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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
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An 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 CiT Galaxy White Mid-Tower PC Gaming Case with 1 x LED Strip 1 x 120mm Rainbow RGB Fan Included Tempered Glass Side PanelAdobe Acrobat Reader DC - The leading PDF Viewer for reliably viewing, printing, signing and commenting on PDF documents. 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 Citrus fruits, members of the Rutaceae family, are widely consumed throughout the world. A prime example is the sweet orange ‘Newhall’ ( C. sinensis), which stands out for its exceptional quality and has its origins in America. Citrus fruits are a rich source of bioactive flavonoids, with the peels often containing a higher concentration of these compounds than pulp and seeds ( Li etal., 2022a; Yu etal., 2022). Previous studies have shown that flavonoid compounds in Citrus peel play a significant role in anti-inflammation, anti-oxidation, immune regulation, and prevention and treatment of multiple respiratory diseases ( Peng etal., 2019; Singh etal., 2020). Unlike most other fruits, Citrus species mainly accumulate flavonone glycosides and polymethoxylated flavones (PMFs) as their main flavonoids ( Zhao etal., 2021). These compounds are highly valued as a source of common Chinese medicines, food, and nutritional supplements due to their abundance of bioactive components. 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. 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
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Increased Air Flow - Top & Front panel incorporate mesh paneling, to improve air flowplastic and mesh steel 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 CiT Level 2 White Micro-ATX Mesh PC Gaming Case with 3 x 120mm RGB Rainbow Fans Included With Tempered Glass Side Panel
CiT LT100 1 Litre USB3.0 Ultra-Thin Mini-ITX Computer Case With 120W Laptop Adpater CPU Cooler WIFI Antenna 5cm Included Bo 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 S8-13 SFF Micro ATX Desktop Case with Mesh Front Panel 8.3 Litre 1x USB3.0 1x USB2.0 1 x 80mm Fan An improved protocol was used to determine the total flavonoid content of citrus peels ( Wang etal., 2007; Yu etal., 2022). Initially, 0.5 g citrus peel powder was weighed and dissolved in 10 mL 70% absolute ethanol at a ratio of 1:20 (w/v). The mixture was then subjected to ultrasonic treatment at 55°C for 40 min, followed by filtration. To 1 mL of the extraction solution, 0.5 mL of 5% NaNO 2 solution was added sequentially and well shaken. The mixture was then left for 5 min. Next, 0.5 mL 10% Al(NO 3) 3 was added to the solution, mixed thoroughly, and left for 6 min. Finally, 5 mL of 1mol/L NaOH was added, and distilled water was added up to 10 mL. The mixture was shaken and left for 10 min to complete the reaction. The absorbance value of solution was measured at 510 nm, with rutin (purity≥98%, sourced from Leaf Shanghai Biological Technology Co., Ltd.) used as the standard product. The flavonoid content (U mol/g) was calculated using the formula (C*V)/W, where C represents the concentration, V represents the volume, and W represents the weight of the sample. To determine the MDA content and SOD activity, Li’s method was followed ( Li etal., 2022b). Metabolomic profile detection and analysis
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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 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 analysis 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 interestIn recent years, analytical methods such as multi-omics have been widely used in the study of food components and functions. To investigate flavonoid compositions in SOPs and elucidate the regulatory mechanism of flavonoid biosynthesis under magnesium stress, transcriptomic and metabolomic analyses were performed. Through these analyses, six hub candidate structural genes and ten hub TF genes involved in flavonoid biosynthesis regulation were identified using WGCNA and CCA. This valuable information enhances our understanding of the nutritional value of SOPs and provides insights into their potential use in food. Materials and methods Plants and sample preparation CiT Luna White Micro-ATX PC Gaming Case with 4 x 120mm Infinity ARGB Fans Included 1 x 4-Port Fan Hub Tempered Glass Side Panel 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. 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
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CiT Terra White Micro-ATX PC Gaming Case with 4 x 120mm Infinity Fans Included Tempered Glass Side PanelProject 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
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