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.

Sodium humate (SH) is the sodium salt of humic acid. Our previous research has demonstrated that SH has the ability to enhance the levels of total flavonoids in various parts of lemons, including the leaves, peels, pulps, and seeds, thereby improving the quality of lemons. In the current study, the regulation effect of SH on the biosynthesis and content of lemon flavonoid compounds was examined using transcriptome sequencing technology and flavonoid metabolomic analysis. Following SH treatment, the transcriptome sequencing analysis revealed 320 differentially expressed genes (DEGs) between samples treated with SH and control (CK) samples, some of which were associated with the phenylalanine pathway by KEGG annotation analysis. The levels of seven flavonoid compounds identified in lemon peels were observed to increase, and eriocitrin and isoorientin were identified as differential metabolites (DMs, VIP > 1) using OPLS-DA analysis. The integrated analysis of transcriptomics and flavonoid metabolomics indicates that SH treatment induces alterations in gene expression and metabolite levels related to flavonoid synthesis. Specifically, SH influences flavonoid biosynthesis by modulating the activity of key enzymes in the phenylalanine pathway, including HCT (O-hydroxycinnamoyltransferase) and F5H (ferulate-5-hydroxylase).