The original contributions presented in the study are publicly available. This data can be found here: NCBI Sequence Read Archive (BioProject: PRJNA934884). Author contributions This study was financially supported by the National Key R&D Program of China (2021YFD1600802-02), the Science and Rechnology Department of Sichuan Province, China (2021ZHCG0084), the 14th- fifth-plan of Breeding in Sichuan Province, China (2021YFYZ0023-14). Conflict of interest To analyze the relative levels of 740 flavonoids and the expression levels of differentially expressed genes in the transcriptome, principal component analysis (PCA) was performed using the default parameters of the Metware data processing platform ( https://www.metware.cn/). Canonical correlation analysis (CCA) was conducted to explore the potential correlation between flavonoid metabolite levels and synthetic genes through metabolomic and transcriptomic data. Specifically, 21 flavonoid content levels and the expression levels of 41 hub genes within the flavonoid synthesis pathway of MS and MD peels at various stages were imported into Canoco 5.0 for CCA analysis, utilizing the default parameters. Quantitative real-time PCR analysis The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note

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CiT LT100 1 Litre USB3.0 Ultra-Thin Mini-ITX Computer Case With 120W Laptop Adpater CPU Cooler WIFI Antenna 5cm Included Increased Air Flow - Top & Front panel incorporate mesh paneling, to improve air flowplastic and mesh steelAn investigation of differentially accumulated flavonoids (DAFs) was conducted in SOPs at different development stages. A total of 740 flavonoids were screened, and 142 DAFs were selected based on a fold change of |log2FC| ≥ 2 or |log2FC| ≤ 0.5 and a variable importance in projection (VIP ≥1) ( Supplementary Table4). Of these, 57 DAFs were identified in MS1 vs. MD1, followed by MS2 vs. MD2 (25) and MS3 vs. MD3 (97) ( Figure4C). The Venn Diagram results revealed two common and unique differential metabolites (Chrysoeriol-7-O-glucoside, Chrysoeriol-7-O-(6’’-feruloyl) glucoside) between MS and MD across all three periods. These flavonoids were flavones, and their change trend was consistent with total flavonoid content. To study the variation of these differential metabolites under magnesium stress, volcano diagrams were performed ( Supplementary Figure5). The results indicated that there were more up-regulated than down-regulated flavonoids in three stages between MS and MD. Specifically, 57 DAFs (53 upregulated and 4 downregulated) were identified during MS1 vs. MD1, 25 DAFs (6 upregulated and 19 downregulated) were identified during MS2 vs. MD2, and 97 DAFs (86 upregulated and 11 downregulated) were identified during MS3 vs. MD3. The majority of DAFs were observed during the development period. There were 278 DAFs (141 upregulated and 137 downregulated) and 261 DAFs (161 upregulated and 100 downregulated) selected from MD1 vs. MD2 and MS1 vs. MS2, respectively. The greater number of DAFs in MD than MS suggested that flavonoids may have been more susceptible to magnesium stress. The interaction of DAFs in SOPs resulted in the formation of different pathways, which were annotated and assigned to the KEGG pathways ( Figure4D). KEGG pathway enrichment analysis showed that flavonoid biosynthesis, phenylpropanoid biosynthesis, flavone and flavonol biosynthesis, secondary metabolites biosynthesis and metabolic pathways were the main enrichment pathways. Therefore, it could be postulated that the differentially accumulated metabolites (DAMs) in the pathways mentioned above may contribute to the variation in flavonoids of SOPs during the developmental process. Differentially expressed gene analysis Project and experiment design, ZW and BX. Experiment execution, QL, JY, ZL, XXY, and XYY. Data analysis, QL, YL, LL, XW, HD, and MZ. Writing, BX and QL. Review, BX and ZW. Project management, BX, GS, and ZW. All authors contributed to the article and approved the submitted version. Funding CiT Pro Android X Gaming Cube White Case with 3 x 120mm Infinity ARGB Fans 1 x 6-Port Fan Hub Tempered Glass Front and Side Panels Mass spectrometry-oriented metabolomics has emerged as a powerful tool for biological research, enabling the systematic identification and quantification of metabolites ( Alseekh etal., 2021). In this study, a widely targeted metabolomic approach combined with transcriptome data was used to explore flavonoid accumulation and its underlying molecular regulation in SOPs under magnesium stress. Although previous studies had explored the flavonoid pathway in citrus, the number of flavonoid components identified remained limited ( Yu etal., 2015; Wang etal., 2019; Yu etal., 2022). In our study, we identified 740 flavonoid components in SOPs, representing a significant supplement to the previous work. These flavonoids were classified into eight categories, with flavones (54.32%) being the most abundant class, followed by flavonols and flavanones ( Figure4A). These results indicated that they are the main flavonoid classes in SOPs and this was consistent with previous research ( Wang etal., 2019). Flavonoid carbonosides, or flavonoid glycosides, which are stable forms of flavonoids with sugar groups bound to an aglycone carbon, were also identified in high numbers (277) in SOPs. These compounds are important phytochemicals in the human diet and have been reported as active components of traditional Chinese medicines with various medicinal properties ( Shen etal., 2022), including anti-inflammatory and antioxidant activities ( Matsia etal., 2022; Wang etal., 2022). The content of Chrysoeriol-7-O-glucoside and Chrysoeriol-7-O-(6’’-feruloyl) glucoside were found to be higher in MD compared to MS during all three stages. In addition, the combined content of all flavonoid carbonosides in MD was also greater than that of MS, which correlated with the overall trend in total flavonoid content ( Figure2C). SOPs under magnesium stress showed higher contents of flavonoid carbonosides, suggesting their potential for functional food production and as a source of bioactive compounds for medication. Furthermore, SOPs also showed higher levels of flavanols, with (-)-Epicatechin-(4β->8)-(-)-epigallocatechin as the predominant component. This suggests that mandarin orange peels under magnesium stress may be a potential source for natural functional beverages and oral liquids. The first two principal components accounted for 50.35% (PC1) and 17.79% (PC2), respectively, and the 18 samples (including 3 replicates) were classified into 6 groups based on their developmental stage along PC1. The sample positions along PC2 were influenced by magnesium stress ( Figure3B). These findings suggest that the observed differences in flavonoid profiles were related to developmental stages and magnesium stress and were consistent with the trend in total flavonoid accumulation that peaked in MS2 or MD2 ( Figures3B, C). In addition, OPLS-DA analysis was utilized to evaluate the differences between MS and MD (Q2 = 0.99) ( Supplementary Figure2). The high Q2 value (>0.9) suggested that the OPLS-DA modules were stable and reliable and that the differences in flavonoid content could be further explored. Hierarchical clustering analysis (HCA) of the flavonoid metabolite accumulation patterns among different samples showed good repeatability within the sample group ( Figures3D). In the HCA, six clusters, corresponding to the successive stages of flavonoid metabolites in SOPs for the 18 samples, were significantly separated. The results of PCA, OPLS-DA, correlation analysis, and HCA reflected large differences between samples, high similarity among the three biological replicates, and high repeatability within samples. Differentially accumulated flavonoids metabolites in SOPs

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AnyDesk - A remote desktop support application that ensures secure and reliable remote desktop connections for our customer support where you may need it. Please note: You will need to generate and provide an ID for our customer support team to make a connection; this application does not allow us to connect without your consent. Correlation analysis and canonical correlation analysis between flavonoids content and expression of synthesis-related genes

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The samples underwent a series of preparation steps, including immersion in liquid nitrogen, grinding to a fine powder, suspension in 70% methanol and centrifugation at 20,000 rpm for 20 minutes at 4°C. The supernatant was then filtered through a 0.22-μm nylon syringe filter before being subjected to UPLC-MS analysis. This involved the use of an Agilent SB-C18 column (2.1 × 100 mm, 1.8 μm), with mobile phase A consisting of water with 0.1% formic acid, while mobile phase B was a solution of 0.1% formic acid in acetonitrile. The elution procedure utilized a gradient of 0-95% B from 0-9 min, followed by a single minute at 95% B and further gradient steps of 95-5% B for 1 minute and 5% B from 11-14 minutes. The flow rate of the system was set at 0.35 mL/min, and the column temperature was held constant at 40°C, with an injection volume of 4 μL. Multiple reaction monitoring (MRM) mode was used to acquire data from the production scan, which was then analyzed with the Metabolites Database (METWARE database) for metabolite identification. Quantitative analysis of metabolites was then performed using Analyst 1.6.3 software, with further examination of the metabolic pathways of these compounds using the KEGG database ( http://www.kegg.jp). Transcriptome analysisBo Xiong *† Qin Li † Junfei Yao Zhuyuan Liu Xinxia Yang Xiaoyong Yu 1 Yuan Li Ling Liao Xun Wang Honghong Deng Mingfei Zhang Guochao Sun Zhihui Wang * CiT Vento White Micro-ATX PC Gaming Case with 4 x 120mm ARGB Fans Included 1 x 6-Port Fan Hub Tempered Glass Side Panel CiT Galaxy White Mid-Tower PC Gaming Case with 1 x LED Strip 1 x 120mm Rainbow RGB Fan Included Tempered Glass Side Panel Environmental factors can regulate gene expression by influencing TFs, which bind specifically to the promoters of their target genes. Among TF families, the MYB family has been shown to play a critical role in regulating gene expression in the flavonoid pathway ( Espley etal., 2007; Zhou etal., 2015; Zhai etal., 2016; Li etal., 2020a). For instance, the MdBBX22–miR858–MdMYB9/11/12 was found to activate the promoters of MdANR and MdLAR in apple, thereby promoting the biosynthesis of proanthocyanidin ( Zhang etal., 2022). In this study, one CitMYB (Cs_ont_1g021030) was identified as highly related to structural genes and seven flavonoids based on WGCNA. Therefore, these ten TF genes were considered important in regulating the flavonoid content of SOPs. Although the results of qRT-PCR showed good consistency with the transcriptome data ( Supplementary Figure10), future studies are needed to elucidate the function of these genes in flavonoid biosynthesis. Conclusion

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CiT Saturn White Micro-ATX PC Gaming Case with 4 x 120mm Infinity ARGB Fans Included 1 x 4-Port Fan Hub Tempered Glass Side Panel CiT Terra White Micro-ATX PC Gaming Case with 4 x 120mm Infinity Fans Included Tempered Glass Side Panel Flavonoids are a significant group of secondary polyphenolic metabolites with a chemical structure of 3-C (C6-C3-C6) ( Li etal., 2020b; Nabavi etal., 2020). These compounds are widely distributed throughout the plant kingdom, and more than 6,000 distinct flavonoids have been identified to date ( Lepiniec etal., 2006; Ferrer etal., 2008). The pathways involved in flavonoid metabolism have been extensively studied in model plants ( Deng and Lu, 2017; Tohge etal., 2017). The flavonoid biosynthesis process begins with the primary glucose produced through photosynthesis, which is then converted into phenylalanine through glycolysis, pentose phosphate, and shikimic acid pathways. Phenylalanine enters the phenylpropane metabolic pathway through the action of phenylalanine ammonylase (PAL). In this pathway, phenylalanine is converted into p-coumaryl CoA through a series of reactions catalyzed by PAL, cinnamate 4-hydroxylase (C4H), and 4-coumaroyl CoA ligase (4CL) ( Forkmann and Martens, 2001; Du etal., 2010; Saito etal., 2013). The flavonoid biosynthesis pathway is initiated by condensation of one molecule of 4-coumaroyl-CoA with three molecules of malonyl CoA by chalcone synthase (CHS) enzyme. Subsequently, chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) lead to the synthesis of anthocyanidin pigments. Flavone synthase (FNS) and isoflavone synthase (IFS) produce flavones and isoflavones, respectively. Flavonol synthase (FLS) catalyzes dihydroflavonols to flavonols, while leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) synthesize cis- or trans-flavan-3-ols, which are precursors of proanthocyanidin (PA) polymers ( Owens etal., 2008; Li etal., 2020a). Regulation of genes involved in flavonoid biosynthesis is controlled through specific mechanisms that vary depending on the species or tissue involved ( Zhang etal., 2022). The accumulation of flavonoids is mainly controlled by structural biosynthetic genes, regulatory MYB transcription factors (TFs) and the MYB-bHLH-WD40 (MBW) complex ( Appelhagen etal., 2011; Hichri etal., 2011; Song etal., 2019; Yang etal., 2023). These factors are responsible for regulating the expression of genes involved in flavonoid biosynthesis, leading to their accumulation. In GO annotation analysis, a total of 38,397 DEGs were annotated in three categories: biological process, molecular function, and cellular component categories ( Supplementary Table7). These DEGs were further divided into 47 categories based on gene function, with 722 genes related to biological processes such as signaling. TopGO analysis revealed that the most enriched molecular function terms were monooxygenase activity (GO0004497), oxidoreductase activity (GO0016705), heme binding (GO0020037), iron ion binding (GO0005506), and tetrapyrrole binding (GO0046906) ( Supplementary Figure7). The most enriched biological process terms were flavonoid metabolic process (GO0009812) and flavonoid biosynthetic process (GO0009813). The most enriched cellular component terms were chloroplast thylakoid (GO0009534) and plastid thylakoid (GO0031976). KEGG enrichment analysis identified the top 20 enriched metabolic pathways, including metabolic pathways (ko01100), biosynthesis of secondary metabolites (ko01110), MAPK signaling pathway-plant (ko04016), plant hormone signal transduction (ko04075), phenylpropanoid biosynthesis (ko00940), and flavonoid biosynthesis (ko00941) under magnesium stress ( Figure5D). These results were presented in a bubble diagram. Metabolic and gene co-expression networks in SOPs at different developmental stages To date, efforts to identify the chemical compositions of citrus, especially flavonoid compounds, have increased significantly ( Barreca etal., 2017; Mahmoud etal., 2019; Yu etal., 2022). However, only a handful of flavonoid components present in citrus, specifically in SOPs, have been successfully identified. This limitation may hinder the growth of SOPs utilization in the food industry. Furthermore, flavonoids are a diverse group of plant metabolites that have diverse structures, wide distribution, and critical roles in plant growth, adaptation, signaling, and response to biotic and abiotic stresses ( Winkel-Shirley, 2001; Treutter, 2005; Rowan etal., 2009; Misra etal., 2010; Zhang etal., 2019). When the environment changes, flavonoid levels may also change. For example, Zanthoxylum bungeanum cv. “Fengjiao” exhibited an increase in total flavonoid content under drought stress ( Hu etal., 2021), while high solar radiation led to an increase in flavonol content in Ginkgo biloba ( Guo etal., 2020). In China, where citrus is mainly grown in subtropical and tropical regions, soil acidification and consequent magnesium (Mg) leaching are major problems ( Long etal., 2017). In addition, improper use of chemicals and inadequate use of organic fertilizers and medium and trace element fertilizers can lead to Mg deficiency in citrus, resulting in a decline in fruit quality and yield. Interestingly, Mg deficiency was found to increase total flavonoid content ( Ramakrishna and Ravishankar, 2011; Bartwal etal., 2013; Li etal., 2017; Khare etal., 2020). However, the underlying mechanism remains unexplored in SOPs.

References

First-strand cDNA was synthesized from each RNA sample (0.2 μg). Specific primers ( Supplementary Table S1), designed by NCBI Primer-BLAST using genome sequences, were used for quantitative real-time PCR (qPCR) cycling on a CFX96 Real-Time PCR Detection System. Real-Time PCR System (Hercules, CA, USA). The qPCR cycling conditions were 95°C for 2 minutes followed by 39 cycles of 95°C for 5 seconds and 57°C for 40 seconds. Actin was utilized as an internal control ( Supplementary Table S1), and biological replicates were triplicated. Statistical analysis Adobe Acrobat Reader DC - The leading PDF Viewer for reliably viewing, printing, signing and commenting on PDF documents.