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CiT Level 2 White Micro-ATX Mesh PC Gaming Case with 3 x 120mm RGB Rainbow Fans Included With Tempered Glass Side Panel 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 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. 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 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 *
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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. ConclusionThe 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 CiT LT100 1 Litre USB3.0 Ultra-Thin Mini-ITX Computer Case With 120W Laptop Adpater CPU Cooler WIFI Antenna 5cm Included 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 Free WiFi Dongle provided to enable WiFi onto the computer. Please note if streaming or gaming online we recommend upgrading the dongle or connecting by Ethernet cable. CiT Level 1 White Micro-ATX PC Gaming Case with 3 x 120mm RGB Rainbow Fans Included With Tempered Glass Front and Side Panel
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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 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. 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 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. FundingCiT 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 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
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CiT Terra White Micro-ATX PC Gaming Case with 4 x 120mm Infinity Fans Included Tempered Glass Side PanelAn 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 The original contributions presented in the study are publicly available. This data can be found here: NCBI Sequence Read Archive (BioProject: PRJNA934884). Author contributions
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