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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 analysis
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Du, H., Huang, Y., Tang, Y. (2010). Genetic and metabolic engineering of isoflavonoid biosynthesis. Appl. Microbiol. Biotechnol. 86, 1293–1312. doi:10.1007/s00253-010-2512-8 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 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 analysisEnvironmental 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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Collins, M. E., Obreza, T. A., Morgan, K. T., Graham, J., Alferez, F. (2020). “Nutrition of Florida citrus trees,” in Chapter 2. production areas, soils, and land preparation, 3rd Edition (University of Florida George A Smathers Libraries).Misra, P., Pandey, A., Tiwari, M., Chandrashekar, K., Sidhu, O. P., Asif, M. H., et al. (2010). Modulation of transcriptome and metabolome of tobacco by arabidopsis transcription factor, AtMYB12, leads to insect resistance. Plant Physiol. 152, 2258–2268. doi:10.1104/pp.109.150979 Correlation analysis and canonical correlation analysis between flavonoids content and expression of synthesis-related genes Zhou, H., Lin-Wang, K., Wang, H., Gu, C., Dare, A. P., Espley, R. V., et al. (2015). Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J. 82, 105–121. doi:10.1111/tpj.12792
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Kwon, M. C., Kim, Y. X., Lee, S., Jung, E. S., Singh, D., Sung, J., et al. (2019). Comparative metabolomics unravel the effect of magnesium oversupply on tomato fruit quality and associated plant metabolism. Metabolites 9. doi:10.3390/metabo9100231 Ahmed, O. M., Hassan, M. A., Abdel-Twab, S. M., Azeem, M. N. A. (2017). Navel orange peel hydroethanolic extract, naringin and naringenin have anti-diabetic potentials in type 2 diabetic rats. Biomed. Pharmacother. 94, 197–205. doi:10.1016/j.biopha.2017.07.094 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 * 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 Song, Z., Luo, Y., Wang, W., Fan, N., Wang, D., Yang, C., et al. (2019). NtMYB12 positively regulates flavonol biosynthesis and enhances tolerance to low pi stress in nicotiana tabacum. Front. Plant Sci. 10. doi:10.3389/fpls.2019.01683
Owens, D. K., Alerding, A. B., Crosby, K. C., Bandara, A. B., Westwood, J. H., Winkel, B. S. (2008). Functional analysis of a predicted flavonol synthase gene family in arabidopsis. Plant Physiol. 147, 1046–1061. doi:10.1104/pp.108.117457
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Khare, S., Singh, N. B., Singh, A., Hussain, I., Niharika, K., Yadav, V., et al. (2020). Plant secondary metabolites synthesis and their regulations under biotic and abiotic constraints. J. Plant Biol. 63, 203–216. doi:10.1007/s12374-020-09245-7 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 Wang, F., Chen, L., Chen, H. P., Chen, S. W., Liu, Y. P. (2019). Analysis of flavonoid metabolites in citrus peels (Citrus reticulata “Dahongpao”) using UPLC-ESI-MS/MS. Molecules 24. doi:10.3390/molecules24152680 Hichri, I., Barrieu, F., Bogs, J., Kappel, C., Delrot, S., Lauvergeat, V. (2011). Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J. Exp. Bot. 62, 2465–2483. doi:10.1093/jxb/erq442 The original contributions presented in the study are publicly available. This data can be found here: NCBI Sequence Read Archive (BioProject: PRJNA934884). Author contributionsAnyDesk - 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. Adobe Acrobat Reader DC - The leading PDF Viewer for reliably viewing, printing, signing and commenting on PDF documents. Forkmann, G., Martens, S. (2001). Metabolic engineering and applications of flavonoids. Curr. Opin. Biotechnol. 12, 155–160. doi:10.1016/s0958-1669(00)00192-0 Li, Y., Chen, Q. Y., Xie, X. D., Cai, Y., Li, J. F., Feng, Y. L., et al. (2020b). Integrated metabolomics and transcriptomics analyses reveal the molecular mechanisms underlying the accumulation of anthocyanins and other flavonoids in cowpea pod (Vigna unguiculata l.). J. Agric. Food Chem. 68, 9260–9275. doi:10.1021/acs.jafc.0c01851 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.
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