Characterization And Metabolic Diversity Of Flavonoids In Citrus Fruits

Flavonoids are widely distributed in plants and play important roles in many biological processes of organisms.It is also the most abundant polyphenolic compound in the human diet and has many functions such as anti-inflammatory,anti-oxidation,anti-cancer,and prevention of cardiovascular diseases.Citrus fruits are rich dietary sources of flavonoids.However,there have been very few reports about the comprehensive metabolic profile and natural diversity of flavonoids in different tissues of various Citrus cultivars.Our study established a database of flavonoid metabolites on 62 Citrus germplasm resources based on non-targeted metabolomics approach and conducted quantitative studies through widely targeted metabolomics approach.Firstly,through non-targeted metabolomics approach detected 7416 metabolic signals in samples of Citrus germplasm.Principal Component Analysis revealed the flavedo has the largest differences from other tissues in metabolite levels.117 different modified types of flavonoids were annotated in the major five species cultivated Citrus germplasms(pummelo,lemon,grapefruit,sweet orange and mandarin),including 7 flavonoid aglycones,10 C-glycosylflavonoids,10 C-/O-glycosylflavonoids,44 O-glycosylflavonoids,39 polymethoxylated flavonoids(PMFs)and 7 newly annotated O-glycosylpolymethoxylated flavonoids.Quantitative result shows different types of flavonoids have different accumulation patterns between tissues.Polymethoxylated flavonoids were specifically enriched in the cortex,especially in the flavedo;flavonoids containing C-glycosylated modification appeared to be higher in flavedo than in other tissues;while O-glycosylated flavonoids were more balanced distribution in various organizations;as flavonoid aglycone,the content is low except luteolin.For different types of citrus,the content of polymethoxylated flavonoids and glycosylated polymethoxylated flavonoids in mandarin citrus was the highest,followed by sweet orange.According to the interspecific metabolic diversity of flavonoids,62 germplasms could be clearly grouped into five distinct clusters via hierarchical clustering analysis,which is highly consistent with the citrus subgenus classification used in our study.The cluster heat map shows that the metabolic profiles of the mandarin and sweet orange are very similar.The metabolic profiles of lemon and grapefruit are special different from those other three varieties.But grapefruit that is highly related to pummelo shows more similar metabolic profiles to sweet orange.In the adjacent tree graph,sweet oranges and mandarin were closely clustered and are distant from the other three species,indicating that the mandarin and sweet orange have very close genetic relationships.Simultaneously,grapefruit also showed a closer relationship with sweet orange,supporting the hypothesis that the mother was pomelo and the father was sweet orange from the metabolic point of view.In summary,there is a significant difference in accumulation of flavonoids in the flavedo of citrus tissues,and the variation of flavonoid content in different populations can serve as an important reference for the evolution of citrus species.