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 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. 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 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

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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 analysisThis 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 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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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. RNA isolation and sequencing were performed by Metware Biotechnology Co., Ltd. (Wuhan, China). The quality of the cDNA libraries was examined, and PCR amplification and sequencing were performed using an Illumina HiSeq™ 2500 platform. The obtained reads were processed to remove contaminants and mapped to the reference genome sequence of C. sinensis v3.0 ( http://www.hzau.edu.cn) using HISAT 2.2.4. StringTie was used for transcript assembly. Fragments per kilobase per million (FPKM) values were calculated to determine gene abundance and normalize data. Differential expression analysis of mRNAs was performed using DESeq2 software. DEGs were identified based on fold change > 2 and false discovery rate (FDR) < 0.05 as cutoff values (P < 0.05). Weighted gene co-expression network analysis 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. 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 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.

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In 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 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 Adobe Acrobat Reader DC - The leading PDF Viewer for reliably viewing, printing, signing and commenting on PDF documents. 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 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 PanelCiT Level 1 White Micro-ATX PC Gaming Case with 3 x 120mm RGB Rainbow Fans Included With Tempered Glass Front and 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. 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 *