidins. Phenylalanine produces p-coumaroyl coenzyme A by the action of phenylalanine lyase (PAL), cinnamic acid-4-hydroxylase (C4H) and 4-coumaryl CoA ligase (4CL). Subsequently, the product is catalyzed to form dihydroflavonols, which are produced as flavonols catalyzed by flavonol synthase (FLS), and leucoanthocyanidin forms from flavonol under the action of dihydroflavonol 4-reductase (DFR). Leucoanthocyanidin also has two branches, one produces flavan-3-alcohol directly under the action of leucoanthocyantin reducase (LAR), and further polymerizes to produce proanthocyanidins. The other branch produces unstable anthocyanins under the action of leucoanthocyanin dioxygenase (LDOX) or anthocyanin synthase (ANS). Anthocyanins also produce proanthocyanidins under the action of anthocyanin reductase. Finally, through the action of UDP-glycosyltransferase (flavonoid 3-O-glucosyltransferase, UFGT), the free anthocyanins form anthocyanins that exist stably in plants and give different tissues the plants’ brilliant colors. After being synthesized in the cytoplasm, anthocyanins are transported to vesicles with the participation of glutathione S-transferase (GST) (Liu et al. 2019b ; Rahim et al. 2014 ; Khan et al. 2022a ). The accumulation of flavonoids in plants is regulated by structural genes, MYB, the basic helix-loop-helix (bHLH), and WD40 transcription factors, particularly in Rosaceae species, such as peaches, apples, and strawberries (Lin-Wang et al. 2010 ; Yan et al. 2021 ). These transcription factors form the MBW complex and bind to the promoters of the structural genes in the flavonoid synthetic pathway, which together regulate the metabolism of flavonoid pathway products (Broucke et al. 2023 ; Xu et al. 2015 ; Dare et al. 2008 ). bHLH transcription factors play an important role in the responses to the external environment, such as light, temperature, and nutrients, and affect flavonoid levels (Feller et al. 2017 ). Some studies have shown that WD40 proteins are not necessary for regulating flavonoid synthesis, and their main role may be in the response to external signals (Feng et al. 2019 ). However, other studies have shown that the diversity of MBW functions is due to MYB, rather than the bHLH or WD40 transcription factors (Chagné et al. 2013 ; Wang et al. 2022 ; Hui et al. 2019 ). As one of the largest families of transcription factors in plants, the MYB family plays an important role in the regulation of plant secondary metabolism (Seo and Kim 2017 ; Liu et al. 2019a ). MYB is also involved in anthocyanin synthesis in fruits, including grapes (Kobayashi et al. 2001 ), apples (An et al. 2020 ), pears (Feng et al. 2010 ), bayberries (Liu et al. 2013 ). MYB plays an important role in the formation of anthocyanins in peaches. Three MYB transcription factors involved in flavonoid biosynthesis in peach fruit have been reported, including PpMYB10.1 , PpMYB10.2 , and PpMYB10.3 (Liu et al. 2019a ). In addition, anthocyanins accumulate in green peach leaves when PpMYB10.4 is transiently expressed (Zhou et al. 2014 ). Peace, an R2R3 MYB transcription factor in peach flowers, promotes the accumulation of anthocyanins in petals, making the peach petals pink (Uematsu et al. 2014 ). PpMYB39 binds to the PpDFR promoter and activates its expression to affect the formation of red flesh (Khan et al. 2022b ). PpMYB108 is closely related to the biosynthesis of peach anthocyanosides and is involved in the formation of red flowers (Khan et al. 2022a ). Taken together, MYB transcription factors play an essential role in regulating the accumulation of flavonoids, particularly anthocyanins. The identification of MYB-specific regulatory genes can guide the development of peach anthocyanins and color-related markers, and provide a basis for the identification, selection, and breeding of peach varieties. In this study, we compared flavonoids of the ‘Super Red’ (SR) peach variety and its bud variant ‘Red Meat Honey Crisp’ (RMHC). The ‘RMHC’ variant accumulated more flavonoids in fruit than ‘SR’, particularly in the flesh during the same period. Because of the increase in flavonoids during fruit development, 79 flavonoid metabolites were identified by metabolomics analysis. These flavonoids, particularly anthocyanins, may be a key factor in the redder flesh of ‘RMHC’ than ‘SR’. Then, transcriptome analysis and RT-qPCR identified key genes, such as PpDFR , PpCHS , PpC4H, PpLDOX , and PpMYB75 which significantly increased during the formation of red flesh. Finally, our study showed that PpMYB75 localizes in the nucleus and we confirmed binding to the PpDFR promoter region to activate its expression. The PpMYB75- PpDFR regulatory pathway of flavonoid biosynthesis may contribute to the red flesh in peach ‘RMHC’ compared to ‘SR’. This study provides new insight into the formation of the red peach color. 2. Materials and Methods 2.1. Plant materials The fresh fruits of ‘RMHC’ and ‘SR’ used in this study were obtained from the experimental base of Shandong Institute of Pomology, Tai 'an, China. Peach samples and three biological copies were collected at different stages on days 90 (S1), 95 (S2), 100 (S3), and 105 (S4) of fruit development after flowering. The fruit was randomly collected at about 2 m above the ground around the crown of the tree, and samples of uniform size and integrity were selected. The peach samples were cut into small pieces and immediately frozen in liquid nitrogen, and then the samples were selected for metabolomics and transcriptome sequencing analyses. S1 period of ‘SR’ (CK1), S2 period of ‘SR’ (CK2), S3 period of ‘SR’ (CK3), S4 period of ‘SR’ (CK4), S1 period of ‘RMHC’ (RF1), S2 period of ‘RMHC’ (RF2), S3 period of ‘RMHC’ (RF3), S4 period of ‘RMHC’ (RF4). 2.2 Flavonoid and anthocyanin measurements Flavonoid content was determined as described previously (Sun et al. 2019 ). Anthocyanin content was determined according to a previous method (Hu et al. 2011 ). SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. Differences between the two groups were detected by one-way analysis of variance and Tukey’s multiple comparison test. A p-value < 0.05 was considered significant. 2.3. Detection and analysis of the metabolomics profile The UPLC-ESI-MS/MS analysis (Metware Co., Wuhan, China) was conducted by a commercial facility. The sample was immersed in liquid nitrogen and ground to a powder with a grinder (MM400, Retsch). A 100 mg portion of powder was dissolved in 70% methanol, centrifuged at 4°C and 12,000 rpm for 10 min, and filtered through a 0.22 µm microporous membrane. All samples were analyzed three times using the UPLC-Qtrap-MS system (SHIMADZU Nexera X2/Applied Biosystems 4500 QTRAP; Tokyo, Japan). The analysis was performed with an Agilent SB-C18 column (2.1 × 100 mm, 1.8 µm). The mobile water phase was ultrapure water containing 0.04% acetic acid, and the mobile organic phase was acetonitrile containing 0.04% acetic acid. The elution gradient was water/acetonitrile (95:5, v/v) for 10 min, 5:95 for 11 min, 5:95 for 12 min, 95:5 for 12.1 min, and 95:5 for 15 min. The flow rate was 0.4 mL/min, the column temperature was held at 40°C, and the injection volume was 5 µl. The raw data were loaded into the MWDB METWARE database to identify the metabolites. Then, qualitative and quantitative analyses of metabolites were performed using Analyst 1.6.3 software. Then, combined with the grouping of specific samples, the fold-changes (fold-change ≥ 2 and fold-change ≤ 0.5 were significantly different) in the quantitative information of the metabolites were compared between the groups. The variable importance in projection (VIP) value of the partial least squares-discriminant analysis model (VIP ≥ 1 was a significant difference) was combined to screen the differentially expressed metabolites. 2.4. RNA extraction and library construction Total RNA was extracted from 24 fresh peach samples using the Total RNA Rapid Extraction kit (Zomanbio, Beijing, China). The purity, concentration, and integrity of the RNA samples were detected by Nanodrop, Qubit 2.0, and Agilent 2100 instruments, respectively. A 20 µg portion of RNA was isolated from each sample for sequencing and to construct the RNA-seq library. The first and second cDNA strands were synthesized using a cDNA synthesis kit (TOYOBO, Osaka, Japan) following the manufacturer’s instructions. The cDNA products were stored temporarily at − 20°C and for the long term at − 80°C. 2.5. Transcriptome sequencing and data analysis Transcriptome sequencing was performed by a commercial company (Novogene, Beijing, China). Three replicates per sample and 24 peach pulp samples were used to construct the library. Total RNA was extracted and assayed as described in Section 2.4 . Qualified DNA samples were randomly interrupted with the Covaris ultrasonic crusher, and the whole library was prepared by terminal repair, A-tail addition, sequencing joint addition, purification, PCR amplification and other steps. The libraries were sequenced with an Illumina sequencer. Subsequently, the different libraries were pooled according to the effective concentration and target downstream data volume required for Illumina sequencing. Raw image data files obtained by high-throughput sequencing were analyzed by CASAVA (Base Calling) and converted into original sequencing sequences, namely Raw Reads. Illunima’s base quality values were expressed as Qphred, and the GC content was counted. The raw reads were filtered to obtain clean reads, and subsequent analyses were based on the clean reads (Jiang et al. 2011 ). The clean reads were mapped to a reference genome sequence and annotated against the reference genome. The HISAT2 tool was used to map the reference genome, and gene transcript levels were calculated using the FKPM fragment mapping method. Differential expression analysis was performed on both samples using edge R with FDR < 0.01 and fold change ≥ 2 as thresholds for significant differential expression. Differentially expressed genes (DEGs) were identified using edge R, and gene functions were annotated according to the KEGG database. The enrichment of DEGs in the KEGG pathway was tested using KOBAS software (Frazee et al. 2015 ). Prunus_persica_GCF_000346465.2 V2.0 ( https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000346465.2/ ) was used as the reference genome. 2.6. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) RT-qPCR was performed as described previously (Zhao et al. 2019 ). We used the NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) and obtained the complete coding region of the selected DEGs. The primers were designed and synthesized at Sangon Biotech’s website (Shanghai, China), and the previously obtained cDNA template was used as the template for RT-qPCR. The data were analyzed using the 2 −ΔΔCt method. We used the TEF2 gene as an internal reference gene for this experiment. Three biological and technical replications were run. All primers used in this experiment are shown in Table S1 . 2.7. Subcellular localization analysis Primers were designed according to the PpMYB75 coding region, and the PpMYB75 open reading frame (ORF) was amplified from the pulp tissue of ‘RMHC’ peaches using PCR with the cDNA library as the template. Then, the PpMYB75 ORF was inserted into the PRI-RFP vector to construct the PpMYB75-RFP vector. The PpMYB75-RFP plasmid was transformed into Agrobacterium tumefaciens LBA4404-injected tobacco for subcellular localization. The primers used are shown in Table S1 . 2.8. Electrophoretic mobility shift assay (EMSA) The EMSA was performed as described previously (Zhao et al. 2019 ). The PpMYB75 coding region was cloned into the pET-32a(+) expression vector, and the PpMYB75-HIS recombinant protein was expressed in Escherichia coli BL21, and purified with the His Microspin Purification kit (Tiangen). The oligonucleotide probe for the PpDFR promoter was labeled by Sangon Biotech. Binding specificity was determined by measuring competition with an excess of unlabeled oligonucleotides. The primers used are shown in Table S1 . 2.9. Yeast one-hybrid (Y1H) assay A yeast one-hybrid assay was performed following the manufacturer’s instructions in the MATCHMAKER One-Hybrid System (Clontech, Palo Alto, CA, USA). The PpDFR promoter and PpMYB75 ORF were inserted into the pHIS2 reporter vector and the PGAD424 vector, respectively. Then, different combinations of the PpMYB75-PGAD424 plasmid and PpDFR -pHIS2 plasmid were co-transformed into the Y187 strain and screened on Leu-, Trp-, and His-deficient medium supplemented with different concentrations of 3-AT (90 mM) for 3–4 days. The primers used are listed in Table S1 . 3. Results 3.1. Flesh reddening of ‘RMHC’ and ‘SR’ peach fruit The most obvious feature of ‘RMHC’ compared with ‘SR’ was the red flesh and pericarp (Fig. 1 A). ‘RMHC’ and ‘SR’ showed varying degrees of anthocyanin content in the pericarp and flesh as they approached maturity (Fig. 1 ). The pericarp of ‘SR’ gradually turned yellow from S1 to S2 with some red, while ‘RMHC’ exhibited a red color. The ‘RMHC’ pericarp had higher anthocyanin content than that of ‘SR’ during the same period, and this difference was particularly evident during S2 and S3 (Fig. 1 ). Dark red appeared in the pericarp during S3, while S1 and S2 were accompanied by a light red feature (Fig. 1 A, B). As shown by the pericarp, the anthocyanin content of the ‘RMHC’ and ‘SR’ flesh also changed significantly (Fig. 1 B, C). Once flesh reddening occurred during S1, the ‘RMHC’ flesh developed rapidly during S2 and the flesh color became deeper during S3 compared to that during S1 and S2 (Fig. 1 B, C). The ‘SR’ flesh remained white, while ‘RMHC’ accumulated some anthocyanin during S2 and S3 (Fig. 1 B, C). The distinctive characteristic of ‘RMHC’ was earlier reddening than ‘SR’, particularly in the peach flesh. The red flesh mainly contributed by anthocyanin may be why ‘RMHC’ has advantages, such as early maturation, high yield, and good taste. Therefore, relevant studies on early red flesh were carried out. 3.2. Flavonoid component analysis The type and content of flavonoids give fruits their different colors, so the flavonoids in peach flesh were analyzed. The flavonoid content in ‘SR’ peach pulp and ‘RMHC’ peach flesh increased during the S1–S2 period, and then began to decrease (Figure S1 ). Flavonoid content in ‘RMHC’ increased significantly during S1–S4 compared to ‘SR’, and content changed significantly during S2–S3 (Figure S1 ). Thus, we analyzed the composition of the flavonoids that occurred during S2–S3 and analyzed the cause of the red flesh. To identify the components in red flesh, 79 flavonoid metabolites from 24 peach flesh samples (RF2, RF3, and CK3) were detected in the flavonoid metabolome material database established by the Metware Company with widely targeted metabolomics based on liquid chromatography-tandem mass spectrometry. We prepared a volcano plot by combining the VIP values and the fold-change data to verify the differences in the expression levels and the statistical differences between the metabolites in the two groups (Figure S2 , Table S2 ). As shown in Figure S2 and Fig. 2 , 27 different metabolites were detected in ‘RMHC’ during S2–S3, among which 26 metabolites were upregulated. Increased expression of flavanols, anthocyanins, flavonoids, dihydroflavonol, isoflavones, and dihydroflavones was observed, including pelargonidin 3-O-glucoside (callistephin), cyanidin O-syringic acid, (+)-gallocatechin, quercetin 3,7-bis-O-β-D-glucoside, chrysoeriol 5-O-hexoside, cyanidin 3-O-galactoside, cyanidin 3-O-glucoside (kuromanin), spiraeoside, quercetin 3-O-glucoside (isotrifoliin), and hesperetin 5-O-glucoside (Figs. 2 A, S2 and Table S2 ). Twenty-five metabolites increased significantly by 80% in ‘RMHC’ compared with ‘SR’ (21 of 25) during the S3 period (Figs. 2 B, S2, Table S2 ). The upregulated metabolites included flavanols, anthocyanins, flavonol, dihydroflavonol, isoflavones, and dihydroflavone. Pelargonidin 3-O-glucoside (callistephin), cyanidin O-syringic acid, (+)-gallocatechin, quercetin 3, 7-bis-o-β-d-glucoside, chrysoeriol 5-O-hexoside, cyanidin 3-o-galactoside, cyanidin 3-O-glucoside (juromanin), spiraeoside, quercetin 3-O-glucoside (isotrifoliin), and hesperetin 5-O-glucoside were metabolites with significant content differences (Figs. 2 , S2 and Table S2 ). As shown in Fig. 4 , quercetin 3, 7-bis-o-β-d-glucoside, (+)-gallocatechin, cyanidin O-syringic acid, chrysoeriol 5-O-hexoside, and pelargonidin 3-O-glucoside (callistephin) increased in the two groups of differentially expressed metabolites. These substances may be the reason for the red flesh in ‘RMHC’ compared to ‘SR’. 3.3. Transcriptome data analysis of the red flesh To investigate the key genes involved in forming red flesh, and to determine why the bud variant ‘RMHC’ is preferred to ‘SR’ peach for early red flesh, we sequenced the transcriptome of the two varieties during the four periods. The results showed that the number of clean reads in each sample was 4.5–7.7 × 10 7 , the base error rate was low (0.3%), and the Q20 and Q30 ratios were 97.09–97.66% and 92.18–93.33%, respectively. The sum of the number of G and C bases to the total number of bases was 0.3%. The ratio of the number of G and C bases to the total number of bases was > 45% (Table S3 ), indicating that the transcriptomic data were of good quality ensuring accurate and reliable results. In addition, the Pearson’s correlation coefficients ( R 2 ) were > 0.8, indicating that the three samples in the groups were reproducible (Fig. 3 A). The principal component analysis results showed that the samples within a group clustered together, while samples between groups were relatively dispersed, indicating that the samples within a group were well reproduced, there were significant differences between the groups, and the sample data were highly reliable (Fig. 3 B). 3.4. Differentially expressed genes in ‘SR’ and ‘RMHC’ We compared the transcriptomes of the four ‘SR’ and ‘RMHC’ developmental stages to identify the DEGs during peach fruit development. The p-value was corrected according to FDR < 0.05 and |log2 (fold-change) ≥ 2.0. The FPKM expression matrix of each sample was obtained by concatenating the DEGs of each combination, and the results showed that the S2 period (RF2) and the S3 period (RF3) of ‘RMHC’ clustered, the S4 period (RF4) of ‘RMHC’ clustered with the S4 period (CK4) of ‘SR’ aggregated, while the S1–S3 periods (CK1, CK2, and CK3) of ‘SR’ collected during S1 of ‘RMHC’ (RF1). In general, the DEGs of each combination were divided into two bundles, the S2–S4 period of ‘RMHC’, the S4 period of 'SR' with the S1 period of ‘RMHC’, and the S1–S3 period of ‘SR’ (Fig. 4 A, Table S4 ). Totals of 2,106, 1,066, 2,018, and 2,253 genes were identified as DEGs during the four developmental stages (Fig. 4 B, Table S4 ). The DEGs analysis among the eight combinations in this experiment revealed that CK4_vs._CK1 differed by 4,631, which was the highest among all groups, with 2,513 up and 2,118 downregulated genes, respectively; followed by RF3_vs._CK1 and RF4_vs._CK1 with 4,343 and 3,631 DEGs, respectively (Fig. 4 B, Table S4 ). The numbers of DEGs for CK2_vs._CK1, RF2_vs._CK2, RF2_vs._RF1, and RF3_vs._RF2 were low, indicating that metabolism was relatively slow before the flesh became red and ripe, and only a few DEGs were present during the different periods (Fig. 4 B, Table S4 ). This result suggests that expression of the relevant DEGs may be the reason why ‘RMHC’ is redder and ripens earlier than ‘SR’. A two-by-two comparison of all samples was made, and the DEGs were enriched in KEGG pathways (Table S5 ). The enriched pathways involved flavonoid biosynthesis, hormone signal transduction, nitrogen metabolism, phenylpropanoid biosynthesis, phenylalanine metabolism, tyrosine and tryptophan biosynthesis, plant-pathogen interactions, and amino sugar and nucleotide sugar metabolism (Table S5 ). To explore the reasons for the formation of red flesh, we analyzed the genes involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, and the phenylalanine metabolic pathway which were expressed at high levels during the same period in ‘SR’ and ‘RMHC’. As shown in Supplementary Figure S5 , the ‘SR’ S3 and S4 periods were involved in flavonoid and phenylalanine metabolism compared to the previous periods (Figure S3 ). Thus, the genes that increased in the flavonoid and phenylalanine metabolic pathways may be critical for red flesh formation. 3.5. The MBW complex and structural gene mining for the formation of red flesh The synthesis of flavonoids and the phenylpropane metabolic pathway are controlled by a variety of structural genes and regulated by the MBW complex. The transcript levels of 104 MYB, 83 bHLH, and 49 WD40 transcription factors changed with the pulp coloration phenotype in this experiment. Among them, the difference in the MYB, bHLH, and WD40 transcript levels was relatively clear from the heatmap, particularly the comparison between the red flesh and non-red flesh samples (Figure S4 , Table S6 ). Studies in the model plant Arabidopsis thaliana and horticultural crops indicate that the function of the MBW complex in anthocyanin biosynthesis is mainly controlled by MYB (Zhou et al. 2016 ). Therefore, studies should focus on the MYB transcription factors involved in the regulation of anthocyanin biosynthesis in the flavonoid pathway of peach flesh during red flesh formation. Among the various MYB transcription factors, the expression level of PpMYB75 during the ‘RMHC’ S3 phase was significantly higher than that in ‘SR’, indicating that PpMYB75 may play an important role in the formation of the red pulp. Structural genes with significantly higher expression levels may also be involved in the production of red meat by ‘RMHC’ and ‘SR’. Therefore, we screened 31 structural genes from the DEGs annotated to 14 enzymes in the flavonoid synthetic pathway, including 2 PpPAL, 14 Pp4CL, 1 PpC4H, 1 PpCHS, 2 PpCHI, 4 PpF3H, 15 PpF3'H, 1 PpF3 '5'H, 3 PpDFR, 5 PpANS, 4 PpFLS, 1 PpLDOX, 52 PpOMT, and 6 PpUFGT genes (Table S6 ). Further analysis revealed that the structural genes PpPAL1 (18772065), PpPAL2 (18784865), Pp4CL1 (18786755), Pp4CL2 (18770881), Pp4CL3 (18781615), PpC4H (18772818), PpCHS (18766147), PpCHI (18784995), PpF3'H (18777306), PpDFR (18788884), PpANS1 (18778549), PpANS2 (187895899), PpLDOX (18777055), PpOMT1 (18783409), PpOMT2 (18789539), PpOMT3 (18771033), and PpUFGT (18785045) were expressed at high levels in red flesh (Fig. 5 , Table S6 ). Therefore, we speculate that they may be essential in the formation of red pulp in ‘RMHC’ and ‘SR’. 3.6. Fluorescent RT-qPCR validation of the DEGs We performed RT-qPCR to verify whether PpMYB75 and the structural genes were essential for the formation of red pulp during the S3 period. The results showed that expression of the PpMYB75 transcription factor and the structural genes PpLDOX , PpDFR , PpC4H , PpCHS, PpOMT , PpANS , and PpUFGT increased significantly in ‘RMHC’ compared to ‘SR’ (Fig. 6 A). In general, the changes in the genes in the RT-qPCR data were consistent with the trend in the transcriptome, which was higher in red meat than in non-red meat. These changes in PpMYB75 and the structural genes were directly involved in the production of red fruit meat. Transcription factors are usually localized in the nucleus to exert their regulatory effects. RFP fluorescence was detected in the nucleus of cells containing the PpMYB75::RFP fusion gene, indicating that the PpMYB75 transcription factor was localized to the nucleus, and suggesting that it may be involved in transcriptional regulation (Fig. 6 C). 3.7. The PpMYB75-PpDFR regulatory network may be important in the redness of ‘RMHC’ compared to ‘SR’ peach Considering the role of the PpMYB75 TF in red flesh, PpMYB75 could affect the accumulation of anthocyanins by regulating the expression of structural genes. A prediction of the interactions between genes revealed the possibility of interactions between PpMYB75 and these structural genes (Fig. 6 B). We selected three genes ( PpDFR , PpCHS , and PpLDOX ) with a high possibility of interaction. Promoter analysis showed that only PpDFR contained the PpMyB75-bound cis-acting element CCGTTG (Fig. 7 A). To investigate the interactions between the PpMYB75 transcription factor and the PpDFR promoter, the amplified PpMYB75 transcription factor was ligated to the pGAD424 vector, the PpDFR promoter sequence was ligated to the pHIS2 vector, respectively, and the yeast strains were transformed. As shown in Fig. 7 B, the PpMYB75 and PpDFR promoter combinations grew on deficient media (SD/-Leu/-Ura) containing 90 mM 3-AT, while the PpDFR promoter and pGAD424 vector combinations in colonies containing 90 mM 3-AT did not. These results show that PpMYB75 was bound to the PpDFR promoter. To further demonstrate binding of PpMYB75 to PpDFR , PpMYB75 was ligated to the PET32a vector, and the protein was induced and purified to obtain the PpMYB75 protein with a HIS tag. Approximately 30 bp of the PpDFR promoter region with the possible PpMYB75 binding site was labeled with biotin, and an EMSA was performed. The results showed that PpMYB75 was bound to the PpDFR gene cis-acting element CCGTTG (Fig. 7 C). However, binding disappeared when the CCGTTG sequence mutated to GGAGGT, indicating that binding was specific. To explore the effect of PpMYB75 on PpDFR transcriptional activation, PpMYB75 was ligated to the pGreenII 62-SK expression vector, and a LUC vector was ligated to the PpDFR promoter. After transforming the plasmid into A. tumefaciens , the tobacco was transiently injected with the infiltrate and left for 2–3 days. The luminescence intensity of the tobacco injection site was measured by in vivo fluorescence imaging, and dual luciferase activity was determined. As results, the transcription factor combination of PpMYB75 on PpDFR co-injected tobacco activated fluorescence by increasing the luminescence density after the co-injection compared to the control (Fig. 7 D). This result indicates that PpMYB75 activated PpDFR expression. PpMYB75 affected the formation of ‘RMHC’ by influencing the expression of structural-related genes, such as PpDFR . 4. Discussion 4.1. The higher content of flavonoids may confer the ‘RMHC’ peach red characteristics Fruit color is an important trait of peaches, as it directly affects their market competitiveness. Anthocyanin, also known as anthocyanidin, is a polyphenolic water-soluble plant pigment widely found in flowers, fruits, leaves, stems, and seeds that determines fruit color (Zhou et al. 2015 ). Apples, grapes, oranges, pears, and peaches accumulate abundant anthocyanosides in ripe fruits (Yan et al. 2021 ). Therefore, the color change produced by accumulating anthocyanin is an important indicator of fruit ripeness. In our study, ‘RMHC’ accumulated more anthocyanin than ‘SR’ during the same period regardless of skin or flesh, particularly in the flesh. The accumulation of higher anthocyanoside content during the same period compared to ‘SR’ was an important factor in the peach flesh of ‘RMHC’. Many anthocyanins affect fruit color and participate in fruit growth and development, such as cyanidin O-syringic acid, cyanidin 3-O-galactoside, and pelargonidin 3-O-glucoside (LaFountain and Yuan 2021 ). We investigated the variations in 79 flavonoids detected in ‘RMHC’ and ‘SR’. As results, the contents of the anthocyanins pelargonidin 3-O-glucoside, cyanidin O-syringic acid, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside were significantly higher in the pulp of ripe ‘RMHC’ than ‘SR’ (S3 period). Wang et al. showed that the accumulation of pelargonidin 3-O-glucoside determines the bright red color of peony flowers (Wang et al. 2023 ). High levels of cyanidin O-syringic acid were associated with reddening of the fruit skin in a study on yellow cherry fruit color (Ji et al. 2021 ). Red-fleshed kiwifruit fruit accumulates cyanidin 3-O-galactoside and cyanidin 3-O-xylo-galactoside (Montefiori et al. 2011 ). (Es-Safi et al. 2002 ) demonstrated that the red compound formed in the presence of cyanidin 3-O-glucoside glycoside is more stable. Therefore, pelargonidin 3-O-glucoside, cyanidin O-syringic acid, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside may be responsible for the red flesh of ‘RMHC’ than ‘SR’. Flavonoids are a class of secondary metabolites widely distributed in plants, and up to several hundred flavonoids have been detected (Tanaka et al. 2008 ). Flavonoids have been divided into yellow chalcones, orange ketones, flavonols and flavones, red to blue anthocyanidins, and brown proanthocyanidin oxidation products. The contents of flavonoids (quercetin 3,7-bis-O-β-D-glucoside, gossypitrin, and spiraeoside) and flavanols, such as (+)-catechin, and flavonoids (chrysoeriol 5-O-hexoside and tricetin), were significantly higher in our study. We speculate that these substances are indirectly related to the formation of flesh color and early ripening of ‘RMHC’. 4.2. Increased expression of flavonoid metabolism-associated genes may be related to flavonoid content and the red flesh in peaches Flavonoids, as secondary metabolites of the phenylalanine pathway, have been well-studied as key enzymes of the synthetic pathway, such as DFR, C4H, CHS, F3H, and LDOX (Cao et al. 2019 ; Sunil and Shetty 2022 ). The function of these structural genes in peach is a major determinant of differences in peach flesh color (Karak 2019 ). In addition, the structural genes encoding flavonoid synthesis-related enzymes determine the fleshy fruit ripening process, which is crucial for the quality and storage capacity of the fruit (Albert et al. 2014 ; Liu et al. 2019b ; Xu et al. 2014 ). DFR is a key enzyme downstream of the anthocyanin biosynthetic pathway. DFR catalyzes the production of geranoside, cornflower, and delphinidin (Springob et al. 2003 ). C4H has high activity in all plant tissues, C4H is a key enzyme in the formation of anthocyanidins in the phenylpropanoid metabolic pathway (Millar et al. 2007 ). CHS is an enzyme in the biosynthetic pathway of flavonoids, such as anthocyanins, flavonoids, and flavonols, that catalyzes the production of yellow tetrahydroxychalcone (Tanaka et al. 2008 ). In our study, 31 structural genes from the DEGs annotated to 14 enzymes in the flavonoid synthetic pathway were analyzed. Transcriptomic and RT-qPCR analyses indicated that PpLDOX / PpDFR / PpC4H / PpCHS increased more significantly in ‘RMHC’ than ‘SR’. Thus, the high expression of PpLDOX / PpDFR / PpC4H / PpCHS may be the main factor for the redder flesh of ‘RMHC’ than ‘SR’. 4.3. PpMYB75 may cause the red flesh of ‘RMHC’ by mediating the expression of PpDFR Numerous studies have shown that MYB, bHLH, and WD repeat proteins are closely associated with the accumulation of secondary metabolites in the flavonoid synthetic pathway (Ying et al. 2019 ). Among them, R2R3-MYBs play a key role in determining the color change in peach pulp (Yan et al. 2021 ). The involvement of MYB transcription factors in the formation mechanism of peach flesh color has been widely reported, and several transcription factors in this family, such as MYB1, MYB3, MYB10.1, MYB18, MYB19, MYB20, and MYB39, have been implicated in the peach flesh red phenotype (Tuan et al. 2015 ; Khan et al. 2022b ). The current study reveals that MYB transcription factors function by binding to promoter regions of structural genes (Liu et al. 2018 ). Our study shows that the transcription levels of many MYB, bHLH, and WD40 transcription factors were altered. Fortunately, the PpMYB75 transcription factor was transcribed at high levels in red pulp. Thus, we speculate that PpMYB75 may be closely related to the formation of ‘RMHC’ red flesh. We demonstrated that PpMYB75 binds to the promoter region of PpDFR and activate its expression. Therefore, these findings led to the hypothesis that PpMYB75 is involved in ‘RMHC’ red pulp formation as a transcriptional activator of PpDFR . The function of MYB75 in several species has been characterized, in Arabidopsis , MYB75 is an important regulator involved in pigment accumulation (Kreynes et al. 2021 ), and our study appears to be the first to determine that MYB75 is associated with the regulation of the flavonoid synthesis in peaches. 5. Conclusions Red flesh is the main distinguishing feature of ‘RMHC’. This study determined by metabolomics analysis that the increase in flavonoid (anthocyanins, flavonols, and flavanols) contents was related to the red pulp of ‘RMHC’. The significantly increased expression of some transcription factors, particularly PpMYB75, and several structural genes, such as PpDFR , PpCHS , PpC4H , and PpLDOX , may play an important role in the formation of red flesh in ‘RMHC’. The transcription factor PpMYB75 localized to the nucleus and activated PpDFR expression by binding to the cis-acting element CCGTTG in the PpDFR promoter region. In conclusion, the increased expression of PpMYB75 and some other structural genes ( PpDFR , PpCHS , PpC4H , and PpLDOX ) in the flavonoid synthetic pathway contributed to the red flesh of ‘RMHC’. Moreover, the PpMYB75- PpDFR module may be the main cause of red pulp formation in ‘RMHC’ shoot variants compared to ‘SR’. Declarations Supplementary Materials: The following supporting information can be downloaded at: Acknowledgments: We thank Bioeditas Technology Corporation (Shaanxi, China) for helping with the transcriptome sequencing analysis and Metware Company for helping with the metabolome analysis. Author Contributions: methodology, XZ; validation, ZL, MC, and YY; formal analysis, ZL, SW, and MS; resources, XX; data curation, CX; writing—original draft preparation, CX; writing—review and editing, XZ; supervision, XZ; project administration, LQ; funding acquisition, WH. All authors have read and agreed to the published version of the manuscript. Funding: This study was financially supported by the National Natural Science Foundation of China (32302508), the Natural Science Foundation of Shandong Province (No. ZR2021QC203), the Basic Research Project of Science, the Education and Industry Integration Pilot Project (2022PY064), Key Innovation Project (2022JBZ01-06) of Qilu University of Technology (Shandong Academy of Sciences), and Development Plan of Youth Innovation Team in Colleges and Universities of Shandong Province 2022KJ127. Data Availability Statement: Data are available on request to the corresponding author. The transcriptome sequencing has been deposited under BioProjectin NCBI. The accession number for these SRA data is PRJNA978508. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Conflicts of Interest: The authors declare no conflicts of interest. 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Identification of the DEGs during ‘SR’ and ‘RMHC’ fruit development at four developmental stages. TableS5.xlsx Table S5. Enrichment of the DEGS in the KEGG pathways at the four developmental stages. TableS6.xlsx Table S6. Structural genes transcriptional expression levels during the four different periods. SupplementaryFigures.doc Cite Share Download PDF Status: Published Journal Publication published 09 Jan, 2024 Read the published version in Journal of Plant Research → Version 1 posted Reviewers agreed at journal 25 Sep, 2023 Reviewers invited by journal 25 Sep, 2023 Editor assigned by journal 23 Sep, 2023 First submitted to journal 22 Sep, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-3378595","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":235995845,"identity":"6c4227d4-7fb3-406a-9f37-873202146ca2","order_by":0,"name":"Chao Xu","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Xu","suffix":""},{"id":235995846,"identity":"a4b65cd9-9048-4699-bd00-78570945ca0b","order_by":1,"name":"Xiaomin Xue","email":"","orcid":"","institution":"Shandong Institute of Pomology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaomin","middleName":"","lastName":"Xue","suffix":""},{"id":235995847,"identity":"4c8ea481-8e3c-432a-b4df-57c7c7431f46","order_by":2,"name":"Zhixing Li","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhixing","middleName":"","lastName":"Li","suffix":""},{"id":235995848,"identity":"ddba3a26-285f-4c64-96e6-cdeb39ec4cb1","order_by":3,"name":"Mingguang Chen","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingguang","middleName":"","lastName":"Chen","suffix":""},{"id":235995849,"identity":"f3c84076-1919-486b-9949-176902f8a98f","order_by":4,"name":"Siyu Wang","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Siyu","middleName":"","lastName":"Wang","suffix":""},{"id":235995850,"identity":"a4cc0476-6045-488f-8a68-14d7d934803d","order_by":5,"name":"Yating Yang","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yating","middleName":"","lastName":"Yang","suffix":""},{"id":235995851,"identity":"8943e36b-be70-4cda-b350-82f136c94d12","order_by":6,"name":"Mingrui Shang","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingrui","middleName":"","lastName":"Shang","suffix":""},{"id":235995852,"identity":"e480d79f-821e-49bf-841d-b5e4508bf019","order_by":7,"name":"Lei Qiu","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Qiu","suffix":""},{"id":235995853,"identity":"9886da44-fe65-44fe-bfcb-f5aec0811471","order_by":8,"name":"Xianyan Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYDACCRBhAGYyPmAoANEJxGthNoAwiNICAWwSRGmRn9388DFPgV2ewfGzxyreGBxm4GfPMWD4uQO3FsY5x4wNZxgkFxucyUu7OQeoRbLnjQFj7xncWpglEswkPhgwJ244kGN2mweoxeBGjgEzYxtuLWwS6d8kEgzqEzecf2NWDNJiT0gLj0QOyJbDiRtu5Jgxg22RIKBFQiKnGOiX44kzb7wxlpxjkM4jceZZwcFePFrkZ6RvfMzzpzqx73yO4Yc3FdZy/O3JGx/8xKMFDhQOgJwJRgwMB4jQALSugQGqfhSMglEwCkYBGgAAmBpONYul/qkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1268-2737","institution":"Qilu University of Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xianyan","middleName":"","lastName":"Zhao","suffix":""},{"id":235995854,"identity":"bcc4342d-14f7-4047-9b23-588ceddab0f9","order_by":9,"name":"Wenxiao Hu","email":"","orcid":"","institution":"Qilu University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenxiao","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2023-09-23 06:00:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3378595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3378595/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10265-023-01512-1","type":"published","date":"2024-01-09T15:00:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":44032640,"identity":"a0344cfc-627f-4129-88ba-b1c0519e231c","added_by":"auto","created_at":"2023-10-03 17:30:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":606585,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype and anthocyanin contents of ‘SR’ and ‘RMHC’.\u003c/p\u003e\n\u003cp\u003e(A) Color morphology of ‘SR’ and ‘RMHC’ during peach fruit development. Fruits were collected at four developmental stages: S1, S2, S3, and S4, on days 90, 95, 100, and 105 after full bloom. (B) Changes in anthocyanin content of ‘SR’ and ‘RMHC’ in pericarp during different periods. (C) Changes in anthocyanin content of ‘SR’ and ‘RMHC’ in flesh during different periods. The same \u003cem\u003eP\u003c/em\u003e\u003csub\u003e0.05\u003c/sub\u003e level letter indicates no difference, while different\u003cem\u003e P\u003c/em\u003e\u003csub\u003e0.05\u003c/sub\u003e levels indicate differences. The error bars indicate the SE of three biological replicates.\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/161e47aa7444940e15116e9e.png"},{"id":44032638,"identity":"fa18fe36-8b7c-4181-a6b1-fe3e9e0861b1","added_by":"auto","created_at":"2023-10-03 17:30:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":175501,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of intermediate products of the flavonoid metabolic pathway. (A) Histogram of flavonoid levels during the S2 (RF2) and S3 periods (RF3) of ‘RMHC’. (B) Histogram of flavonoid levels during the S3 period of ‘SR’ (CK3) vs. the S3 period of ‘RMHC’ (RF3).Error bars indicate the SE of three biological replicates, while * indicates a significant difference at \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 by Student’s \u003cem\u003et\u003c/em\u003e-test, while ** indicates significant difference at \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/0d37f00d8cdab82693cc0680.png"},{"id":44033420,"identity":"a55df7e1-4610-467c-b599-daf6bd49084b","added_by":"auto","created_at":"2023-10-03 17:38:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":420384,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential expression analysis between samples. (A) Pearson’s correlation analysis between samples. (B) Principal component analysis between samples.\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/46256b3fc20741558627fd6d.png"},{"id":44035056,"identity":"4b18ee31-3b79-4108-b94f-e4417fb46373","added_by":"auto","created_at":"2023-10-03 17:46:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":384230,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs analysis between samples in different periods. (A) Cluster analysis of the ‘SR’ and ‘RMHC’ DEGS during the four periods. (B) DEGS comparison between the groups.\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/1e184aa87598c7613e6d21d9.png"},{"id":44032639,"identity":"b40c6b40-cdb2-4e91-8378-f3a90c78c434","added_by":"auto","created_at":"2023-10-03 17:30:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":463321,"visible":true,"origin":"","legend":"\u003cp\u003eSynthetic pathway of anthocyanin in ‘SR’ and ‘RMHC’. The green and red colors in the heatmap represent the transcript levels of the structural genes in the anthocyanin pathway of the ‘SR’ and ‘RMHC’ flesh tissues, and the S2 and S3 periods were selected for analysis.\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/bccfd507ec281efd14b28e79.png"},{"id":44033421,"identity":"88636565-7604-42ba-bb45-494c91bec3df","added_by":"auto","created_at":"2023-10-03 17:38:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":537939,"visible":true,"origin":"","legend":"\u003cp\u003e(A)RT-qPCR analysis of the genes. RT-qPCR analysis of \u003cem\u003ePpC4H\u003c/em\u003e, \u003cem\u003ePpMYB75\u003c/em\u003e, \u003cem\u003ePpCHI\u003c/em\u003e, \u003cem\u003ePp4CL\u003c/em\u003e, \u003cem\u003ePpCHS\u003c/em\u003e, \u003cem\u003ePpANS\u003c/em\u003e, \u003cem\u003ePpDFR\u003c/em\u003e, \u003cem\u003ePpF3H\u003c/em\u003e, \u003cem\u003ePpOMT\u003c/em\u003e, \u003cem\u003ePpLDOX\u003c/em\u003e, and \u003cem\u003ePpUFGT\u003c/em\u003e from ‘SR’ and ‘RMHC’ during the S3 period. The same \u003cem\u003eP\u003c/em\u003e\u003csub\u003e0.05\u003c/sub\u003e level letter indicates no difference, while different \u003cem\u003eP\u003c/em\u003e\u003csub\u003e0.05\u003c/sub\u003e levels indicate differences. Error bars show the SE of three biological replicates. (B) Predicting the interactions between proteins and genes. Different colored circles rep-resent different genes or proteins. (C) Subcellular localization analysis. Subcellular lo-calization of the PpMYB75 protein. Laser-scanning confocal-microscopy-observed Ni-cotiana tabacum leaves expressing PpMYB75-RFP. Micrographs show that PpMYB75-RFP was localized in the nucleus. DAPI is a nuclear dye. Scale bar represents 10 μM.\u003c/p\u003e","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/8c47feda265b936a542c7639.png"},{"id":44032644,"identity":"23929d7a-6d8d-4354-9cc9-a71c6da19251","added_by":"auto","created_at":"2023-10-03 17:30:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":424052,"visible":true,"origin":"","legend":"\u003cp\u003e(A) \u003cem\u003ePpDFR\u003c/em\u003epromoter schematic showing potential PpMYB75 binding sites. The black line represents the CCGTTG sequence. The CCGTTG sequence in the mutation probe mutates to GGAGGT. (B) Yeast one-hybrid assay (Y1H) detected the interaction between the \u003cem\u003ePpDFR\u003c/em\u003e promoter and PpMYB75. PGAD424-PpMYB75 and pHIS2-\u003cem\u003ePpDFR\u003c/em\u003epro constructs were co-transformed into yeast Y187. The horizontal axis represents the addition of pHIS2-\u003cem\u003ePpDFR\u003c/em\u003epro, and the vertical axis represents the addition of pGAD424 and PGAD424-PpMYB75. (C) EMSA detected binding of the PpMYB75-\u003cem\u003eHIS\u003c/em\u003e protein to the cis-acting element CCGTTG of the \u003cem\u003ePpDFR\u003c/em\u003epromoter \u003cem\u003ein vitro\u003c/em\u003e. PpMYB75-HIS was combined with each biotin-labeled DNA probe. The CCGTTG sequence mutated to GGAGGT. (D) \u003cem\u003e35Spro\u003c/em\u003e:PpMYB75 and pro\u003cem\u003ePpDFR:LUC\u003c/em\u003e were transiently transformed into tobacco leaves to detect luminescence, and \u003cem\u003e35Spro:62-SK+LUC\u003c/em\u003e, \u003cem\u003e35Spro:62-SK+proPpDFR:LUC\u003c/em\u003e, \u003cem\u003e35Spro:\u003c/em\u003ePpMYB75+\u003cem\u003eLUC\u003c/em\u003e were used as controls.\u003c/p\u003e","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/231b11b4fb9a25d07cc749bb.png"},{"id":49628379,"identity":"20bcbcb0-107b-4494-899c-1f16f322ef87","added_by":"auto","created_at":"2024-01-15 15:03:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2510881,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/daf1731f-ba03-4c0d-80d5-9fe7e929ad1d.pdf"},{"id":44032633,"identity":"f7ea6970-f4b1-43f4-9af7-1cf586bdf94b","added_by":"auto","created_at":"2023-10-03 17:30:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1. \u003c/strong\u003ePrimers used in this study.\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/889ffa131e7c437ddc8484f8.docx"},{"id":44033419,"identity":"5b1f050f-45b3-4cce-9059-02bc038314f8","added_by":"auto","created_at":"2023-10-03 17:38:55","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":79091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2. \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003e79 flavonoid metabolites detected in peaches (RF2, RF3, and CK3). S2 period of ‘RMHC’ (RF2), S3 period of ‘RMHC’ (RF3), S3 period of ‘SR’ (CK3).\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/72e129fd821dc246c1965f2b.xlsx"},{"id":44032635,"identity":"9b6d5364-dbab-49ef-bdc7-4223775cc992","added_by":"auto","created_at":"2023-10-03 17:30:55","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3. \u003c/strong\u003eTranscriptome assembly and Illumina HiSeq quality control information. Three biological replicates were used for each sample.\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/22527fa623a949b2ef4770be.xlsx"},{"id":44032646,"identity":"dbc5688e-08a5-43e6-92e7-25dfaf2c2112","added_by":"auto","created_at":"2023-10-03 17:30:56","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3938926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4. \u003c/strong\u003eIdentification of the DEGs during ‘SR’ and ‘RMHC’ fruit development at four developmental stages.\u003c/p\u003e","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/26bce77544dfc33e2ec49ffd.xlsx"},{"id":44032637,"identity":"397c6c2b-12f9-4b85-b796-0568afef686d","added_by":"auto","created_at":"2023-10-03 17:30:56","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":61185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S5. \u003c/strong\u003eEnrichment of the DEGS in the KEGG pathways at the four developmental stages.\u003c/p\u003e","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/a424198340a3ba7186a34c73.xlsx"},{"id":44032643,"identity":"33aefd3b-5366-4727-8b64-4c09f5d6fef4","added_by":"auto","created_at":"2023-10-03 17:30:56","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":137370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S6. \u003c/strong\u003eStructural genes transcriptional expression levels during the four different periods.\u003c/p\u003e","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/e16f136acc2c9ce3f775786b.xlsx"},{"id":44032647,"identity":"90763a35-346f-4c65-961b-bec8aa649d19","added_by":"auto","created_at":"2023-10-03 17:30:56","extension":"doc","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":9157632,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.doc","url":"https://assets-eu.researchsquare.com/files/rs-3378595/v1/ba6321707c23b08ac11ede34.doc"}],"financialInterests":"","formattedTitle":"The PpMYB75-PpDFR module reveals the difference between ‘SR’ and its Bud Variant ‘RMHC’ in Peach Red Flesh","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePeach is a fruit with high commercial and nutritional value that has a wide market demand worldwide (Zhao et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consumers and cultivators have a very intuitive feeling about the coloring of peaches when choosing fruit. Peach varieties have been divided into white, yellow, and red flesh color categories. The type and content of flavonoids, carotenoids, and chlorophyll in the plants are the main reasons for the different colored fruits and leaves (Drogoudi et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Many studies have confirmed that yellow pulp is due to the accumulation of carotenoids, while white pulp has a lower pigment content (Falchi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The red-fleshed peach, which is mainly determined by the accumulation of flavonoids, such as anthocyanins, flavonoids, and flavonols, is a rare peach germplasm resource with important potential value in production and breeding (Cao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, studies on the mechanism for the formation of red peach pulp are significant.\u003c/p\u003e \u003cp\u003eThe flavonoid biosynthetic pathway in peaches as well as other plants is highly conserved (Araguirang and Richter \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The enzymes and related structural genes associated with the flavonoid biosynthetic pathway have been well studied. First, phenylalanine serves as a precursor for the biosynthesis of many plant secondary metabolites, such as flavonols, anthocyanins, and proanthocyanidins. Phenylalanine produces p-coumaroyl coenzyme A by the action of phenylalanine lyase (PAL), cinnamic acid-4-hydroxylase (C4H) and 4-coumaryl CoA ligase (4CL). Subsequently, the product is catalyzed to form dihydroflavonols, which are produced as flavonols catalyzed by flavonol synthase (FLS), and leucoanthocyanidin forms from flavonol under the action of dihydroflavonol 4-reductase (DFR). Leucoanthocyanidin also has two branches, one produces flavan-3-alcohol directly under the action of leucoanthocyantin reducase (LAR), and further polymerizes to produce proanthocyanidins. The other branch produces unstable anthocyanins under the action of leucoanthocyanin dioxygenase (LDOX) or anthocyanin synthase (ANS). Anthocyanins also produce proanthocyanidins under the action of anthocyanin reductase. Finally, through the action of UDP-glycosyltransferase (flavonoid 3-O-glucosyltransferase, UFGT), the free anthocyanins form anthocyanins that exist stably in plants and give different tissues the plants\u0026rsquo; brilliant colors. After being synthesized in the cytoplasm, anthocyanins are transported to vesicles with the participation of glutathione S-transferase (GST) (Liu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Rahim et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe accumulation of flavonoids in plants is regulated by structural genes, MYB, the basic helix-loop-helix (bHLH), and WD40 transcription factors, particularly in Rosaceae species, such as peaches, apples, and strawberries (Lin-Wang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These transcription factors form the MBW complex and bind to the promoters of the structural genes in the flavonoid synthetic pathway, which together regulate the metabolism of flavonoid pathway products (Broucke et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Dare et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). bHLH transcription factors play an important role in the responses to the external environment, such as light, temperature, and nutrients, and affect flavonoid levels (Feller et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Some studies have shown that WD40 proteins are not necessary for regulating flavonoid synthesis, and their main role may be in the response to external signals (Feng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, other studies have shown that the diversity of MBW functions is due to MYB, rather than the bHLH or WD40 transcription factors (Chagn\u0026eacute; et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hui et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs one of the largest families of transcription factors in plants, the MYB family plays an important role in the regulation of plant secondary metabolism (Seo and Kim \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). MYB is also involved in anthocyanin synthesis in fruits, including grapes (Kobayashi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), apples (An et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), pears (Feng et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), bayberries (Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). MYB plays an important role in the formation of anthocyanins in peaches. Three MYB transcription factors involved in flavonoid biosynthesis in peach fruit have been reported, including \u003cem\u003ePpMYB10.1\u003c/em\u003e, \u003cem\u003ePpMYB10.2\u003c/em\u003e, and \u003cem\u003ePpMYB10.3\u003c/em\u003e (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). In addition, anthocyanins accumulate in green peach leaves when PpMYB10.4 is transiently expressed (Zhou et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Peace, an R2R3 MYB transcription factor in peach flowers, promotes the accumulation of anthocyanins in petals, making the peach petals pink (Uematsu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). PpMYB39 binds to the \u003cem\u003ePpDFR\u003c/em\u003e promoter and activates its expression to affect the formation of red flesh (Khan et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). PpMYB108 is closely related to the biosynthesis of peach anthocyanosides and is involved in the formation of red flowers (Khan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Taken together, MYB transcription factors play an essential role in regulating the accumulation of flavonoids, particularly anthocyanins.\u003c/p\u003e \u003cp\u003eThe identification of MYB-specific regulatory genes can guide the development of peach anthocyanins and color-related markers, and provide a basis for the identification, selection, and breeding of peach varieties. In this study, we compared flavonoids of the \u0026lsquo;Super Red\u0026rsquo; (SR) peach variety and its bud variant \u0026lsquo;Red Meat Honey Crisp\u0026rsquo; (RMHC). The \u0026lsquo;RMHC\u0026rsquo; variant accumulated more flavonoids in fruit than \u0026lsquo;SR\u0026rsquo;, particularly in the flesh during the same period. Because of the increase in flavonoids during fruit development, 79 flavonoid metabolites were identified by metabolomics analysis. These flavonoids, particularly anthocyanins, may be a key factor in the redder flesh of \u0026lsquo;RMHC\u0026rsquo; than \u0026lsquo;SR\u0026rsquo;. Then, transcriptome analysis and RT-qPCR identified key genes, such as \u003cem\u003ePpDFR\u003c/em\u003e, \u003cem\u003ePpCHS\u003c/em\u003e, \u003cem\u003ePpC4H, PpLDOX\u003c/em\u003e, and PpMYB75 which significantly increased during the formation of red flesh. Finally, our study showed that PpMYB75 localizes in the nucleus and we confirmed binding to the \u003cem\u003ePpDFR\u003c/em\u003e promoter region to activate its expression. The PpMYB75-\u003cem\u003ePpDFR\u003c/em\u003e regulatory pathway of flavonoid biosynthesis may contribute to the red flesh in peach \u0026lsquo;RMHC\u0026rsquo; compared to \u0026lsquo;SR\u0026rsquo;. This study provides new insight into the formation of the red peach color.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant materials\u003c/h2\u003e \u003cp\u003eThe fresh fruits of \u0026lsquo;RMHC\u0026rsquo; and \u0026lsquo;SR\u0026rsquo; used in this study were obtained from the experimental base of Shandong Institute of Pomology, Tai 'an, China. Peach samples and three biological copies were collected at different stages on days 90 (S1), 95 (S2), 100 (S3), and 105 (S4) of fruit development after flowering. The fruit was randomly collected at about 2 m above the ground around the crown of the tree, and samples of uniform size and integrity were selected. The peach samples were cut into small pieces and immediately frozen in liquid nitrogen, and then the samples were selected for metabolomics and transcriptome sequencing analyses. S1 period of \u0026lsquo;SR\u0026rsquo; (CK1), S2 period of \u0026lsquo;SR\u0026rsquo; (CK2), S3 period of \u0026lsquo;SR\u0026rsquo; (CK3), S4 period of \u0026lsquo;SR\u0026rsquo; (CK4), S1 period of \u0026lsquo;RMHC\u0026rsquo; (RF1), S2 period of \u0026lsquo;RMHC\u0026rsquo; (RF2), S3 period of \u0026lsquo;RMHC\u0026rsquo; (RF3), S4 period of \u0026lsquo;RMHC\u0026rsquo; (RF4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Flavonoid and anthocyanin measurements\u003c/h2\u003e \u003cp\u003eFlavonoid content was determined as described previously (Sun et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Anthocyanin content was determined according to a previous method (Hu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. Differences between the two groups were detected by one-way analysis of variance and Tukey\u0026rsquo;s multiple comparison test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Detection and analysis of the metabolomics profile\u003c/h2\u003e \u003cp\u003eThe UPLC-ESI-MS/MS analysis (Metware Co., Wuhan, China) was conducted by a commercial facility. The sample was immersed in liquid nitrogen and ground to a powder with a grinder (MM400, Retsch). A 100 mg portion of powder was dissolved in 70% methanol, centrifuged at 4\u0026deg;C and 12,000 rpm for 10 min, and filtered through a 0.22 \u0026micro;m microporous membrane. All samples were analyzed three times using the UPLC-Qtrap-MS system (SHIMADZU Nexera X2/Applied Biosystems 4500 QTRAP; Tokyo, Japan). The analysis was performed with an Agilent SB-C18 column (2.1 \u0026times; 100 mm, 1.8 \u0026micro;m). The mobile water phase was ultrapure water containing 0.04% acetic acid, and the mobile organic phase was acetonitrile containing 0.04% acetic acid. The elution gradient was water/acetonitrile (95:5, v/v) for 10 min, 5:95 for 11 min, 5:95 for 12 min, 95:5 for 12.1 min, and 95:5 for 15 min. The flow rate was 0.4 mL/min, the column temperature was held at 40\u0026deg;C, and the injection volume was 5 \u0026micro;l. The raw data were loaded into the MWDB METWARE database to identify the metabolites. Then, qualitative and quantitative analyses of metabolites were performed using Analyst 1.6.3 software. Then, combined with the grouping of specific samples, the fold-changes (fold-change\u0026thinsp;\u0026ge;\u0026thinsp;2 and fold-change\u0026thinsp;\u0026le;\u0026thinsp;0.5 were significantly different) in the quantitative information of the metabolites were compared between the groups. The variable importance in projection (VIP) value of the partial least squares-discriminant analysis model (VIP\u0026thinsp;\u0026ge;\u0026thinsp;1 was a significant difference) was combined to screen the differentially expressed metabolites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. RNA extraction and library construction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 24 fresh peach samples using the Total RNA Rapid Extraction kit (Zomanbio, Beijing, China). The purity, concentration, and integrity of the RNA samples were detected by Nanodrop, Qubit 2.0, and Agilent 2100 instruments, respectively. A 20 \u0026micro;g portion of RNA was isolated from each sample for sequencing and to construct the RNA-seq library. The first and second cDNA strands were synthesized using a cDNA synthesis kit (TOYOBO, Osaka, Japan) following the manufacturer\u0026rsquo;s instructions. The cDNA products were stored temporarily at \u0026minus;\u0026thinsp;20\u0026deg;C and for the long term at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Transcriptome sequencing and data analysis\u003c/h2\u003e \u003cp\u003eTranscriptome sequencing was performed by a commercial company (Novogene, Beijing, China).\u003c/p\u003e \u003cp\u003eThree replicates per sample and 24 peach pulp samples were used to construct the library. Total RNA was extracted and assayed as described in Section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e. Qualified DNA samples were randomly interrupted with the Covaris ultrasonic crusher, and the whole library was prepared by terminal repair, A-tail addition, sequencing joint addition, purification, PCR amplification and other steps. The libraries were sequenced with an Illumina sequencer. Subsequently, the different libraries were pooled according to the effective concentration and target downstream data volume required for Illumina sequencing. Raw image data files obtained by high-throughput sequencing were analyzed by CASAVA (Base Calling) and converted into original sequencing sequences, namely Raw Reads. Illunima\u0026rsquo;s base quality values were expressed as Qphred, and the GC content was counted. The raw reads were filtered to obtain clean reads, and subsequent analyses were based on the clean reads (Jiang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The clean reads were mapped to a reference genome sequence and annotated against the reference genome. The HISAT2 tool was used to map the reference genome, and gene transcript levels were calculated using the FKPM fragment mapping method. Differential expression analysis was performed on both samples using edge R with FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 as thresholds for significant differential expression. Differentially expressed genes (DEGs) were identified using edge R, and gene functions were annotated according to the KEGG database. The enrichment of DEGs in the KEGG pathway was tested using KOBAS software (Frazee et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Prunus_persica_GCF_000346465.2 V2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000346465.2/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000346465.2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used as the reference genome.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Quantitative reverse transcription polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eRT-qPCR was performed as described previously (Zhao et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We used the NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) and obtained the complete coding region of the selected DEGs. The primers were designed and synthesized at Sangon Biotech\u0026rsquo;s website (Shanghai, China), and the previously obtained cDNA template was used as the template for RT-qPCR. The data were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. We used the TEF2 gene as an internal reference gene for this experiment. Three biological and technical replications were run. All primers used in this experiment are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Subcellular localization analysis\u003c/h2\u003e \u003cp\u003ePrimers were designed according to the PpMYB75 coding region, and the PpMYB75 open reading frame (ORF) was amplified from the pulp tissue of \u0026lsquo;RMHC\u0026rsquo; peaches using PCR with the cDNA library as the template. Then, the \u003cem\u003ePpMYB75\u003c/em\u003e ORF was inserted into the PRI-RFP vector to construct the PpMYB75-RFP vector. The PpMYB75-RFP plasmid was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e LBA4404-injected tobacco for subcellular localization. The primers used are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Electrophoretic mobility shift assay (EMSA)\u003c/h2\u003e \u003cp\u003eThe EMSA was performed as described previously (Zhao et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The PpMYB75 coding region was cloned into the pET-32a(+) expression vector, and the PpMYB75-HIS recombinant protein was expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21, and purified with the His Microspin Purification kit (Tiangen). The oligonucleotide probe for the \u003cem\u003ePpDFR\u003c/em\u003e promoter was labeled by Sangon Biotech. Binding specificity was determined by measuring competition with an excess of unlabeled oligonucleotides. The primers used are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Yeast one-hybrid (Y1H) assay\u003c/h2\u003e \u003cp\u003eA yeast one-hybrid assay was performed following the manufacturer\u0026rsquo;s instructions in the MATCHMAKER One-Hybrid System (Clontech, Palo Alto, CA, USA). The \u003cem\u003ePpDFR\u003c/em\u003e promoter and \u003cem\u003ePpMYB75\u003c/em\u003e ORF were inserted into the pHIS2 reporter vector and the PGAD424 vector, respectively. Then, different combinations of the PpMYB75-PGAD424 plasmid and \u003cem\u003ePpDFR\u003c/em\u003e-pHIS2 plasmid were co-transformed into the Y187 strain and screened on Leu-, Trp-, and His-deficient medium supplemented with different concentrations of 3-AT (90 mM) for 3\u0026ndash;4 days. The primers used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Flesh reddening of \u0026lsquo;RMHC\u0026rsquo; and \u0026lsquo;SR\u0026rsquo; peach fruit\u003c/h2\u003e \u003cp\u003eThe most obvious feature of \u0026lsquo;RMHC\u0026rsquo; compared with \u0026lsquo;SR\u0026rsquo; was the red flesh and pericarp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). \u0026lsquo;RMHC\u0026rsquo; and \u0026lsquo;SR\u0026rsquo; showed varying degrees of anthocyanin content in the pericarp and flesh as they approached maturity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The pericarp of \u0026lsquo;SR\u0026rsquo; gradually turned yellow from S1 to S2 with some red, while \u0026lsquo;RMHC\u0026rsquo; exhibited a red color. The \u0026lsquo;RMHC\u0026rsquo; pericarp had higher anthocyanin content than that of \u0026lsquo;SR\u0026rsquo; during the same period, and this difference was particularly evident during S2 and S3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Dark red appeared in the pericarp during S3, while S1 and S2 were accompanied by a light red feature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). As shown by the pericarp, the anthocyanin content of the \u0026lsquo;RMHC\u0026rsquo; and \u0026lsquo;SR\u0026rsquo; flesh also changed significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Once flesh reddening occurred during S1, the \u0026lsquo;RMHC\u0026rsquo; flesh developed rapidly during S2 and the flesh color became deeper during S3 compared to that during S1 and S2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). The \u0026lsquo;SR\u0026rsquo; flesh remained white, while \u0026lsquo;RMHC\u0026rsquo; accumulated some anthocyanin during S2 and S3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). The distinctive characteristic of \u0026lsquo;RMHC\u0026rsquo; was earlier reddening than \u0026lsquo;SR\u0026rsquo;, particularly in the peach flesh. The red flesh mainly contributed by anthocyanin may be why \u0026lsquo;RMHC\u0026rsquo; has advantages, such as early maturation, high yield, and good taste. Therefore, relevant studies on early red flesh were carried out.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.2. Flavonoid component analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe type and content of flavonoids give fruits their different colors, so the flavonoids in peach flesh were analyzed. The flavonoid content in \u0026lsquo;SR\u0026rsquo; peach pulp and \u0026lsquo;RMHC\u0026rsquo; peach flesh increased during the S1\u0026ndash;S2 period, and then began to decrease (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Flavonoid content in \u0026lsquo;RMHC\u0026rsquo; increased significantly during S1\u0026ndash;S4 compared to \u0026lsquo;SR\u0026rsquo;, and content changed significantly during S2\u0026ndash;S3 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Thus, we analyzed the composition of the flavonoids that occurred during S2\u0026ndash;S3 and analyzed the cause of the red flesh.\u003c/p\u003e \u003cp\u003eTo identify the components in red flesh, 79 flavonoid metabolites from 24 peach flesh samples (RF2, RF3, and CK3) were detected in the flavonoid metabolome material database established by the Metware Company with widely targeted metabolomics based on liquid chromatography-tandem mass spectrometry. We prepared a volcano plot by combining the VIP values and the fold-change data to verify the differences in the expression levels and the statistical differences between the metabolites in the two groups (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). As shown in Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, 27 different metabolites were detected in \u0026lsquo;RMHC\u0026rsquo; during S2\u0026ndash;S3, among which 26 metabolites were upregulated. Increased expression of flavanols, anthocyanins, flavonoids, dihydroflavonol, isoflavones, and dihydroflavones was observed, including pelargonidin 3-O-glucoside (callistephin), cyanidin O-syringic acid, (+)-gallocatechin, quercetin 3,7-bis-O-β-D-glucoside, chrysoeriol 5-O-hexoside, cyanidin 3-O-galactoside, cyanidin 3-O-glucoside (kuromanin), spiraeoside, quercetin 3-O-glucoside (isotrifoliin), and hesperetin 5-O-glucoside (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, S2 and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwenty-five metabolites increased significantly by 80% in \u0026lsquo;RMHC\u0026rsquo; compared with \u0026lsquo;SR\u0026rsquo; (21 of 25) during the S3 period (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, S2, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The upregulated metabolites included flavanols, anthocyanins, flavonol, dihydroflavonol, isoflavones, and dihydroflavone. Pelargonidin 3-O-glucoside (callistephin), cyanidin O-syringic acid, (+)-gallocatechin, quercetin 3, 7-bis-o-β-d-glucoside, chrysoeriol 5-O-hexoside, cyanidin 3-o-galactoside, cyanidin 3-O-glucoside (juromanin), spiraeoside, quercetin 3-O-glucoside (isotrifoliin), and hesperetin 5-O-glucoside were metabolites with significant content differences (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, S2 and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e, quercetin 3, 7-bis-o-β-d-glucoside, (+)-gallocatechin, cyanidin O-syringic acid, chrysoeriol 5-O-hexoside, and pelargonidin 3-O-glucoside (callistephin) increased in the two groups of differentially expressed metabolites. These substances may be the reason for the red flesh in \u0026lsquo;RMHC\u0026rsquo; compared to \u0026lsquo;SR\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Transcriptome data analysis of the red flesh\u003c/h2\u003e \u003cp\u003eTo investigate the key genes involved in forming red flesh, and to determine why the bud variant \u0026lsquo;RMHC\u0026rsquo; is preferred to \u0026lsquo;SR\u0026rsquo; peach for early red flesh, we sequenced the transcriptome of the two varieties during the four periods. The results showed that the number of clean reads in each sample was 4.5\u0026ndash;7.7 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e, the base error rate was low (0.3%), and the Q20 and Q30 ratios were 97.09\u0026ndash;97.66% and 92.18\u0026ndash;93.33%, respectively. The sum of the number of G and C bases to the total number of bases was 0.3%. The ratio of the number of G and C bases to the total number of bases was \u0026gt;\u0026thinsp;45% (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), indicating that the transcriptomic data were of good quality ensuring accurate and reliable results.\u003c/p\u003e \u003cp\u003eIn addition, the Pearson\u0026rsquo;s correlation coefficients (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e) were \u0026gt;\u0026thinsp;0.8, indicating that the three samples in the groups were reproducible (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The principal component analysis results showed that the samples within a group clustered together, while samples between groups were relatively dispersed, indicating that the samples within a group were well reproduced, there were significant differences between the groups, and the sample data were highly reliable (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Differentially expressed genes in \u0026lsquo;SR\u0026rsquo; and \u0026lsquo;RMHC\u0026rsquo;\u003c/h2\u003e \u003cp\u003eWe compared the transcriptomes of the four \u0026lsquo;SR\u0026rsquo; and \u0026lsquo;RMHC\u0026rsquo; developmental stages to identify the DEGs during peach fruit development. The p-value was corrected according to FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2 (fold-change)\u0026thinsp;\u0026ge;\u0026thinsp;2.0. The FPKM expression matrix of each sample was obtained by concatenating the DEGs of each combination, and the results showed that the S2 period (RF2) and the S3 period (RF3) of \u0026lsquo;RMHC\u0026rsquo; clustered, the S4 period (RF4) of \u0026lsquo;RMHC\u0026rsquo; clustered with the S4 period (CK4) of \u0026lsquo;SR\u0026rsquo; aggregated, while the S1\u0026ndash;S3 periods (CK1, CK2, and CK3) of \u0026lsquo;SR\u0026rsquo; collected during S1 of \u0026lsquo;RMHC\u0026rsquo; (RF1). In general, the DEGs of each combination were divided into two bundles, the S2\u0026ndash;S4 period of \u0026lsquo;RMHC\u0026rsquo;, the S4 period of 'SR' with the S1 period of \u0026lsquo;RMHC\u0026rsquo;, and the S1\u0026ndash;S3 period of \u0026lsquo;SR\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTotals of 2,106, 1,066, 2,018, and 2,253 genes were identified as DEGs during the four developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). The DEGs analysis among the eight combinations in this experiment revealed that CK4_vs._CK1 differed by 4,631, which was the highest among all groups, with 2,513 up and 2,118 downregulated genes, respectively; followed by RF3_vs._CK1 and RF4_vs._CK1 with 4,343 and 3,631 DEGs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). The numbers of DEGs for CK2_vs._CK1, RF2_vs._CK2, RF2_vs._RF1, and RF3_vs._RF2 were low, indicating that metabolism was relatively slow before the flesh became red and ripe, and only a few DEGs were present during the different periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). This result suggests that expression of the relevant DEGs may be the reason why \u0026lsquo;RMHC\u0026rsquo; is redder and ripens earlier than \u0026lsquo;SR\u0026rsquo;.\u003c/p\u003e \u003cp\u003eA two-by-two comparison of all samples was made, and the DEGs were enriched in KEGG pathways (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The enriched pathways involved flavonoid biosynthesis, hormone signal transduction, nitrogen metabolism, phenylpropanoid biosynthesis, phenylalanine metabolism, tyrosine and tryptophan biosynthesis, plant-pathogen interactions, and amino sugar and nucleotide sugar metabolism (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). To explore the reasons for the formation of red flesh, we analyzed the genes involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, and the phenylalanine metabolic pathway which were expressed at high levels during the same period in \u0026lsquo;SR\u0026rsquo; and \u0026lsquo;RMHC\u0026rsquo;. As shown in Supplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e, the \u0026lsquo;SR\u0026rsquo; S3 and S4 periods were involved in flavonoid and phenylalanine metabolism compared to the previous periods (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Thus, the genes that increased in the flavonoid and phenylalanine metabolic pathways may be critical for red flesh formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. The MBW complex and structural gene mining for the formation of red flesh\u003c/h2\u003e \u003cp\u003eThe synthesis of flavonoids and the phenylpropane metabolic pathway are controlled by a variety of structural genes and regulated by the MBW complex. The transcript levels of 104 MYB, 83 bHLH, and 49 WD40 transcription factors changed with the pulp coloration phenotype in this experiment. Among them, the difference in the MYB, bHLH, and WD40 transcript levels was relatively clear from the heatmap, particularly the comparison between the red flesh and non-red flesh samples (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e, Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). Studies in the model plant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and horticultural crops indicate that the function of the MBW complex in anthocyanin biosynthesis is mainly controlled by MYB (Zhou et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, studies should focus on the MYB transcription factors involved in the regulation of anthocyanin biosynthesis in the flavonoid pathway of peach flesh during red flesh formation. Among the various MYB transcription factors, the expression level of PpMYB75 during the \u0026lsquo;RMHC\u0026rsquo; S3 phase was significantly higher than that in \u0026lsquo;SR\u0026rsquo;, indicating that PpMYB75 may play an important role in the formation of the red pulp.\u003c/p\u003e \u003cp\u003eStructural genes with significantly higher expression levels may also be involved in the production of red meat by \u0026lsquo;RMHC\u0026rsquo; and \u0026lsquo;SR\u0026rsquo;. Therefore, we screened 31 structural genes from the DEGs annotated to 14 enzymes in the flavonoid synthetic pathway, including 2 PpPAL, 14 Pp4CL, 1 PpC4H, 1 PpCHS, 2 PpCHI, 4 PpF3H, 15 PpF3'H, 1 PpF3 '5'H, 3 PpDFR, 5 PpANS, 4 PpFLS, 1 PpLDOX, 52 PpOMT, and 6 PpUFGT genes (Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurther analysis revealed that the structural genes \u003cem\u003ePpPAL1\u003c/em\u003e (18772065), \u003cem\u003ePpPAL2\u003c/em\u003e (18784865), \u003cem\u003ePp4CL1\u003c/em\u003e (18786755), \u003cem\u003ePp4CL2\u003c/em\u003e (18770881), \u003cem\u003ePp4CL3\u003c/em\u003e (18781615), \u003cem\u003ePpC4H\u003c/em\u003e (18772818), \u003cem\u003ePpCHS\u003c/em\u003e (18766147), \u003cem\u003ePpCHI\u003c/em\u003e (18784995), \u003cem\u003ePpF3'H\u003c/em\u003e (18777306), \u003cem\u003ePpDFR\u003c/em\u003e (18788884), \u003cem\u003ePpANS1\u003c/em\u003e(18778549), \u003cem\u003ePpANS2\u003c/em\u003e (187895899), \u003cem\u003ePpLDOX\u003c/em\u003e (18777055), \u003cem\u003ePpOMT1\u003c/em\u003e (18783409), \u003cem\u003ePpOMT2\u003c/em\u003e (18789539), \u003cem\u003ePpOMT3\u003c/em\u003e (18771033), and \u003cem\u003ePpUFGT\u003c/em\u003e (18785045) were expressed at high levels in red flesh (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). Therefore, we speculate that they may be essential in the formation of red pulp in \u0026lsquo;RMHC\u0026rsquo; and \u0026lsquo;SR\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Fluorescent RT-qPCR validation of the DEGs\u003c/h2\u003e \u003cp\u003eWe performed RT-qPCR to verify whether PpMYB75 and the structural genes were essential for the formation of red pulp during the S3 period. The results showed that expression of the PpMYB75 transcription factor and the structural genes \u003cem\u003ePpLDOX\u003c/em\u003e, \u003cem\u003ePpDFR\u003c/em\u003e, \u003cem\u003ePpC4H\u003c/em\u003e, PpCHS, \u003cem\u003ePpOMT\u003c/em\u003e, \u003cem\u003ePpANS\u003c/em\u003e, and \u003cem\u003ePpUFGT\u003c/em\u003e increased significantly in \u0026lsquo;RMHC\u0026rsquo; compared to \u0026lsquo;SR\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In general, the changes in the genes in the RT-qPCR data were consistent with the trend in the transcriptome, which was higher in red meat than in non-red meat. These changes in PpMYB75 and the structural genes were directly involved in the production of red fruit meat.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTranscription factors are usually localized in the nucleus to exert their regulatory effects. RFP fluorescence was detected in the nucleus of cells containing the PpMYB75::RFP fusion gene, indicating that the PpMYB75 transcription factor was localized to the nucleus, and suggesting that it may be involved in transcriptional regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003ch2\u003e3.7. The PpMYB75-PpDFR regulatory network may be important in the redness of \u0026lsquo;RMHC\u0026rsquo; compared to \u0026lsquo;SR\u0026rsquo; peach\u003c/h2\u003e \u003cp\u003eConsidering the role of the PpMYB75 TF in red flesh, PpMYB75 could affect the accumulation of anthocyanins by regulating the expression of structural genes. A prediction of the interactions between genes revealed the possibility of interactions between PpMYB75 and these structural genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We selected three genes (\u003cem\u003ePpDFR\u003c/em\u003e, \u003cem\u003ePpCHS\u003c/em\u003e, and \u003cem\u003ePpLDOX\u003c/em\u003e) with a high possibility of interaction. Promoter analysis showed that only \u003cem\u003ePpDFR\u003c/em\u003e contained the PpMyB75-bound cis-acting element CCGTTG (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). To investigate the interactions between the PpMYB75 transcription factor and the \u003cem\u003ePpDFR\u003c/em\u003e promoter, the amplified PpMYB75 transcription factor was ligated to the pGAD424 vector, the \u003cem\u003ePpDFR\u003c/em\u003e promoter sequence was ligated to the pHIS2 vector, respectively, and the yeast strains were transformed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, the PpMYB75 and \u003cem\u003ePpDFR\u003c/em\u003e promoter combinations grew on deficient media (SD/-Leu/-Ura) containing 90 mM 3-AT, while the \u003cem\u003ePpDFR\u003c/em\u003e promoter and pGAD424 vector combinations in colonies containing 90 mM 3-AT did not. These results show that PpMYB75 was bound to the \u003cem\u003ePpDFR\u003c/em\u003e promoter. To further demonstrate binding of PpMYB75 to \u003cem\u003ePpDFR\u003c/em\u003e, PpMYB75 was ligated to the PET32a vector, and the protein was induced and purified to obtain the PpMYB75 protein with a HIS tag. Approximately 30 bp of the \u003cem\u003ePpDFR\u003c/em\u003e promoter region with the possible PpMYB75 binding site was labeled with biotin, and an EMSA was performed. The results showed that PpMYB75 was bound to the \u003cem\u003ePpDFR\u003c/em\u003e gene cis-acting element CCGTTG (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). However, binding disappeared when the CCGTTG sequence mutated to GGAGGT, indicating that binding was specific.\u003c/p\u003e \u003cp\u003eTo explore the effect of PpMYB75 on \u003cem\u003ePpDFR\u003c/em\u003e transcriptional activation, PpMYB75 was ligated to the pGreenII 62-SK expression vector, and a LUC vector was ligated to the \u003cem\u003ePpDFR\u003c/em\u003e promoter. After transforming the plasmid into \u003cem\u003eA. tumefaciens\u003c/em\u003e, the tobacco was transiently injected with the infiltrate and left for 2\u0026ndash;3 days. The luminescence intensity of the tobacco injection site was measured by \u003cem\u003ein vivo\u003c/em\u003e fluorescence imaging, and dual luciferase activity was determined. As results, the transcription factor combination of PpMYB75 on \u003cem\u003ePpDFR\u003c/em\u003e co-injected tobacco activated fluorescence by increasing the luminescence density after the co-injection compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). This result indicates that PpMYB75 activated \u003cem\u003ePpDFR\u003c/em\u003e expression. PpMYB75 affected the formation of \u0026lsquo;RMHC\u0026rsquo; by influencing the expression of structural-related genes, such as \u003cem\u003ePpDFR\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.1. The higher content of flavonoids may confer the \u0026lsquo;RMHC\u0026rsquo; peach red characteristics\u003c/h2\u003e \u003cp\u003eFruit color is an important trait of peaches, as it directly affects their market competitiveness. Anthocyanin, also known as anthocyanidin, is a polyphenolic water-soluble plant pigment widely found in flowers, fruits, leaves, stems, and seeds that determines fruit color (Zhou et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Apples, grapes, oranges, pears, and peaches accumulate abundant anthocyanosides in ripe fruits (Yan et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, the color change produced by accumulating anthocyanin is an important indicator of fruit ripeness. In our study, \u0026lsquo;RMHC\u0026rsquo; accumulated more anthocyanin than \u0026lsquo;SR\u0026rsquo; during the same period regardless of skin or flesh, particularly in the flesh. The accumulation of higher anthocyanoside content during the same period compared to \u0026lsquo;SR\u0026rsquo; was an important factor in the peach flesh of \u0026lsquo;RMHC\u0026rsquo;.\u003c/p\u003e \u003cp\u003eMany anthocyanins affect fruit color and participate in fruit growth and development, such as cyanidin O-syringic acid, cyanidin 3-O-galactoside, and pelargonidin 3-O-glucoside (LaFountain and Yuan \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We investigated the variations in 79 flavonoids detected in \u0026lsquo;RMHC\u0026rsquo; and \u0026lsquo;SR\u0026rsquo;. As results, the contents of the anthocyanins pelargonidin 3-O-glucoside, cyanidin O-syringic acid, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside were significantly higher in the pulp of ripe \u0026lsquo;RMHC\u0026rsquo; than \u0026lsquo;SR\u0026rsquo; (S3 period). Wang et al. showed that the accumulation of pelargonidin 3-O-glucoside determines the bright red color of peony flowers (Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). High levels of cyanidin O-syringic acid were associated with reddening of the fruit skin in a study on yellow cherry fruit color (Ji et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Red-fleshed kiwifruit fruit accumulates cyanidin 3-O-galactoside and cyanidin 3-O-xylo-galactoside (Montefiori et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). (Es-Safi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) demonstrated that the red compound formed in the presence of cyanidin 3-O-glucoside glycoside is more stable. Therefore, pelargonidin 3-O-glucoside, cyanidin O-syringic acid, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside may be responsible for the red flesh of \u0026lsquo;RMHC\u0026rsquo; than \u0026lsquo;SR\u0026rsquo;. Flavonoids are a class of secondary metabolites widely distributed in plants, and up to several hundred flavonoids have been detected (Tanaka et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Flavonoids have been divided into yellow chalcones, orange ketones, flavonols and flavones, red to blue anthocyanidins, and brown proanthocyanidin oxidation products. The contents of flavonoids (quercetin 3,7-bis-O-β-D-glucoside, gossypitrin, and spiraeoside) and flavanols, such as (+)-catechin, and flavonoids (chrysoeriol 5-O-hexoside and tricetin), were significantly higher in our study. We speculate that these substances are indirectly related to the formation of flesh color and early ripening of \u0026lsquo;RMHC\u0026rsquo;.\u003c/p\u003e \u003ch2\u003e4.2. Increased expression of flavonoid metabolism-associated genes may be related to flavonoid content and the red flesh in peaches\u003c/h2\u003e \u003cp\u003eFlavonoids, as secondary metabolites of the phenylalanine pathway, have been well-studied as key enzymes of the synthetic pathway, such as DFR, C4H, CHS, F3H, and LDOX (Cao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sunil and Shetty \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The function of these structural genes in peach is a major determinant of differences in peach flesh color (Karak \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, the structural genes encoding flavonoid synthesis-related enzymes determine the fleshy fruit ripening process, which is crucial for the quality and storage capacity of the fruit (Albert et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). DFR is a key enzyme downstream of the anthocyanin biosynthetic pathway. DFR catalyzes the production of geranoside, cornflower, and delphinidin (Springob et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). C4H has high activity in all plant tissues, C4H is a key enzyme in the formation of anthocyanidins in the phenylpropanoid metabolic pathway (Millar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). CHS is an enzyme in the biosynthetic pathway of flavonoids, such as anthocyanins, flavonoids, and flavonols, that catalyzes the production of yellow tetrahydroxychalcone (Tanaka et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, 31 structural genes from the DEGs annotated to 14 enzymes in the flavonoid synthetic pathway were analyzed. Transcriptomic and RT-qPCR analyses indicated that \u003cem\u003ePpLDOX\u003c/em\u003e/\u003cem\u003ePpDFR\u003c/em\u003e/\u003cem\u003ePpC4H\u003c/em\u003e/\u003cem\u003ePpCHS\u003c/em\u003e increased more significantly in \u0026lsquo;RMHC\u0026rsquo; than \u0026lsquo;SR\u0026rsquo;. Thus, the high expression of \u003cem\u003ePpLDOX\u003c/em\u003e/\u003cem\u003ePpDFR\u003c/em\u003e/\u003cem\u003ePpC4H\u003c/em\u003e/\u003cem\u003ePpCHS\u003c/em\u003e may be the main factor for the redder flesh of \u0026lsquo;RMHC\u0026rsquo; than \u0026lsquo;SR\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.3. PpMYB75 may cause the red flesh of \u0026lsquo;RMHC\u0026rsquo; by mediating the expression of PpDFR\u003c/h2\u003e \u003cp\u003eNumerous studies have shown that MYB, bHLH, and WD repeat proteins are closely associated with the accumulation of secondary metabolites in the flavonoid synthetic pathway (Ying et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among them, R2R3-MYBs play a key role in determining the color change in peach pulp (Yan et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The involvement of MYB transcription factors in the formation mechanism of peach flesh color has been widely reported, and several transcription factors in this family, such as MYB1, MYB3, MYB10.1, MYB18, MYB19, MYB20, and MYB39, have been implicated in the peach flesh red phenotype (Tuan et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe current study reveals that MYB transcription factors function by binding to promoter regions of structural genes (Liu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our study shows that the transcription levels of many MYB, bHLH, and WD40 transcription factors were altered. Fortunately, the PpMYB75 transcription factor was transcribed at high levels in red pulp. Thus, we speculate that PpMYB75 may be closely related to the formation of \u0026lsquo;RMHC\u0026rsquo; red flesh. We demonstrated that PpMYB75 binds to the promoter region of \u003cem\u003ePpDFR\u003c/em\u003e and activate its expression. Therefore, these findings led to the hypothesis that PpMYB75 is involved in \u0026lsquo;RMHC\u0026rsquo; red pulp formation as a transcriptional activator of \u003cem\u003ePpDFR\u003c/em\u003e. The function of MYB75 in several species has been characterized, in \u003cem\u003eArabidopsis\u003c/em\u003e, MYB75 is an important regulator involved in pigment accumulation (Kreynes et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and our study appears to be the first to determine that MYB75 is associated with the regulation of the flavonoid synthesis in peaches.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eRed flesh is the main distinguishing feature of \u0026lsquo;RMHC\u0026rsquo;. This study determined by metabolomics analysis that the increase in flavonoid (anthocyanins, flavonols, and flavanols) contents was related to the red pulp of \u0026lsquo;RMHC\u0026rsquo;. The significantly increased expression of some transcription factors, particularly PpMYB75, and several structural genes, such as \u003cem\u003ePpDFR\u003c/em\u003e, \u003cem\u003ePpCHS\u003c/em\u003e, \u003cem\u003ePpC4H\u003c/em\u003e, and \u003cem\u003ePpLDOX\u003c/em\u003e, may play an important role in the formation of red flesh in \u0026lsquo;RMHC\u0026rsquo;. The transcription factor PpMYB75 localized to the nucleus and activated \u003cem\u003ePpDFR\u003c/em\u003e expression by binding to the cis-acting element CCGTTG in the \u003cem\u003ePpDFR\u003c/em\u003e promoter region. In conclusion, the increased expression of PpMYB75 and some other structural genes (\u003cem\u003ePpDFR\u003c/em\u003e, \u003cem\u003ePpCHS\u003c/em\u003e, \u003cem\u003ePpC4H\u003c/em\u003e, and \u003cem\u003ePpLDOX\u003c/em\u003e) in the flavonoid synthetic pathway contributed to the red flesh of \u0026lsquo;RMHC\u0026rsquo;. Moreover, the PpMYB75-\u003cem\u003ePpDFR\u003c/em\u003e module may be the main cause of red pulp formation in \u0026lsquo;RMHC\u0026rsquo; shoot variants compared to \u0026lsquo;SR\u0026rsquo;.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u0026nbsp;\u003c/strong\u003eThe following supporting information can be downloaded at:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Bioeditas Technology Corporation (Shaanxi, China) for helping with the transcriptome sequencing analysis and Metware Company for helping with the metabolome analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e methodology, XZ; validation, ZL, MC, and YY; formal analysis, ZL, SW, and MS; resources, XX; data curation, CX; writing\u0026mdash;original draft preparation, CX; writing\u0026mdash;review and editing, XZ; supervision, XZ; project administration, LQ; funding acquisition, WH. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was financially supported by the National Natural Science Foundation of China (32302508), the Natural Science Foundation of Shandong Province (No. ZR2021QC203), the Basic Research Project of Science, the Education and Industry Integration Pilot Project (2022PY064), Key Innovation Project (2022JBZ01-06) of Qilu University of Technology (Shandong Academy of Sciences), and Development Plan of Youth Innovation Team in Colleges and Universities of Shandong Province 2022KJ127.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Data are available on request to the corresponding author. The transcriptome sequencing has been deposited under BioProjectin NCBI. The accession number for these SRA data is PRJNA978508.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbert NW, Davies KM, Lewis DH, Zhang H, Montefiori M, Brendolise C, Boase MR, Ngo H, Jameson PE, Schwinn KE (2014) A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. 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BMC Plant Biol 14:388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12870-014-0388-y\u003c/span\u003e\u003cspan address=\"10.1186/s12870-014-0388-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"SR, RMHC, Flavonoids, Flesh reddening, Anthocyanins","lastPublishedDoi":"10.21203/rs.3.rs-3378595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3378595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u0026lsquo;Red Meat Honey Crisp (RMHC)\u0026rsquo; has been widely cultivated by growers in recent years due to its early maturity, and red meat type characteristics. As a bud variant of \u0026lsquo;Super Red (SR)\u0026rsquo; peach, red flesh is the most distinctive characteristic of \u0026lsquo;Red Meat Honey Crisp (RMHC)\u0026rsquo;. However, the mechanism of red flesh formation in \u0026lsquo;RMHC\u0026rsquo; remains unclear. In this study, 79 differentially produced metabolites were identified by metabolomics analysis. The anthocyanin content in \u0026lsquo;RMHC\u0026rsquo; was significantly higher than that in \u0026lsquo;SR\u0026rsquo; during the same period, such as cyanidin O-syringic acid and cyanidin 3-O-glucoside. Other flavonoids also increased during the formation of red flesh, including flavonols (6-hydroxykaempferol-7-O-glucoside, hyperin), flavanols (protocatechuic acid, (+)-gallocatechin), and flavonoids (chrysoeriol 5-O-hexoside, tricetin). In addition, transcriptomic analysis and RT-qPCR showed that the expression levels of the flavonoid synthesis pathway transcription factor MYB75 and some structural genes, such as \u003cem\u003ePpDFR\u003c/em\u003e, \u003cem\u003ePpCHS\u003c/em\u003e, \u003cem\u003ePpC4H\u003c/em\u003e, and \u003cem\u003ePpLDOX\u003c/em\u003e increased significantly in \u0026lsquo;RMHC\u0026rsquo;. Subcellular localization analysis revealed that MYB75 was localized to the nucleus. Yeast single hybridization assays showed that MYB75 bound to the cis-acting element CCGTTG of the \u003cem\u003ePpDFR\u003c/em\u003e promoter region. The MYB75-\u003cem\u003ePpDFR\u003c/em\u003e regulatory network was identified to be a key pathway in the reddening of \u0026lsquo;RMHC\u0026rsquo; flesh. Moreover, this is the first study to describe the cause for red meat reddening in \u0026lsquo;RMHC\u0026rsquo; compared to \u0026lsquo;SR\u0026rsquo; peaches using transcriptomics, metabolomics and molecular methods. Our study identified a key transcription factor involved in the regulation of the flavonoid synthetic pathway and contributes to peach breeding-related efforts as well as the identification of genes involved in color formation in other species.\u003c/p\u003e","manuscriptTitle":"The PpMYB75-PpDFR module reveals the difference between ‘SR’ and its Bud Variant ‘RMHC’ in Peach Red Flesh","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-03 17:30:50","doi":"10.21203/rs.3.rs-3378595/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-09-26T02:56:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-09-26T01:06:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-23T06:00:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Research","date":"2023-09-22T21:15:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a16c610e-36ae-4209-ac87-00b31fab43d9","owner":[],"postedDate":"October 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-01-15T15:02:21+00:00","versionOfRecord":{"articleIdentity":"rs-3378595","link":"https://doi.org/10.1007/s10265-023-01512-1","journal":{"identity":"journal-of-plant-research","isVorOnly":false,"title":"Journal of Plant Research"},"publishedOn":"2024-01-09 15:00:33","publishedOnDateReadable":"January 9th, 2024"},"versionCreatedAt":"2023-10-03 17:30:50","video":"","vorDoi":"10.1007/s10265-023-01512-1","vorDoiUrl":"https://doi.org/10.1007/s10265-023-01512-1","workflowStages":[]},"version":"v1","identity":"rs-3378595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3378595","identity":"rs-3378595","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}