The Effect and Potential Mechanism of Fulvic Acid on Flavonoids in Lemon Leaves

Citrus limon (L.) Burm. f. is a horticultural crop known for its abundance of valuable secondary metabolites, including flavonoids, which are found in its fruits and leaves. Our previous research has shown that treating C. limon with fulvic acid (FA) can enhance the levels of vitamin C, total acid, total sugar, total flavonoids, and phenols in its fruits as well as the volatiles and total flavonoids in its leaves. In this current study, we established a method to analyze eight specific flavonoids in lemon leaves and evaluated the impact of irrigation with FA on the content of these flavonoids over a six-month period using HPLC-DAD/MS analysis. Moreover, we investigated the potential mechanisms of FA through ELISA and q-PCR methods. The results indicated that FA increased the contents of four flavonoids, namely, isoorientin, eriocitrin, vitexin, and rutin, and promoted the activity and gene expression of phenylalanineammonialyase (PAL), 4-coumaric acid coenzyme A ligase (4CL), chalcone synthase (CHS), flavonoid 3′-hydroxylase (F3′H), and flavonol synthase (FLS). Furthermore, the relationship between flavonoid content and the activities of biosynthetic enzymes was analyzed using orthogonal partial least squares discriminant analysis (OPLS-DA), which revealed a positive correlation between seven flavonoid levels and the activity of five biosynthetic enzymes under FA treatment.