Integrative analysis of metabolome and transcriptome reveals the mechanism of color formation in pepper fruit (Capsicum annuum L.)

Food Chemistry 306 (2020) 125629

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Integrative analysis of metabolome and transcriptome reveals the mechanism of color formation in pepper fruit (Capsicum annuum L.)

T

Yuhua Liua,b,1, Junheng Lva,b,1, Zhoubin Liua,b,1, Jing Wanga, Bozhi Yangb, Wenchao Chenb, ⁎ ⁎ ⁎ Lijun Ouc, Xiongze Daic, , Zhuqing Zhangb, , Xuexiao Zoua,c, a

Longping Branch, Graduate School of Hunan University, Changsha, Hunan 410125, China Vegetable Institution of Hunan Academy of Agricultural Science, Changsha, Hunan 410125, China c College of Horticulture and Landscape, Hunan Agricultural University, Changsha, Hunan 410128, China b

A R T I C LE I N FO

A B S T R A C T

Chemical compounds: Apigenin (PubChem CID5280443) Quercetin (PubChem CID5280343) Chrysoeriol (PubChem CID5280666) Delphinidin (PubChem CID128853) Capsanthin (PubChem CID5281228) Lutein (PubChem CID5281243) Violaxanthin (PubChem CID448438) Zeaxanthin (PubChem CID5280899) Antheraxanthin (PubChem CID5281223) β-Cryptoxanthin (PubChem CID5281235)

To understand the mechanism of the color formation of pepper fruit, integrative analysis of the metabolome and transcriptome profiles was performed in pepper varieties with 4 different fruit colors. A total of 188 flavonoids were identified, and most of the anthocyanins, flavonols and flavones showed markedly higher abundances in purple variety than in other varieties, which was linked to the high expression of flavonoid synthesis and regulatory genes. Using weighted gene co-expression network analyses, modules related to flavonoid synthesis and candidate genes that regulate flavonoid synthesis and transport were identified. Furthermore, the analysis of 12 carotenoids showed that the content of xanthophylls at 50 days after anthesis was significantly different between the four pepper varieties, which was resulted from the differential expressions of genes downstream of the carotenoid pathway. Our results provide new insights into the understanding of the synthesis and accumulation of flavonoids and carotenoids in pepper fruit.

Keywords: Pepper Fruit color Flavonoid Carotenoid Metabolome Transcriptome

1. Introduction As one of the important characteristics of pepper, fruit color has long been concerned by breeders and consumers (Timberlake, 1989). Bright colors not only increase the commercial value of the fruit but also serve as an essential indicator of fruit ripeness (Facteau, Chestnut, & Rowe, 1983). Previous studies have shown that the color of pepper fruit is mainly determined by the type and content of chlorophyll, carotenoids, and flavonoids (Lightbourn et al., 2008). Among these pigments, flavonoids are widely distributed in plants as important secondary metabolites, and there are about 9000 flavonoids identified in plants so far (Ferrer, Austin, Stewart, & Noel, 2008). Flavonoids are divided into six categories, including flavonols, flavones,

flavanones, flavan-3-ols, isoflavones, and anthocyanins (Chen et al., 2014). Flavonoids are synthesized from phenylalanine via the phenylpropanoid and flavonoid pathways. First, phenylalanine catabolism is catalyzed by phenylalanine lyase (PAL), cinnamic acid hydroxylase (C4H) and coumadin CoA ligase (4CL) to form the starting substrate for flavonoid synthesis, p-coumaroyl-CoA. Following the desaturation and isomerization reactions catalyzed by chalcone synthase (CHS) and chalcone isomerase (CHI), naringenin is produced from p-coumaroylCoA and 3 malonyl-CoA. Naringenin is a core intermediate for flavonoid biosynthesis, which is catalyzed to form different types of flavonoids by a series of enzymes, such as flavonoid 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′5′-hydroxylase (F3′5′H), flavonoid Synthase (FNS), flavonol synthase (FLS), dihydroflavonol 4-

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Corresponding authors at: Longping Branch, Graduate School of Hunan University, Changsha, Hunan 410125, China (X. Zou). E-mail addresses: [email protected] (Y. Liu), [email protected] (J. Lv), [email protected] (X. Dai), [email protected] (Z. Zhang), [email protected] (X. Zou). 1 Yuhua Liu, Junheng Lv and Zhoubin Liu contributed equally to this work. https://doi.org/10.1016/j.foodchem.2019.125629 Received 8 May 2019; Received in revised form 27 September 2019; Accepted 30 September 2019 Available online 09 October 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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of pepper fruit (Lightbourn et al., 2008), and the vast accumulation of anthocyanins in immature fruits is responsible for the purple or black color of pepper fruits (Lightbourn et al., 2008). Previous studies have demonstrated that the synthesis of anthocyanins is controlled by the A locus, which encodes a MYB transcription factor that is homologous to the petunia Anthocyanin2 (An2) protein (Borovsky, Oren-Shamir, Ovadia, & Paran, 2004). With the development of pepper fruit, flavonoids and chlorophyll gradually degrade in the plastid, accompanied by the synthesis and accumulation of carotenoids (Hugueney et al., 1996). Three loci y, c1 and c2 were proposed to explain the variation in carotenoid composition, resulting in the different color of pepper fruit (Odland, 1938). Genetic analysis indicated that the c2 locus corresponds to the PSY gene (Huh et al., 2001), while the y locus was identified as the CCS gene which is involved in the synthesis of capsanthin (Popovsky & Paran, 2000). Although some genes related to fruit color have been cloned in peppers, it is still a mystery how these genes regulate the synthesis of flavonoids and carotenoids because of the lack of effective mutants. Fruit color, as an important appearance and quality character of pepper fruit, has been studied extensively. However, these studies mainly focus on one or several flavonoids/carotenoids, and there are rare studies on large-scale identification and quantification on flavonoids and carotenoids in pepper. In this study, we used liquid chromatography tandem mass spectrometry (LC–MS/MS) to detect and quantify flavonoids and carotenoids of pepper fruits. 188 flavonoids and 12 carotenoids were identified and quantified in the fruits of pepper with four different color varieties. The differential regulation of flavonoids and carotenoid structural genes was analyzed by transcriptional data and verified by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, through weighted gene co-expression network analysis (WGCNA), we obtained co-expression gene modules, and screened some key genes involved in flavonoid synthesis. This work not only clarified the accumulation of flavonoids and carotenoids in pepper fruits with different colors, but also provided important insights in the molecular network of flavonoid synthesis.

reductase (DFR), anthocyanidin synthase (ANS) and anthocyanin reductase (ANR). However, the anthocyanins produced in the cytoplasm are unstable, require further glycosylation modification by UDP-glucosyltransferase (UGT), and are finally transfered into the vacuole for storage by glutathione S-transferase (GST) (Jaakola, 2013; WinkelShirley, 2001). Flavonoids are widely involved in the physiological processes of plants as polyphenols, such as the attraction of pollinators, the interaction between plants, microorganisms and animals, the protection of plants from UV damage (Ferrer et al., 2008), and the formation of flowers, fruits and seeds colors (Forkmann, 1991). In addition to structural genes, several transcription factors have been reported to be involved in the synthesis of flavonoids by regulating the expression of flavonoid biosynthetic genes, such as R2R3 MYB transcription factors, basic helix-loop-helix proteins (bHLH), WDrepeat proteins (WD), MADS-box and zinc finger protein (Koes & Verweij, 2005). In Arabidopsis thaliana, 13 R2R3 MYB transcription factors that control flavonoids synthesis have been identified, 9 of which are positively correlated with the synthesis of flavonoids, and the other four MYB transcription factors inhibit the synthesis of flavonoids by down-regulating the expression of phenylpropanoid pathway and flavonoid synthesis downstream genes (Dubos et al., 2010). bHLH and WD proteins are involved in the synthesis of flavonoids more as a partner of MYB, which interacts with MYB to form MBW complex to regulate the accumulation of flavonoids (Koes & Verweij, 2005). By analyzing the Arabidopsis mutant tt1, Sagasser et al found that the zinc finger protein AtTT1 can activate the expression of ANR and control proanthocyanidins synthesis in testa (Sagasser, Lu, Hahlbrock, & Weisshaar, 2002). Carotenoids are 40-carbon isoprenoids with multiple conjugated double bonds, and the conjugated double bonds enable carotenoids to absorb visible light to produce yellow, orange and red colors, giving bright colors to fruits, flowers, and vegetables (Yuan, Zhang, Nageswaran, & Li, 2015). Furthermore, carotenoids also serve as precursors to many flavor-related substances, conferring sensory attributes to the consumers (Vogel et al., 2010). Due to the visibility of carotenoids and their important role in plant growth and development, the main pathway of carotenoids was described as early as 1971 (Goodwin, 1971). Like other isoprenoids, carotenoids are synthesized from the 5carbon compound isopentenyl diphosphate (IPP). First, the precursor IPP is catalyzed by IPP isomerase (IPI) and geranylgeranyl diphosphate synthase (GGPS) to form geranylgeranyl diphosphate (GGPP) in plastids. Then, the condensation of two GGPP molecules by phytoene synthase (PSY) produces the first colorless carotenoid. Subsequently, Colorless phytoene produces red lycopene through a series of desaturation and isomerization by phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), ζ-carotene isomerase (Z-ISO) and carotenoid isomerase (CRTISO). After lycopene, the synthesis of carotenoids differentiates into two branches: β, ε-carotene and β, β-carotene. In the β, ε-carotene branch, lycopene is sequentially catalyzed to finally generate yellow lutein by ζ-lycopene cyclase (LCYE), β-lycopene cyclase (LCYB), cytochrome P450-type monooxygenase 97A and cytochrome P450-type monooxygenase 97C. The synthesis of carotenoids in the β,β-carotene branch requires the participation of β-lycopene cyclase (LCYB), β-carotene hydroxylase (CHYB), and zeaxanthin epoxidase (ZEP), violaxanthin de-epoxidase (VDE) and capsanthin-capsorubin synthase (CCS) (Hirschberg, 2001; Yuan et al., 2015). Capsicum is rich in fruit color, and the color changes across the distinct developmental stages of the fruit. In the immature period, the color of the pepper fruit mainly includes white, green, purple and black and gradually changes to yellow, orange, red and brown as the fruit develops (Matsufuji, Ishikawa, Nunomura, Chino, & Takeda, 2007; Wahyuni, Ballester, Sudarmonowati, Bino, & Bovy, 2011). The difference in the color of pepper fruit is mainly due to the differential accumulation of flavonoids and carotenoids (Delgado-Vargas and Paredes-Lopez (2002)). Anthocyanin, as the final product of the flavonoid synthesis pathway, is mainly accumulated in the outer epidermis

2. Materials and methods 2.1. Plant material and sampling Four pepper varieties were planted in Changsha experimental station (N 28°11′49″, E 112°58′429″) of Vegetable Institution of Hunan Academy of Agricultural Science. The fruit color of the four pepper varieties, HJ10-1, HJ11-3-1, CJ12-17-1 and 0622-1-3-2-1-3-1, was green, white, purple and green at 30 days after anthesis (30 DAA) respectively, and the fruit color was yellow, orange, red and red at 50 days after flowering (50 DAA) (Fig. 1A). As many varieties were involved in this study and two developmental stages of the pepper fruit were studied, the HJ10-1, HJ11-3-1, CJ12-17-1, and 0622-1-3-2-1-3-1 varieties were re-named A, B, C, and D, respectively, for easy following. Four pepper varieties were sampled at 30 and 50 days after anthesis. Six fruit peels were collected and pooled from three individual plants, and three replicates were performed for each pepper variety at each time point. These samples were frozen in liquid nitrogen immediately and stored at −80 °C for subsequent metabolite extraction, transcriptome sequencing, and real-time PCR analysis. Samples of HJ10-1, HJ11-3-1, CJ12-17-1, and 0622-1-3-2-1-3-1 were named A1-G, B1-W, C1-P and D1-G at 30 DAA, and designated as A2-Y, B2-O, C2-R, and D2-R at 50 DAA, respectively (G, Green; W, White; P, Purple; Y, Yellow; O, Orange; R, Red). 2.2. Sample preparation and extraction The flavonoids were extracted as described previously (Chen et al., 2013) with some modifications. The freeze-dried pericarp was crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 2

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Fig. 1. Fruit colors and number of differentially accumulated flavonoids between four pepper varieties. A: Fruit colors of the four varieties at 30 days after anthesis (30 DAA, left) and 50 DAA (right). HJ10-1, HJ11-3-1, CJ12-17-1 and 0622-1-3-2-1-3-1 varieties were re-named as A, B, C, D respectively. Number of differentially accumulated flavonoids between four pepper varieties at 30 DAA (B) and 50 DAA (C), DAFs, differentially accumulated flavonoids.

(UPLC, Shim-pack UFLC SHIMADZU CBM30A system; MS, Applied Biosystems 6500 Q TRAP). Metabolite quantification was performed using a scheduled multiple reaction monitoring (MRM) method, which has been previously described (Chen et al., 2013). The identified metabolites were subjected to orthogonal partial least squares discriminant analysis (OPLS-DA), and metabolites with |Log2 (fold change)| ≥1, pvalue < 0.05, and VIP (variable importance in project) ≥ 1 were considered as differentially accumulated flavonoids (DAFs).

30 Hz. 100 mg powder was weighted and extracted with 1.0 mL 70% aqueous methanol. The sample was then stored at 4 °C overnight. During this time the sample was vortexed six times to increase the extraction rate. Following centrifugation at 10,000g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 mL; ANPEL, China) and filtrated (SCAA-104, 0.22 μm pore size; ANPEL, China) before LC–MS analysis. The carotenoids were extracted as described previously (Ma et al., 2017) with some modifications. The lyophilized peel was ground into powder in a mixer mill (MM 400, Retsch) with a zirconia bead for 1 min at 30 Hz. 50 mg powder was extracted with 1.0 mL mixed reagent of nhexane: acetone: ethanol (2:1:1, V/V/V) (containing 0.01% butylated hydroxytoluene (BHT)), with internal standard added. The extract was vortexed (30 s), and ultrasound-assisted extraction was carried out for 20 min at room temperature. After centrifugation at 12,000g for 5 min, the supernatants were collected, and the extraction steps above were repeated. The supernatant from the two centrifugations was combined and then evaporated to dryness under nitrogen gas stream, reconstituted in methanol: methyl tert-butyl ether (1:1, V/V). The solution was vortexed (30 s), and ultrasound-assisted dissolution was carried out for 2 min. Following centrifugation at 12,000g for 2 min, the supernatant was filtered (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China) before metabolomics analysis.

2.4. Carotenoid identification and quantification Carotenoid extracts were analyzed using an LC-APCI-MS/MS system (UPLC, ExionLC™ AD; MS, Applied Biosystems 6500 Triple Quadrupole). Chromatographic separations were performed on a YMC C30 (3 µm, 2 mm × 100 mm) column operating at 28 °C. The flow rate was set at 0.8 mL/min and the mobile phase consisted of solvent A (acetonitrile: methanol (1:3, V/V) containing 0.01% butylated hydroxytoluene (BHT) and 0.1% formic acid) and solvent B (methyl tert-butyl ether containing 0.01% BHT). The gradient programs (solvent A: solvent B) were as follows, 100:0 V/V at 0 min, 100:0 V/V at 3 min, 58:42 V/V at 6 min, 20:80 V/V at 8 min, 5:95 V/V at 9.0 min, 100:0 V/V at 9.1 min, 100:0 V/V at 11 min; The injection volume for each sample was 2 μL. A high-resolution tandem mass spectrometer APCI-triple quadrupole-linear ion trap (Q TRAP)-MS was used to detect metabolites eluted form the column. API 6500 Q TRAP LC-MS/MS System, equipped with an atmospheric pressure chemical ionization (APCI) Turbo Ion-Spray

2.3. Flavonoid identification and quantification Flavonoid extracts were analyzed using an LC-ESI-MS/MS system 3

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Fig. 2. Heat map of metabolites of flavonoid synthesis pathway at 30 DAA. This pathway is constructed based on the KEGG pathway and literary references. Each colored cell represents the normalized intensity of each compound ion according to the color scale (three biological replicates × four cultivars, n = 12). Box-andwhisker plots are shown for changes of flavonoids in each pepper variety. Maximum and minimum values of a metabolite among three biological replicates are represented at the upper and lower ends of the whisker, respectively. CHI, chalcone isomerase; F3H, flavonoid 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; FLS, flavonol synthase; FNS, flavonoid synthase.

2018), and clean reads were then mapped to the pepper genome (Zunla1 version) using HISAT2 (Kim, Langmead, & Salzberg, 2015). FPKM was used for gene/transcript level quantification. Based on the raw count data, differential expression analysis between samples was performed by DESeq2 software (Varet, Brillet-Guéguen, Coppée, & Dillies, 2016). Genes satisfying |log2Fold Change| > = 1, and False Discovery Rate (FDR) < 0.05 were defined as differentially expressed genes (DEGs) and subjected to Gene Ontology (GO) enrichment analysis.

interface, operating in a positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The APCI source operation parameters were as follows: ion source, APCI+; source temperature, 350 °C; curtain gas (CUR) were set at 25.0 psi; the collision gas (CAD) was medium. Declustering potential (DP) and collision energy (CE) for individual MRM transitions were obtained with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the plant carotenoids eluted within this period. The MRM analysis was performed by Metware Biotechnology Co., Ltd. (Wuhan, China). The quantification of the carotenoids was achieved using calibration curves for 12 standards.

2.6. Co-expression network analysis for construction of modules For co-expression network analysis, the weighted gene co-expression network analysis (WGCNA) package (Langfelder & Horvath, 2008) was used. To obtain the genes related to flavonoid synthesis, differentially expressed genes (DEGs) and differentially accumulated flavonoids (DAFs) detected in the two fruit developmental stages were selected for integrative analysis. The DEGs with DAFs Pearson correlation coefficient (PCC) ≥ 0.90 or ≤ −0.90 were selected for subsequent WGCNA

2.5. RNA-seq analysis The total RNA was extracted from frozen peel, and the mRNA library of each sample was constructed and sequenced in the Illumina HiSeq4000 platform. The adaptor and low-quality sequence were removed using Fastp with default parameters (Chen, Zhou, Chen, & Jia, 4

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3.2. Transcriptome analysis of the fruits of four pepper varieties

with default parameters and subjected to KEGG enrichment analysis. The co-expression network was visualized using the free software Cytoscape.

After removing the adaptor and low-quality sequence, each library received 43041634–74948366 clean reads. These clean reads were mapped to the reference genome with match ratios in the range of 92.73–94.48 %, and 27,132 genes predicted from the genome were found to be expressed in at least one sample (with FPKM > 0). Through the differential expression analysis between 4 pepper varieties, a total of 15,227 DEGs were identified, and the DEG numbers ranged from 1782 to 6420 between the sample groups (Fig. S2B-C). GO enrichment analysis of these DEGs illustrated that they were widely distributed in the three functional groups, i.e., biological processes, molecular functions, and cellular components (Fig. S2A). According to the GO analysis, the cellular process is the largest group in the biological process, followed by the metabolic process and response to stimulus (Fig. S2A). The genes related to “cell”, “cell part”, and “organelle” are predominant in the category of cellular components (Fig. S2A). In the molecular function category, the genes were mostly involved in catalytic activity and binding (Fig. S2A).

2.7. qRT-PCR analysis The total RNA was extracted from the Peels of four pepper varieties at 30 DAA according to the Magen kit (China) instructions. The first strand cDNA was synthesized using reverse transcriptase (Vazyme, China). Twelve genes related to fruit color were selected for qRT-PCR analysis, and the CPActin gene was used as a reference gene to correct gene expression. All primers used in this study are listed in Supplementary Table S1. Reactions were carried out on a LightCycler® 96 quantitative Real-Time PCR detection system (Roche, Switzerland) using a Real Master Mix (SYBR Green) kit (Vazyme, China). Three replicates were performed for each sample. Quantitative data were analyzed using the 2−ΔCT method.

3. Results and discussion 3.3. Expression of flavonoid synthesis-related genes in four pepper varieties 3.1. Identification of flavonoids in the fruits of four pepper varieties To further explore the mechanism of accumulating flavonoids in pepper fruits, the expression patterns of genes in the phenylpropane and flavonoid pathways were analyzed. In addition to the ANS, the genes in the flavonoid and phenylpropanoid pathways at 30 DAA had higher levels of transcription compare to those at 50 DAA (Fig. S3), suggesting that flavonoids were mainly synthesized in the early stages of fruit development. Flavonoids are derived from the phenylpropanoid pathway. The high expression of PAL, C4H and 4CL in C1-P ensured the production of p-coumaroyl-CoA and provided sufficient precursor compounds for the synthesis of flavonoids in C1-P (Fig. S3), which may be one of the reasons for higher anthocyanin, flavonol and flavone content in C1-P. Chalcone isomerase (CHI) catalyzes the conversion of chalcone to naringenin, which is the core material for flavonoid synthesis. In the present study, the high expression of CHI in D1-G led to the highest accumulation of naringenin in D1-G, but flavones, flavonols, and anthocyanins downstream of naringenin showed less accumulation in D1-G (Fig. 2 and Fig. S3), which could be resulted from the low expression of the flavonoid 3-hydroxylase (F3H) and flavonoid 3′5′hydroxylase (F3′5′H) genes in D1-G (Fig. 2 and Fig. S3). F3′5′H, F3H and F3′H are important branching enzymes in the synthesis of flavonoids, catalyzing the formation of hydroxylated derivatives such as luteolin, dihydroquercetin and dihydromyricetin (Wang et al., 2014). In this study, although F3′H was weakly expressed in C1-P, C1-P fruit showed a high content of luteolin, chrysoeriol (downstream flavonoids of luteolin), quercetin, myricetin and delphinidin derivatives (Fig. 2 and Fig. S3), suggesting that F3′5′H and F3H were the main hydroxylases in pepper and affected the synthesis of flavonoids by regulating the flux through luteolin, dihydroquercetin and dihydromyricetin. Dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS) use dihydroflavonol as the substrate to produce colored anthocyanins and colorless flavonols respectively, and the expression of DFR and FLS determines the content of anthocyanins and flavonols (Tian et al., 2015). The transcription levels of FLS1 and FLS2 in D1-G were significantly higher than that of the other three varieties, but the content of quercetin and myricetin derivatives in D1-G was significantly lower than that in C1-P (Fig. 2 and Fig. S3), indicating that other FLS genes are present in the pepper to catalyze the production of flavonol by dihydroflavonol. The unstable anthocyanin needs to be modified and eventually transferred to vacuoles for storage (Jaakola, 2013). Interestingly, We found that the anthocyanin synthesis, modification and transport genes (DFR, UGT and GST2) were only highly expressed in C1P (Fig. S3), implying that the high expression of DFR, UFGT, and GST2 is essential for the accumulation of anthocyanins in pepper fruits.

To compare the content of flavonoids in pepper fruits of different periods and colors, we analyzed the fruit samples of four varieties by LC-ESI-Q TRAP-MS/MS. A total of 188 kinds of flavonoids were detected in 4 pepper varieties, including 94 flavones (51 flavones and 43 flavone C-glycosides), 38 flavonols, 8 catechin derivatives, 2 flavonolignan, 20 flavanones, 10 isoflavones and 16 anthocyanins (Tables S2). Setting |Log2 (fold change)| ≥1, p-value < 0.05, and VIP(variable importance in project) ≥ 1 as thresholds for differentially accumulated flavonoids (DAFs), the DAF numbers ranged from 43 to 72 between 4 pepper varieties at 30 and 50 DAA, and these DAFs are mainly distributed in three major categories of anthocyanins, flavones and flavonols (Fig. 1B-C, Table S3-4). Among these DAFs, most of compounds (anthocyanins, flavones, and flavonols) were significantly higher in C than in the other three varieties, including delphinidin, luteolin, chrysoeriol and quercetin derivatives (Fig. 1B-C, Table S3-4). Furthermore, we also found that the DAFs between the four pepper varieties at 30DAA were similar to those detected at 50 DAA, which may be related to the transcription level of the flavonoid structural gene (Fig. 2, Fig. S1 and Table S3-4). The significant down-regulation of flavonoid synthesis genes in four pepper cultivars at 50 DAA slowed the synthesis or degradation of flavonoids, which eventually led to the content of flavonoids in pepper fruits at 50 DAA close to that of 30 DAA. Anthocyanin as an important pigment plays a vital role in the formation of immature pepper fruit color. The accumulation of anthocyanin causes immature pepper fruit to appear purple-black (Lightbourn et al., 2008). In this study, 16 anthocyanins were detected from the fruits of four pepper varieties. Among these anthocyanins, delphinidin, cyanidin and malvidin derivatives were the major anthocyanins (Fig. 2). The content of delphinidin and its derivatives as the sources of purple and dark color was significantly higher in C1-P than that in the other three varieties, of which delphinidin 3,5-diglucoside and delphinidin 3-O-rutinoside were specifically accumulated in C (Fig. 2 and Fig. S1). However, the levels of malvidin derivatives (the delphinidin methylated derivatives) are similar in the four varieties (Fig. 2 and Fig. S1). The cyanidin derivative is a red-purple pigment, and its accumulation pattern is more complicated in the fruits of the four varieties. Kuromanin and keracyanin were higher in C1-P, with no or only a small accumulation in the other three fruits, while cyanidin 3O-malonylhexoside and cyanidin O-acetylhexoside displayed higher levels in B1-W and D1-G (Fig. 2 and Fig. S1). Thus, the high accumulation of cyanidin and delphinidin derivatives in C1-P leads to the purple appearance of C at 30 DAA. 5

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Fig. 3. Weighted gene co-expression network analysis (WGCNA) of 4308 DEGs (with DAFs Pearson correlation coefficient (PCC) ≥ 0.90 or ≤−0.90). (A) Hierarchical clustering tree (cluster dendrogram) showing 11 modules of co-expressed genes by WGCNA. Each leaf of tree corresponds to one gene. The major tree branches constitute 11 modules, labeled with different colors. (B-D) Module–flavonoid relationship. Each row represents a module, and the number of genes in each module is shown on the left. Each column represents a specific flavonoid (detailed information reference Table S2). The value in each cell at the row-column intersection represents the correlation coefficient between the module and the flavonoid and is displayed according to the color scale on the right. The value in parentheses in each cell represents the P value. B, module-Anthocyanin association; C, module-flavone association; D, module-flavonol association.

3.4. Co-expression network analysis identified flavonoid-related DEGs

two main clusters. Genes of cluster I showed higher levels in C than in the other three varieties, while genes of cluster II were highly expressed in the other three varieties (Fig. 4B). In the pink module, the co-expressed genes were highly expressed only in C1-P, exhibiting significant variety specificity (Fig. 4A). Although both the red and pink modules show a high correlation with the synthesis of flavonoids, the genes in the two modules exhibit different expression patterns, suggesting that the genes in the pink and red modules may perform different functions in the synthesis of flavonoids. To further determine the function of the genes in the module, KEGG analysis was performed. KEGG enrichment analysis revealed that the genes in the pink module were primarily linked to the synthesis and metabolism of primary and secondary metabolites, with metabolic pathways and biosynthesis of secondary metabolites being the two most prominent pathways for enrichment (Fig. 4C). In addition, nine flavonoid-synthesized structural genes such as F3H, CHS, and C4H were enriched into phenylpropanoid biosynthesis, flavonoid biosynthesis and flavone and flavonol biosynthesis (Fig. 4C). The flavonoid synthetic structural gene was abundantly enriched in the pink module, which

To investigate the gene regulatory network of flavonoid synthesis in pepper fruit, we performed a weighted gene co-expression network analysis (WGCNA) using non-redundant 4308 DEGs (with DAFs PCC ≥ 0.90 or ≤ − 0.90) (Table S5). These non-redundant DEGs were clustered into 11 major tree branches, and each of which represents a module (labeled with different colors) (Fig. 3A and Table S6). Modules are clusters of genes with high correlation, and genes within the same module are co-expressed. The analysis of the module-flavonoid relationships revealed the pink and red modules have a high positive correlation with most of the anthocyanins, flavonols, catechin derivatives, flavone and flavone-C-glycosides (correlation coefficient > 0.5 and 0 < P < 0.05) (Fig. 3B-D and Fig. S4), indicating that the genes in the pink and red modules play an important role in the synthesis of flavonoids in pepper fruits. To determine the expression pattern of genes in the pink and red modules, heat maps were performed with the FPKM values of the genes in the module (Table S7). The heat map results showed that genes in the red module could be clearly grouped into

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Fig. 4. Heat map and KEGG analysis of genes in the module. Heat map of genes in the pink (A) and red (B) modules. Red represents high expression and green corresponds to low expression. The KEGG enrichment analysis of the genes in the pink (C) and red (D) modules showed only the top 20 pathways with the most significant enrichment. HJ10-1, HJ11-3-1, CJ12-17-1 and 0622-1-3-2-1-3-1 varieties were re-named by A1-G, B1-W, C1-P, D1-G at 30 DAA respectively, and A2-Y, B2-O, C2-R, D2-R at 50 DAA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

red modules and 10 for pink modules) were predicted to encode transporters and were highly expressed in C1-P (except for ABC transporter G family member 7 (ABCG7), lipid transport superfamily protein (LTP) and sodium/metabolite cotransporter (BASS3)) (Fig. S5). Among the 26 genes, the solute carrier family protein, ABC transporter family, and multidrug and toxic compound extrusion protein families have the most members (members 5, 3, and 3, respectively). Through genetic and physiological analysis, Debeaujon et al. found that multidrug and toxic efflux transporter protein (TT12) is involved in the transport of proanthocyanidins in Arabidopsis seed coat vacuoles (Debeaujon, Peeters, Léon-Kloosterziel, & Koornneef, 2001). ATP-binding cassette (ABC) transporter protein is an important class of transporters in plants. An ABC-type transporter protein ZmMRP3 (multidrug resistance–associated protein 3) has been reported to be involved in the transport of flavonoids in Zea may (Goodman, Casati, & Walbot, 2004). In addition, we also found a proton transporter H+-ATPase (ATP2) in the red module. Previous studies have shown that H+-ATPase is involved in the acid balance between vacuoles and cytoplasm, while the vacuole and cytoplasmic pH gradient determines the flavonoid uptake efficiency (Grotewold, 2006). In Arabidopsis, mutations in P-type H+ATPase (AHA10) result in vacuolar morphology defects and reduced proanthocyanidin content in seeds (Baxter et al., 2005). Although only

further proved that the co-expressed gene in this module involved the accumulation of flavonoids in the pepper fruit. In the red module, the genes are significantly enriched for protein synthesis, plant photosynthesis, and plant-pathogen interaction pathways (Fig. 4D), which is also well linked to previous studies that flavonoids involved in plant photosynthesis and plant-pathogen interactions (Ferrer et al., 2008). We hypothesize that flavonoids can induce or inhibit the expression of genes in photosynthesis and plant-pathogen pathways when involved in photosynthesis and plant-pathogen interactions, and gene expression abundance in these pathways can be used as a signal feedback to plants to inhibit or promote the synthesis of flavonoids. Interestingly, MYB113 (A locus, Capana10g001433), a major regulator of flavonoid synthesis in pepper, was also clustered into red module, suggesting that the genes in this module may regulate the synthesis of flavonoids. 3.5. Genes involved in flavonoid transport Flavonoids are considered highly reactive and are potentially toxic in the cytoplasm. To avoid toxicity, cytoplasmic synthetic flavonoids are transported to vacuoles via transporters for storage or isolation (Grotewold, 2006). The red and pink modules serve as two modules highly associated with flavonoid synthesis, of which 26 genes (16 for 7

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the H+-ATPase, multidrug and toxic compound extrusion protein and ABC-type transporter families of the 26 transporter genes have been reported to be involved in flavonoid transport, these transport genes are significantly enriched in the pink and red modules, and it is reasonable to speculate that these transport genes may be involved in the transmembrane transport of flavonoids.

and COL2 (Fig. 5A and Table S8). For the above reasons, FH11, Capana01g003186, and WDR68 were identified as key genes for flavonoid synthesis. The network diagram of the red module is composed of 23 co-expressed genes (with edge weight ≥0.35), in which genes such as the solute carrier family 40 member 1 (SLC40), MYB113, late blight resistance protein B16 (R1B16) and late blight resistance protein A3(R1A-3) have the highest degree of connectivity (Fig. 5B and Table S9). The role of SLC40 and MYB113 in flavonoid synthesis has been described above. The high degree of connectivity further demonstrates that SLC40 and MYB113 may play a key role in the synthesis of flavonoids. In addition, R1B16 and R1A-3, two members of the late blight resistance protein family, showed high correlation with ERF109 (ethylene-responsive transcription factor 109), SLC40, MYB113 and other genes in this network (Fig. 5B and Table S9), suggesting that the late blight resistance protein family may participate in the synthesis of flavonoids by affecting the expression of ERF109, SLC40, and MYB113, which requires further verification.

3.6. Transcription factors involved in the synthesis of flavonoids In addition to structural and transport genes, transcription factors are also a key factor for the synthesis of flavonoids in plants. In the red module, 13 genes were identified to encode transcription factors, including two MYB transcription factors (MYB and MYB113) and five zinc finger proteins (YY1, DOF3.3, Y4919, CIR and ZFP4). By analyzing the expression patterns of these transcription factors, we found that except for eukaryotic translation initiation factor 4G (EIF4G), NAC transcription factor-like 9 (NAC9) and YY1, transcription factors such as MYB113 have higher expression abundance in C (Fig. S6). MYB-113, a major regulator of flavonoid synthesis in pepper, has been reported to be involved in the anthocyanin synthesis of pepper fruits, flowers and leaves (Borovsky et al., 2004). Although there is no report on the regulation of flavonoid synthesis by zinc finger proteins in pepper, the zinc finger protein TT1 interacts with TT2 (MYB transcription factor) to regulate the expression of flavonoid synthetic structural genes in Arabidopsis (Appelhagen et al., 2011). In the pink module, 10 genes encoding transcription factors were highly expressed in C1-P, including 2 WD (WD68 and WD44), 1 bHLH (bHLH143), and 1 MADS-box protein (AGL16). In Arabidopsis, TTG1 (TRANSPARENT TESTA GLABRA1), homologous to WD68, interacts with MYB and bHLH to form MBW complexes that regulate the synthesis of anthocyanins in seeds (Koes & Verweij, 2005). MADS-box protein is an important transcription factor in plants. Some MADS-box proteins have been reported to be involved in the synthesis of flavonoids, such as AtTT16 (MADS-box protein), which regulates the synthesis of proanthocyanidins in Arabidopsis seed coats (Nesi et al., 2002), VmTDR (MADS-box protein) participate in the accumulation of anthocyanins in bilberry fruits (Jaakola et al., 2010). In the present study, although the molecular and physiological functions of some transcription factors in the pink and red modules are unclear, these transcription factors are co-expressed with MYB, WD and bHLH, suggesting that these transcription factors may regulate the expression of MYB, WD, bHLH or structural genes in the same module to affect the synthesis of flavonoids in pepper fruits.

3.8. Identification of carotenoids in the fruits of four pepper varieties We determined the contents of 12 carotenoids in the fruits of 4 pepper varieties. The results showed that the carotenoid level of fruit was low at 30 DAA, and the accumulation was mainly of violaxanthin, neoxanthin, β-carotene and lutein. The green fruit showed higher content of violaxanthin, neoxanthin, β-carotene and lutein than other colors at 30 DAA (D1-G or A1-G > B1-W > C1-P) (Fig. 6A). Although carotenoids were accumulated in the fruits of four varieties at 30 DAA, the types and contents were limited, indicating that the synthesis of carotenoids mainly occurs in the middle and late stages of fruit development. As previously reported, the ripening of pepper fruit is accompanied by an increase in the content of most carotenoids (Matsufuji et al., 2007). Consistently, the content of phytoene in four varieties reached 8.5–45.2 μg/g FW at 50 DAA, and the highest accumulation was in B2-O (Fig. 6A). The accumulation of phytoene provides sufficient precursors for the synthesis of downstream carotenes and xanthophylls. After phytoene, the synthesis of carotenoids is divided into two branches, β, ε-carotene and β,β-carotene. In the β, ε-carotene branch, the contents of α-carotene and lutein in A2-Y and B2-O were significantly higher than those in C2-R and D2-R. Compared with the β, ε-carotene branch, the carotenoids in the β, β-carotene branch have more complex accumulation patterns in the four varieties. γ-Carotene and β-carotene act as orange pigments in the β, β-carotene branch, and their contents in C2-R and D2-R were higher than those of A2-Y and B2O. Yellow xanthophylls (β-cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin and neoxanthin) mainly accumulated in A2-Y and B2-O. Capsanthin (red pigment) is the most typical xanthophylls in pepper, and its content in C2-R and D2-R is significantly higher than that of A2Y and B2-O (Fig. 6A). The distinct accumulation pattern of these carotenoids leads to the difference in fruit color among the varieties.

3.7. Candidate Hub genes related to flavonoid synthesis To further determine the relationship between genes within the module and to screen for the Hub gene (highly connected gene), a correlation network was constructed. In the pink module, 58 genes showed a high correlation (with edge weight ≥0.4), including 7 transcription factors, 6 flavonoid synthesis genes, 6 transporters, and 3 plant hormone related genes (Fig. 5A and Table S8). Forin-like protein 11 (FH11), zinc finger protein CONSTANS-LIKE 2 (COL2), Capana01g003186 (unknown Protein), auxin-induced in root cultures protein 12 (AIR12), Capana02g003118 (unknown Protein), disease resistance protein 2 (RPS2) and homeobox-leucine zipper protein (HDZIP) have the highest degree of connectivity in network (Fig. 5A and Table S8). Among these highest degree of connectivity genes, FH11 and Capana01g003186 genes have high correlation with flavonoid synthesis genes: F3H, C4H, flavonol 3′-O-methyltransferase(OMT1), transcription factors: COL2, DIV (DIVARICATA) and transporter: MATE16 (multidrug and toxic compound extrusion protein 16), VPS41 (Vacuolar protein sorting-associated protein 41), indicating FH11 and Capana01g003186 genes may play an important role in the synthesis of flavonoids. In addition, a WD68 protein homologous to Arabidopsis TTG1 is also present in this network and has a high correlation with OMT1, POPTR (potassium transport system protein), RPS2, HD-ZIP, Capana02g003118

3.9. Expression of carotenoid synthesis genes in four pepper varieties PSY, the first catalytic enzyme for carotenoid synthesis, was previously reported to control the synthesis of carotenoids in chromoplasts and determine the color of mature pepper fruits (Huh et al., 2001). In the present study, the Psy1 (Capana04g002519) gene was highly expressed in A2-Y, B2-O and C2-R, resulting in a significantly higher content of phytoene in A2-Y B2-O and C2-R than in D2-R (Fig. 6). Although phytoene was highly accumulated in A2-Y, B2-O and C2-R, some carotenoids (β-carotene, zeaxanthin and antheraxanthin) downstream of phytoene in D2-R are higher than in A2-Y, B2-O and C2-R, indicating Psy1 mainly regulates the accumulation of phytoene to provide a precursor for the downstream carotenoid synthesis and is not directly involved in the color formation of the pepper fruit. Lycopene βcyclase (LCYB) and lycopene ε-cyclase (LCYE) are the key enzymes in 8

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Fig. 5. The correlation networks of genes in the pink (A) and red (B) modules, in which only edges with weight above a threshold of 0.4 and 0.35 are displayed, respectively. Red, genes with the highest degree of connectivity (top 6); Purple, transcription factors; Blue, flavonoid synthetic structural genes; Green: transporter genes; Yellow, hormone synthesis or signaling genes; Dark yellow, sugar metabolism genes; Light blue, residual genes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

γ-carotene and β-carotene in C2-R and D2-R was derived from high expression of LCYB2 (Fig. 6). At the same time, we also uncovered that the lutein of β, ε-carotene branch showed high accumulation in A2-Y (low levels of lutein in C2-R and D2-R) (Fig. 6). These result indicates that LCYE and LCYB synergistically regulate the flux in the β, β-carotene and β, ε-carotene branches of different color pepper fruits, and

the carotenoid synthesis that mediate carbon influx into β, β-carotene and β, ε-carotene branches (Yuan et al., 2015). In this study, we found that the expression of four varieties of LCYE and LCYB genes was related to the accumulation of α-carotene, γ-carotene, and β-carotene (Fig. 6). Specifically, high expression of LCYE and LCYB1 gene in A2-Y resulted in high α-carotene content in A2-Y, while the accumulation of 9

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Fig. 6. Carotenoid levels and heat map of carotenoid synthesis genes. This pathway is constructed based on the KEGG pathway and literary references. A: The carotenoid levels of four varieties at 30 and 50 DAA. Each bar represents the average of three biological replicates plus standard deviation. B: Expression of carotenoid synthesis genes at 30 and 50 DAA. Red indicates high expression, and green indicates low expression. GGPP, geranylgeranyl diphosphates; PSY, phytoene synthase; PDS, phytoene desaturase; Z-ISO, ζ-carotene isomerase; ZDS,ζ-carotene desaturase; CRTISO, carotenoid isomerase; LCYE, lycopeneε-cyclase; LCYB, lycopeneβ-cyclase; CHYB, β-carotene hydroxylase; CYP97A, cytochrome P450-type monooxygenase 97A; CYP97C, cytochrome P450-type monooxygenase 97C; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; CCS, capsanthin-capsorubin synthase; NXS, neoxanthin synthase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2001). High expression of cytochrome P450-type monooxygenase 97C (CYP97C) and cytochrome P450-type monooxygenase 97C (CYP97A) gene in A2-Y at 50 DAA resulted in the highest accumulation of lutein in A2-Y (Fig. 6). In addition to CYP97C and CYP97A, two other hydroxylation enzymes, CHYB1 and CHYB2, are present in the pepper, which act on the β, β-carotene branch. Although CHYB1 and CHYB2 have the

that there may be functional differentiation between the two LCYBs in the process. LCYB1 exerts its function in the β, ε-carotene branch, while LYCB2 acts on the β, β-carotene branch. In addition to the regulation of LCYB and LCYE genes, cytochrome P450-type monooxygenase 97C (CYP97C) and cytochrome P450-type monooxygenase 97C (CYP97A) are particularly critical for the accumulation of lutein (Hirschberg, 10

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Acknowledgements

highest expression in C2-R and D2-R, respectively (CHYB1 and CHYB2 were lowly expressed in D2-R and C2-R, respectively), the contents of βcryptoxanthin and zeaxanthin in C2-R and D2-R are similar to those of A2-Y or B2-O. These data indicate that the two CHYB genes are functionally complementary in carotenoid synthesis, which is in line with the previous research that the two CHYB (HYD) genes are also confirmed to be involved in the accumulation of carotenoids in Arabidopsis (Tian, Magallanes-Lundback, Musetti, & DellaPenna, 2003). Capsanthin-capsorubin synthase (CCS), as a unique enzyme in pepper and tiger lily, converts antheraxanthin and violaxanthin into capsanthin and capsorubin respectively (Yuan et al., 2015). In this study, the CCS genes of four varieties were significantly up-regulated at 50 DAA whereas low level of capsanthin was detected in B2-O and A2-Y (Fig. 6), suggesting that the accumulation of capsanthin does not solely depend on the expression of the CCS gene. Previous studies have demonstrated that the lack of capsanthin in yellow and orange pepper fruits is due to the absence or mutation of CCS gene that results in the inability to translated normal CCS (Li, Wang, Gui, Chang, & Gong, 2013). Interestingly, we observed no significant difference in the expression of zeaxanthin epoxidase (ZEP) and violaxanthin de-epoxidase (VDE) genes between four varieties at 50 DAA, but a significant difference in the content of corresponding antheraxanthin and violaxanthin (Fig. 6). Although the exact mechanism is under investigation, it is possible that the highly active CCS drives antheraxanthin to be converted into capsanthin in C2-R and D2-R, which reduces the flux to violaxanthin, eventually resulting in significantly lower levels of antheraxanthin and violaxanthin in C2-R and D2-R than in A2-Y and B2O.

This work was financially supported by China Agriculture Research System (Grant No. CARS–24-A-05). Conflicts of interest The authors declare no competing financial interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125629. References Appelhagen, I., Lu, G. H., Huep, G., Schmelzer, E., Weisshaar, B., & Sagasser, M. (2011). TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds. The Plant Journal, 67(3), 406–419. Baxter, I. R., Young, J. C., Armstrong, G., Foster, N., Bogenschutz, N., Cordova, T., ... Harper, J. F. (2005). A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America, 102, 2649–2654. Borovsky, Y., Oren-Shamir, M., Ovadia, R. J. W., & Paran, I. (2004). The A locus that controls anthocyanin accumulation in pepper encodes a MYB transcription factor homologous to Anthocyanin2 of Petunia. Theoretical & Applied Genetics, 109(1), 23–29. Chen, S., Zhou, Y., Chen, Y., & Jia, G. (2018). fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 34(17), i884–i890. Chen, W., Gao, Y., Xie, W., Gong, L., Lu, K., Wang, W., ... Luo, J. (2014). Genome-wide association analyses provide genetic and biochemical insights into natural variation in rice metabolism. Nature Genetics, 46(7), 714–721. Chen, W., Gong, L., Guo, Z., Wang, W., Zhang, H., Liu, X., ... Luo, J. (2013). A novel integrated method for large-scale detection, identification, and quantification of widely targeted metabolites: Application in the study of rice metabolomics. Molecular plant, 6(6), 1769–1780. Debeaujon, I., Peeters, A. J., Léon-Kloosterziel, K. M., & Koornneef, M. (2001). The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Plant Cell, 13(4), 853–871. Delgado-Vargas, F., & Paredes-Lopez, O. (2002). Natural colorants for food and nutraceutical uses. CRC Press. Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., & Lepiniec, L. (2010). MYB transcription factors in Arabidopsis. Trends in Plant Science, 15(10), 573–581. Facteau, T. J., Chestnut, N. E., & Rowe, K. E. (1983). Relationship between fruit weight, firmness, and leaf/fruit ratio in L. Canadian Journal of Plant Science, 63(3), 763–765. Ferrer, J. L., Austin, M. B., Stewart, C., & Noel, J. P. (2008). Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiology and Biochemistry, 46(3), 356–370. Forkmann, G. (1991). Flavonoids as flower pigments: The formation of the natural spectrum and its extension by genetic engineering. 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3.10. Verification of RNA sequencing results by qRT-PCR To verify the accuracy of RNA-seq data, we selected 12 differentially expressed genes (6 flavonoid synthesis-related genes and 6 carotenoid synthesis genes) (Tables S1). qRT-PCR was used to analyze the expression levels of these genes in four varieties at 30 DAA (Fig. S7). The results showed that the expression patterns of these genes were consistent with the RNA-seq results, indicating that the RNA data are valid and reliable.

4. Conclusion In this study, targeted metabolome and transcriptome comparisons were carried out using pepper varieties of four different fruit colors. The high expression of genes in the phenylpropane and flavonoid pathways in purple varieties resulted in significantly higher levels of flavonoids (especially quercetin, lutein, chrysoeriol, delphinidin derivatives) in purple varieties than that in the other three varieties. Through WGCNA, two modules positively correlated with flavonoid accumulation were identified, in which transcription factors or transporters such as MYB113, WD68 and SLC40 were predicted to be involved in flavonoid synthesis or transport. Moreover, carotenoids content analysis showed that the accumulation of carotenoids displayed significant variety specificity at 50 DAA. Red and orange carotenoids (except for α-carotene) accumulate mainly in C2-R and D2-R, while yellow lutein has a higher content in A2-Y and B2-O. Together, this study not only provides new insights into the understanding of the synthesis and accumulation of flavonoids and carotenoids in pepper fruit, but also lays a solid biological foundation for the breeding of high pigmented pepper varieties.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 11

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Lightbourn, G. J., Griesbach, R. J., Novotny, J. A., Clevidence, B. A., Rao, D. D., & Stommel, J. R. (2008). Effects of anthocyanin and carotenoid combinations on foliage and immature fruit color of Capsicum annuum L. Journal of heredity, 99(2), 105–111. Ma, G., Zhang, L., Iida, K., Madono, Y., Yungyuen, W., Yahata, M., ... Kato, M. (2017). Identification and quantitative analysis of β-cryptoxanthin and β-citraurin esters in Satsuma mandarin fruit during the ripening process. Food Chemistry, 234, 356–364. Matsufuji, H., Ishikawa, K., Nunomura, O., Chino, M., & Takeda, M. (2007). Anti-oxidant content of different coloured sweet peppers, white, green, yellow, orange and red (Capsicum annuum L.). International Journal of Food Science & Technology. 42(12), 1482–1488. Nesi, N., Debeaujon, I., Jond, C., Stewart, A. J., Jenkins, G. I., Caboche, M., & Lepiniec, L. (2002). The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat. The Plant Cell, 14(10), 2463–2479. Odland, M. L. (1938). Inheritance of the immature fruit color of peppers. Proceedings of the American Society for Horticultural Science, 36, 647–651. Popovsky, S., & Paran, I. (2000). Molecular genetics of the y locus in pepper: Its relation to capsanthin-capsorubin synthase and to fruit color. Theoretical & Applied Genetics, 101(1–2), 86–89. Sagasser, M., Lu, G. H., Hahlbrock, K., & Weisshaar, B. (2002). A. thaliana TRANSPARENT TESTA 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins. Genes & Development, 16(1), 138–149. Tian, J., Han, Z.-Y., Zhang, J., Hu, Y., Song, T., & Yao, Y. (2015). The Balance of Expression of Dihydroflavonol 4-reductase and Flavonol Synthase Regulates

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