Metabolome and Transcriptome Analysis of Flavor Components and Flavonoid Biosynthesis in Fig Fruit (Ficus Carica L.) After Bagging

Background: Bagging can improve the appearance of fruits and increase the food safety and commodication, it also has effects on intrinsic quality of the fruits, which was commonly reported negative changes. Fig can be regarded as a new model fruit with its relatively small genome size and long fruit season. Results: In this study, widely targeted metabolomics based on HPLC MS/MS and RNA-seq of the fruit tissue of the ‘Zibao’ g before and after bagging were analyzed to reveal the metabolites changes of the edible part of gs and the underneath gene expression network changes. A total of 771 metabolites were identied in the metabolome analysis using g female ower tissue. Of these, 88 metabolites (including one carbohydrate, eight organic acids, seven amino acids, and two vitamins) showed signicant differences in fruit tissue before and after bagging. Changes in 16 structural genes, 13 MYB transcription factors, and endogenous hormone (ABA, IAA, and GA) metabolism and signal transduction-related genes in the biosynthesis pathway of avonoids after bagging were analyzed by transcriptome analysis. KEGG enrichment analysis also determined signicant differences in avonoid biosynthesis pathways in female ower tissue before and after bagging. Conclusions: This work provided comprehensive information on the composition and abundance of metabolites in the female ower tissue of g. The results showed that the differences in avor components of the fruit before and after bagging could be explained by changes in the composition and abundance of carbohydrates, organic acids, amino acids, and phenolic compounds. This study provides new insights into the effects of bagging on changes in the intrinsic and appearance quality of fruit.


Background
addition to providing sugar and energy, g fruit is also rich in dietary ber and is considered a good source of minerals [1], sterols [2], carotenoids [3], anthocyanins [4], and polyphenols [5]. The development of g fruit demonstrates a typical double-S-shaped curve, with two rapid growth phases (phases I and III) and one slow growth phase (phase II) between them. Fig is a female ower hermaphrodite plant with diverse fruit color, the edible part is the compound fruit formed from the enlarged in orescence and receptacle of female owers, botanically, it is a false fruit termed 'Syconium'. The coloration of g pericarp and female ower tissue shows obvious space-time and expression differences in that the coloration of female ower tissue initiates very early, while the accumulation of anthocyanin in the pericarp is signi cantly later. The process of anthocyanin accumulation in the pericarp is very fast compared with that of female ower tissue, and the concentration is also signi cantly higher. Fruit color is an important index to evaluate the fresh food quality and commodity value of fruit because a bright and attractive fruit color is one of the most important factors affecting the choice of growers and consumers [7,8].
Anthocyanins, water-soluble avonoids, are important secondary metabolites in plants and have the physiological function of resisting UV damage, pests and diseases, and attracting insect pollination [9]. They also have a strong free radical scavenging effect and have many biological activities, such as cardiovascular protection [10] and anti-tumor properties [11]. Anthocyanins are classi ed according to the location of phenolic hydroxyl and methyl groups, there are at least 13 types of anthocyanins in nature, six of which are common and include petunidin, peonidin, cyanidin, delphinidin, pelargonidin, and malvidin.
Currently, these six types of anthocyanins and their derivatives are about 95% of the total amount of anthocyanins in nature. The biosynthesis pathway of anthocyanins has been studied in horticultural plants in detail. These studies have identi ed important upstream structural genes, such as chalcone synthase (CHS) and chalcone isomerase (CHI), and downstream structural genes, such as dihydro avonol reductase (DFR), anthocyanidin synthase (ANS), and avonoid 3-O-glycosyl transferase (UFGT), in the avonoid biosynthesis pathway. The expression of anthocyanin synthetic structural genes is regulated by a variety of transcription factors (TFs). According to the different conserved domains contained in TFs, they are roughly divided into MYB, bHLH, WD40, MADS-box, and bZIP families [12], among which the most studied is the MYB family. MYB is one of the most characteristic families of TFs, widely distributed in all eukaryotes, including animals, plants, and fungi. MYB proteins can be divided into four types based on the number of MYB repeats:1 R-MYB, R2R3-MYB, R1R2R3-MYB, and 4 R-MYB [13]. The studies on the regulation of anthocyanin biosynthesis in the MYB TF family mainly focus on the R2R3-MYB TF, and the study on the regulation of anthocyanin in fruit trees by the R2R3-MYB TF began late. In 2006, Espley et al. found a light-induced anthocyanin synthesis gene MdMYB1 in apple, followed by two types of genes regulating anthocyanin biosynthesis in pulp, MdMYB10 [14] and MdMYB110a [15]. With the separation and functional validation of the key TFs, MdMYB1 and MdMYB10, of anthocyanin in apple, the homologous genes were isolated successively in plants such as strawberry [16], peach [17], pear [18], sweet cherry [19], and pomegranate [20]. R2R3-MYB regulation can be divided into activation and inhibition, for example, FaMYB1 in strawberries [21], AtMYBL2 in Arabidopsis [22], and VvMYBC in grapes [23], which inhibit anthocyanin synthesis.
Bagging is used frequently in the cultivation and management of fruit trees and is widely applied in apple, pear, grape, and other fruit trees. Fruit bagging can improve the appearance and intrinsic quality of fruit to a certain extent, reduce pesticide residues, and improve the food safety and commodi cation of fruit. When there is a difference in light sensitivity between the pericarp and pulp coloration, bagging can change the in uence of external environmental factors, especially the light signal, on anthocyanin synthesis. Bagging also has effects on intrinsic quality of the fruits, which was commonly reported negative changes. Fig can be regarded as a new model fruit with its relatively small genome size and long fruit season. As a non-photosensitive part, the anthocyanin synthesis regulation gene and the appearance of avor components of the female ower tissue of g may have another regulatory mechanism. Arabidopsis with mutant cop1 can maintain anthocyanin synthesis under dark conditions, PAP1 and PAP2 activate structural genes PALs, CHS, CHI, F3H, F3 H, ANS, and DFR, promoting anthocyanin biosynthesis [24]. Bagging has no great in uence on anthocyanin accumulation in nonphotosensitive fruits, but it has a great impact on the formation of avor components and taste. The appearance of the fruit after bagging improves signi cantly, however, the taste and avor of the fruit is another important factor affecting customer choice. Fruit odor (caused by volatile compounds such as phenols and alcohols) and taste (determined by the proportion and concentration of sugars, organic acids, and amino acids) contribute to the formation of fruit avor [25]. Previous studies have focused on the identi cation of color, soluble solids, total sugar, and total acid components of the fruit after bagging, however, research on the changes of internal transcription and metabolite networks in the fruit after bagging is lacking.
In this study, the g variety 'Zibao' with both colored pericarp and pulp was used as the research material. To better understand the effect of bagging treatment on fruit color quality and avor components, the transcriptome and widely targeted metabolome were used jointly to analyze bagged fruit. The changes of related secondary metabolites including carbohydrates, organic acids, and amino acids in female ower tissues before and after bagging in g fruit were identi ed by the metabolome, and the differential changes of related MYB TFs, structural genes and endogenous hormone (ABA, IAA, and GA) metabolism and signal transduction-related genes in the anthocyanin synthesis pathway were analyzed through the transcriptome. This study investigated how the molecular mechanism of bagging affects the color quality and the formation of avor components in g fruit, which provides a theoretical basis for in-depth analysis into the gene regulation mechanism of g fruit coloration and further delivers a reference to produce high-quality g fruit.

Plant material and treatment
The common g cultivar 'Zibao' (the formal identi cation was approved by the State Forestry Administration of China, the new variety right number [20150145]) was planted at the Shangzhuang Experimental Station (40°23 N, 116°49 W) of China Agricultural University, Haidian District, Beijing, China. The original source of the g materials used from Weihai Changshoukang Food Co., Ltd. in Shandong province. Experimental research on g comply with China and Shandong province local legislation. The trees have been cultivated for 5 years and planted in a greenhouse with a row spacing and plant spacing of 2 m × 3 m. Double-layer opaque paper bags with a black inside and light brown outside (150 mm × 150 mm, Zhengguo Paper Bag, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences) were used for shading the gs. Figs were sampled in the late stage of phase III and termed CK (natural growth of female owers) and BF (bagged female owers). There were three biological replicates per sample each with 30 fruits collected randomly from ve trees. We took the gs back to the laboratory, and the female owers (about 10 g in weight) were carefully excised with a razor blade. The female owers were immediately frozen in liquid nitrogen and stored at -80 °C for further use.

Sample preparation and metabolite extraction
Fig female ower samples were further ground and thoroughly mixed with a mortar and pestle in liquid nitrogen. The freeze-dried tissue was crushed using a mixer mill (MM 400, Retsch) with Zirconia beads for 1.5 min at 30 Hz. The powdered sample (100 mg) was weighed and extracted overnight at 4 °C with 1.0 mL 70% aqueous methanol. Following centrifugation at 10,000 × g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 mL; ANPEL, Shanghai, China, www.anpel.com.cn/cnw) and ltrated (SCAA-104, 0.22 µm pore size; ANPEL) before LC-MS analysis.

Metabolite identi cation and quanti cation
LIT and triple quadrupole (QQQ) scans were acquired on a QQQ-linear ion trap mass spectrometer (Q TRAP), API 6500 Q TRAP LC/MS/MS system, equipped with an ESI Turbo Ion-Spray interface, operating in a positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature, 500 °C; ion spray voltage (IS) 5500 V; ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 µmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. Declustering potentials (DP) and collision energies (CE) for individual MRM transitions were done with further DP and CE optimization. A speci c set of MRM transitions were monitored for each period according to the metabolites eluted within this period. Metabolite data analysis was conducted with Analyst 1.6.1 software (AB SCIEX, Ontario, Canada). Metabolite quanti cation was carried out using MRM. Partial least squares discriminant analysis (PLS-DA) was carried out with the metabolites identi ed. Metabolites with signi cant differences in content were set with thresholds of variable importance in projection (VIP) ≥ 1 and fold change ≥ 2 or ≤ 0.5 [27,28].

RNA-seq and annotation
Six libraries representing the two female ower samples and the three replicates were constructed for transcriptome sequencing. Total RNA extraction from g material was performed using the CTAB method [29]. RNA concentration and purity were measured by NanoDrop 2000 (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA), respectively. After RNA integrity was determined by 1% agarose gel electrophoresis, RNA concentration was adjusted to the same level. mRNA was isolated from total RNA using magnetic beads with oligo (dT); cDNA was synthesized using a cDNA Synthesis Kit (TaKaRa, Japan) and linking the sequencing adapter to both ends [30]. The library preparations were sequenced on an Illumina HiSeq 4000 platform and the unigene sequences obtained from our laboratory transcriptome database by RSEM software were integrated for annotation [29].

Transcriptome data analysis
Raw reads were processed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) to lter out adapters and low-quality sequences. For gene expression analysis, counts were mapped to the reading of each gene by HTSeq (v0.5.4p3) and then normalized to fragments per kilobase of transcript per million mapped reads (FPKM). EdgeR software (http://www.bioconductor.org/packages/2.12/bioc/html/edgeR.html) was used to analyze differentially expressed genes (DEGs). Screening of signi cant DEGs was performed with p-value (p-FDR) ≤ 0.05 and |log2FC| ≥ 1 as the criteria. Enrichment analyses were performed using GOatools (https://github.com/tanghaibao/GOatools) and Fisher's exact test. To control the calculated false positive rate, the p-values were corrected using four multiple test methods, and signi cant differences in GO functionalization were performed on the differential genes at p ≤ 0.05. KEGG pathway enrichment analysis was performed using KOBAS software (http://kobas.cbi.pku.edu.cn/home.do) with a corrected P-value ≤ 0.05 [27].

Veri cation by using RT-qPCR
Based on the transcriptome data of CK and BF g female owers, the expression level of 15 DEGs were validated. The PCR was performed with an ABI 7500 Fast Real-Time Detection System (Applied Biosystems) using the Ultra SYBR Mix kit (TaKaRa, Japan). The ampli cation system consisted of 10 µL Ultra SYBR Premix System II, 0.5 µL of 10 µmol/L upstream primer, 0.5 µL of 10 µmol/L downstream primer, 2 µL template, and double distilled water to a total volume of 20 µL. The ampli cation program was 95 °C for 10 min, followed by 40 cycles of 95 °C for 5 s and 58 °C for 30 s. Relative quantitative analysis of data was performed by the 2 −ΔΔCT method with β-actin as the reference gene. The primers used for RT-qPCR are listed as Additional le 1: Table S1.

Widely targeted metabolome and KEGG classi cation analysis of differential metabolites
The coloration of mature g pericarp must be illuminated to promote anthocyanin accumulation under pulsed light irradiation [31]. The subsequent experiments showed that the coloration of fruit pericarp was regulated by the light signal, and the accumulation of anthocyanin stopped almost completely under bagging conditions. However, the coloration of the female ower tissue of g was not affected by the light signal, and the development of anthocyanin in the female ower was barely affected under the condition of fruit bagging, and the content of anthocyanins of female ower in the mature stage was basically the same as that of the control group, for the red variety; the female ower tissue in the mature stage is bright red (Fig. 1). Although there is no signi cant difference in the color of female ower tissue, the avor and taste are still slightly different. To explore the effect of bagging on fruit avor components, the secondary metabolites of samples before and after bagging were analyzed by a widely targeted metabolome. A total of 771 compounds in 16 classes were detected (Table S2). Principal component analysis (PCA) was conducted with 771 metabolites, 49.21% of PC1 and 16.02% of PC2. PCA separated the two varieties and the quality control (QC) samples, the signi cance was 0.01 ( Fig. 2A). With Log2FC ≥ 2 and ≤ 0.5, and VIP ≥ 1, 43 upregulated and ve downregulated metabolites were identi ed (Fig. 2B).
Using metabolite concentration data for the cluster analysis of a strati ed heat map of the samples, it was observed that all biological replicates were grouped together (top of the gure), which indicates a high reliability of the resulting metabolome data (Fig. 2C). Interestingly, a clear distinction between the unbagged fruit samples (CK) and the bagged fruit samples (BF) were observed, suggesting a distinct difference in metabolite characteristics in both samples. The metabolites (left side of the gure) were also clustered into two main groups, showing opposite accumulation levels between red female ower tissue samples before and after bagging. A total of 771 metabolites were mapped to the KEGG database and the results indicated that most metabolites were associated with "metabolism". Some metabolites were classi ed as "biological systems" or "human diseases", which suggested that some metabolites in the female ower tissues of g may have potential health effects. KEGG enrichment analysis showed that there were mainly 35 groups, among which, the metabolites of "biosynthesis of plant secondary metabolites", " avone and avonol biosynthesis", and "metabolic pathway" were three groups with signi cantly different female ower tissues before and after bagging (p < 0.05) (Fig. 2D).

Phenylpropanoids, avone, avonol, avanone, and anthocyanins
A total of 234 substances were detected by avonoid metabolite analysis, of which 32 reached a level of signi cant difference. For further mining differential metabolites, data was analyzed by PLS-DA ( Figure  S1). The PLS-DA score map exhibited a distinct separation between groups and obvious clustering within the group, which further suggested that the difference between the two groups is signi cant. The quality parameters of the model with two principal components were as follows: R2X = 0.643, R2Y R2X = 0.995, and Q2R2X = 0.739, which indicated that the current model had a better ability to interpret and predict data. Using VIP ≥ 1.0 and | Log 2 FC | ≥ 1 as a threshold for signi cant differences, 32 avonoid metabolites were identi ed from 'CK vs. BF' samples as signi cantly differentially expressed, including 12 avones, nine phenylpropanoids, ve avonols, three anthocyanins, and one avanone ( Table 1). The cyanidin O-acetylhexoside and cyanidin 3-O-malonylhexoside were found to be upregulated by 1.13 times and 1.06 times compared with CK in the female ower tissue of the bagged fruit, which explained the slightly darker color of the bagged fruit compared with CK. The phenylpropanoid biosynthesis pathway is upstream of the anthocyanin and avonoid biosynthesis pathway. Nine phenylpropanoid secondary metabolites were identi ed, among which the expression of six was upregulated and three were downregulated. The phenethyl caffeate, angelicin, and methyl p-coumarate contents in female ower tissues increased 8.26 times, 2.87 times, and 2.20 times in 'CK vs. BF', while the contents of 3,4,5-trimethoxycinnamic acid, ioacteoside, and 3,4-dihydrocoumarin decreased 2.26,1.48, and 1.09 times, respectively. Twelve avonoids were detected in female ower tissue, 11 of which were upregulated. The biggest differences were shown in three groups: apigenin, nobiletin, and tangeretin. Among them, the upstream substrate apigenin of luteolin increased signi cantly in BF and was upregulated by 11.11 times. VIP ≥ 1, and fold change ≥ 1.6 and ≤ 0.6 was set as the threshold of signi cant difference, a total of 19 signi cantly differentially expressed avor-related metabolites were identi ed from the 'CK vs. BF' samples, including one carbohydrate, seven amino acids and derivatives, eight organic acids and derivatives, and three vitamins and derivatives (Table 2). D(-)-Threose was upregulated 3.70 times more than that of CK in the female ower tissues of bagged fruit. Seven substances of amino acids and derivatives were screened from the female ower tissue before and after bagging, two of which were upregulated, while ve were downregulated. In the signi cantly downregulated metabolites, L-aspartic acid, CYS-GLY, Lhomocysteine, phenylacetyl-L-glutamine, and aspartic acid all reached more than ve times the signi cant difference level, which indicates that the bagged fruit did not have a rich proportion of sugar and acid compared with the CK, leading to a reduction in the avor. Among them, the upstream substrate apigenin of luteolin increased signi cantly in BF and was upregulated by 11.11 times. The phenylpropanoid biosynthesis pathway is upstream of the anthocyanin and avonoid biosynthesis pathway. In 'CK vs. BF', eight signi cantly different organic acids and derivatives were screened, of which seven were upregulated and one was down-regulated. Among these, the α-hydroxyisobutyric acid, (S)-(-)-2-hydroxyisocaproic acid, and isochlorogenic acid B contents increased 4.61 times, 3.05 times, and 2.28 times, respectively, while the phosphoenolpyruvate trisodium salt content decreased 1.46 times.  (Table S3). Different genes were identi ed between the two samples and ltrated and corrected by FDR < 0.05 and | log2FC | ≥ 2. By comparing the number of different genes between the control and bagged group between the pericarp and the female ower, 2389 signi cantly different genes were found in the 'CK vs. BF', among which the number of up-regulated genes was slightly higher than that of down-regulated genes, 1208 and 1181, respectively ( Figure S2 A). Gene Ontology (GO, http://www.geneontology.org/) annotation of the DEGs found that 1,154 unigenes were annotated to 'Biological Process', 674 unigenes were annotated to 'Cellular Component', and 572 unigenes were annotated to 'Molecular Function' (Figure S2 B). To identify the biological pathways activated in g female owers, the normative reference pathway for annotation sequences to the KEGG database was chosen. In KEGG pathways, protein processing in the endoplasmic reticulum, plant hormone signal transduction, and plant-pathogen interaction pathways were signi cantly changed in the 'CK vs. BF' group with corrected P-value ≤ 0.05 (Table S4, Figure S2 C).
Gene expression at the transcriptional level plays an important role in regulating and controlling many biological processes, TFs are the key to the regulation of secondary metabolite genes. After bagging, the original red pulp of the g 'Zibao' did not change, showing non-light dependency. In 'CK vs. BF', 13 MYB and 17 bHLH family members showed signi cant differences. MYB TFs are widely found in plants and are involved in almost all aspects of plant development and metabolism. In these MYB families, the expression of 12 genes was upregulated, of which c43673_g1, c12586_g1, c44925_g1, and c36728_g1 were different by more than four times, 4.87, 4.81, 4.24, and 4.14 times, respectively, while c66005_g2 was downregulated 3.8 times (Fig. 3B). The basic helix-loop-helix (bHLH) family is the second largest family of TFs in plants and has many functions, including regulating ower organ development, photomorphogenesis, epidermal hair, and stomatal formation, plant hormone response, and avonoid metabolism [12] Eight genes from the bHLH family were signi cantly upregulated in the 'CK vs. BF', c66694_g1(4.94), c81283_g1(3.68), c72970_g1(3.66), c43099_g1(3.42), c25473_g1(2.92), c43008_g2(2.72), c39854_g1(2.16), and c33397_g1(2.12), while nine genes showed a trend of signi cant downregulation, c43844_g1 and c40266_g1 were different by more than three times, 3.71 and 4.39 times, respectively (Fig. 3B). The bHLH family members are associated with anthocyanin biosynthesis in fruit trees and have been shown to interact with MYB TFs to regulate fruit color. The co-expression of bHLH transcription factor VvMYC1 and VvMYBA1 can accumulate anthocyanins in grape suspension cells [32].
The interaction of MdbHLH3 and MdbHLH33 with MYB TFs is involved in the regulation of anthocyanin synthesis in apple fruit [33].

Changes of endogenous plant hormone metabolism and signal transduction genes after bagging
The endogenous hormones GA and ABA are transformed from glucose molecules, a direct product of photosynthesis, through plant isoprene biosynthesis, which regulates anthocyanin biosynthesis [34]. In ABA biosynthesis, except for c36086_g2, which was signi cantly upregulated, all of the other four 9-cisepoxycarotene dioxygenase (NCED) genes were signi cantly downregulated. Eight genes were annotated as possible protein phosphatase 2 C (PP2C), signi cantly upregulated, and two genes (c2285_g1 and c25449_g1) were annotated as ABA-activated protein kinase 2(SNRK2) in female owers, and four ABRE binding factor (ABF) genes were signi cantly downregulated after bagging. One ABA 8 -hydroxylase (ABA 8 -h) gene (c23609_g1) was signi cantly downregulated 3.17 times for ABA catabolism (Fig. 4A). In IAA biosynthetic genes, three were annotated as indole-3-acetic acid-inducible protein (ARG7) genes: one signi cantly upregulated gene c39732_g4, two signi cantly downregulated genes c1801_g1 and c45831_g4, three auxin response factors (ARF) were upregulated, c20025_g1 and c20025_g2, and downregulated, c32996_g1. Moreover, there was one upregulated Gretchen Hagen3 (GH3) gene c78527_g1, one upregulated signal transduction auxin-instream vector (AUX1) gene, and two IAA-amino acid hydrolase (IAH) genes c32134_g2 and c32501_g1 (Fig. 4B). Gibberellin GA plays an important role in the ripening of strawberries and sweet cherries [35]. The GA degradation gene GA2 oxidase (GA2ox) c32275_g2 was signi cantly up-regulated by 2.88 times in the 'CK vs. BF', and the GA stimulus transcript (GASTI) gene c27193_1 was inversely regulated, which decreased 2.32 times in the bagged female ower tissue (Fig. 4C).

The qRT-PCR validation
To verify the key results of the RNA-seq, the number of 15 avonoid biosynthesis pathways structural genes and endogenous hormone signal transduction pathway genes were selected for validation and their expression levels in CK and BF were analyzed by qRT-PCR ( Figure S3). The results con rmed that the expression level of these structural genes was similar to that of the RNA-seq results, and there was good agreement with the up-and downregulated gene expression at the RNA-seq level.

Discussion
Bagging can improve the sensitivity of the fruit to light and affect the intrinsic and appearance quality of the fruit while changing the microenvironment of fruit development. The avor components of fruit are generally related to the contents of sugar, organic acids, and phenols. Previous studies have focused on the effect of bagging on pericarp color, while studies on avor changes have just focused on several speci c types of metabolites, such as sugars (fructose, glucose, and sucrose), organic acids, amino acids, and alcohols [36,37]. To date, the overall variation of secondary metabolites of g fruits by bagging has not been studied. In this study, an LC-MS/MS-based widely targeted metabolomics approach was used to understand the avor changes of g fruit after bagging. A total of 771 metabolites were identi ed, 88 of which were accumulated in female ower tissues after bagging in a differentially expressed manner. A large amount of carbohydrates were con rmed in the tissues of female g owers (Table S2), including 20 sugars, 12 of which had signi cantly reduced concentrations in female ower tissue after bagging, including D-glucose 6-, D-fructose 6-, ribulose-5-phosphate, glucose-1-phosphate, and D-fructose 6-phosphate-disodium salt. These ve reduced sugar types are the main sugar types that form the avor of g fruit, while 115 types of organic acids were identi ed, the concentration of half (52) were signi cantly reduced in female ower tissue after bagging, which can partly explain the change in the avor of the fruit. The composition and richness of amino acids are the key indexes of nutritional quality and are also important for determining avor [38]. Previous studies have identi ed 12 amino acids in g fruit [39]. However, 99 amino acids were identi ed by widely targeted metabolome analysis, seven of these accumulated differences between CK and SF ( Table 2). Five of these amino acids (L-aspartic acid, CYS-GLY, L-homocysteine, phenylacetyl-L-glutamine, and aspartic acid) were signi cantly reduced after bagging (Table 2). Therefore, the results show that differences in amino acid composition and abundance can also lead to changes in avor matter.  [35]. The GO enrichment analysis of the DEGs described above indicates that, functionally, GA participates in photo-mediated anthocyanin biosynthesis (Figs. 4). Meanwhile, HY5 and PP2CA, as well as PIF3 and PYL10, are co-expressed with other TFs and structural genes involved in anthocyanin biosynthesis. Similarly, members of possible interaction pathways HY5-SLY1-GASA3 and PIF3-GID1A are co-expressed with anthocyanin biosynthetic genes. At present, it has been found that ABA plays an important role in the development, maturation, and postharvest stage of g female ower tissue.
ABA is produced rapidly in female ower tissue in gs before color conversion and reaches the highest level before the commercial maturation period, whereas the anthocyanin content changes later than ABA. The ABA synthesis inhibitor NDGA inhibits the accumulation of anthocyanin, which indicates that ABA is one of the important factors to promote fruit coloring during the normal development of g [35]. Hence, this study proposes a co-expression network that involves ABA-HY5-MYB in the biosynthesis of anthocyanin in female ower tissue. To understand whether the change of the expression level of numerous hormone metabolism and synthesis-related genes in fruit affects the change of avor components in a later stage of fruit, future studies will be carried out on hormone metabolites in fruit after bagging. This study will lay the foundation for exploring the molecular and metabolic mechanisms of plant hormones and fruit avor component formation.

Conclusions
In this study, HPLC-MS/MS-based metabolome and transcriptome analysis were successfully performed to systematically compare avor component differences and avonoid biosynthesis changes before and after bagging in g fruit. This work provided comprehensive information on the composition and abundance of metabolites in the female ower tissue of g. It also preliminarily explored the difference in patterns of the space-time coloration of different g tissues. To identify the various regulatory patterns of structural genes and MYB TFs involved in avonoid biosynthesis pathways, as well as component differences and concentration changes of carbohydrates, organic acids, amino acids, phenols, and alcohols in female ower tissue, the underlying causes of changes in fruit avor quality after bagging were determined.