Integrated Metabolomic-Transcriptomic Analyses of Flavonoid Accumulation in Citrus Fruit under Exogenous Melatonin Treatment.

The flavonoids in citrus fruits are crucial physiological regulators and natural bioactive products of high pharmaceutical value. Melatonin is a pleiotropic hormone that can regulate plant morphogenesis and stress resistance and alter the accumulation of flavonoids in these processes. However, the direct effect of melatonin on citrus flavonoids remains unclear. In this study, nontargeted metabolomics and transcriptomics were utilized to reveal how exogenous melatonin affects flavonoid biosynthesis in "Bingtangcheng" citrus fruits. The melatonin treatment at 0.1 mmol L-1 significantly increased the contents of seven polymethoxylated flavones (PMFs) and up-regulated a series of flavonoid pathway genes, including 4CL (4-coumaroyl CoA ligase), FNS (flavone synthase), and FHs (flavonoid hydroxylases). Meanwhile, CHS (chalcone synthase) was down-regulated, causing a decrease in the content of most flavonoid glycosides. Pearson correlation analysis obtained 21 transcription factors co-expressed with differentially accumulated flavonoids, among which the AP2/EREBP members were the most numerous. Additionally, circadian rhythm and photosynthesis pathways were enriched in the DEG (differentially expressed gene) analysis, suggesting that melatonin might also mediate changes in the flavonoid biosynthesis pathway by affecting the fruit's circadian rhythm. These results provide valuable information for further exploration of the molecular mechanisms through which melatonin regulates citrus fruit metabolism.

Functional characterization of two flavone synthase II members in citrus.

Polymethoxylated flavones (PMFs), the main form of flavones in citrus, are derived from the flavone branch of the flavonoid biosynthesis pathway. Flavone synthases (FNSs) are enzymes that catalyze the synthesis of flavones from flavanones. However, the FNS in citrus has not been characterized yet. Here, we identified two type II FNSs, designated CitFNSII-1 and CitFNSII-2, based on phylogenetics and transcriptome analysis. Both recombinant CitFNSII-1 and CitFNSII-2 proteins directly converted naringenin, pinocembrin, and liquiritigenin to the corresponding flavones in yeast. In addition, transient overexpression of CitFNSII-1 and CitFNSII-2, respectively, in citrus peel significantly enhanced the accumulation of total PMFs, while virus-induced CitFNSII-1 and CitFNSII-2 genes silencing simultaneously significantly reduced the expression levels of both genes and total PMF content in citrus seedlings. CitFNSII-1 and CitFNSII-2 presented distinct expression patterns in different cultivars as well as different developmental stages. Methyl salicylate (MeSA) treatment reduced the CitFNSII-2 expression as well as the PMFs content in the peel of Citrus sinensis fruit but did not affect the CitFNSII-1 expression. These results indicated that both CitFNSII-1 and CitFNSII-2 participated in the flavone biosynthesis in citrus while the regulatory mechanism governing their expression might be specific. Our findings improved the understanding of the PMFs biosynthesis pathway in citrus and laid the foundation for further investigation on flavone synthesis regulation.

A multifunctional true caffeoyl coenzyme A O-methyltransferase enzyme participates in the biosynthesis of polymethoxylated flavones in citrus.

Polymethoxylated flavones (PMFs) have received extensive attention due to their abundant bioactivities. Citrus peels specifically accumulate abundant PMFs, and methylation modification is a key step in PMF biosynthesis; however, the function of reported O-methyltransferase (OMT) in citrus is insufficient to elucidate the complete methylation process of PMFs. In this study, we analyzed the accumulation pattern of PMFs in the flavedo of the sweet orange (Citrus sinensis) cultivar "Bingtangcheng" at different developmental stages. We found that accumulation of PMFs was completed at the early stage of fruit development (60-d after flowering). Furthermore, we characterized a true caffeoyl-CoA O-methyltransferase (named CsCCoAOMT1) from C. sinensis. Functional analysis in vitro showed that CsCCoAOMT1 preferred flavonoids to caffeoyl-CoA and esculetin. This enzyme efficiently methylated the 6-, 7- 8-, and 3'-OH of a wide array of flavonoids with vicinal hydroxyl groups with a strong preference for quercetin (flavonol) and flavones. The transient overexpression and virus-induced gene silencing experiments verified that CsCCoAOMT1 could promote the accumulation of PMFs in citrus. These results reveal the function of true CCoAOMTs and indicate that CsCCoAOMT1 is a highly efficient multifunctional O-methyltransferase involved in the biosynthesis of PMFs in citrus.