Integrative transcriptomics and metabolomics reveal the biosynthesis of flavonoid metabolites in Tilia miqueliana Maxim. leaves

<i>Tilia miqueliana</i> Maxim. is renowned for its rich bioactive compounds, including flavonoids, phenolic acids, coumarins, and other secondary metabolites, which possess antioxidant, anticancer, antidepressant, and analgesic effects. This study aims to investigate the seasonal dynamic changes of secondary metabolites in <i>T. miqueliana</i> leaves and their biosynthetic regulatory mechanisms. The leaves of <i>T. miqueliana</i> were sampled at four different growth stages. Total flavonoids, phenolic acids, amino acids, coumarins, and terpenoid contents were determined using UV spectrophotometry, and enzyme activities of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate: CoA ligase (4CL) were measured. Flavonoid monomers such as quercetin and kaempferol, along with endogenous hormones, were quantitatively analyzed using high-performance liquid chromatography (HPLC). Widely targeted metabolomic analysis via UPLC-MS/MS and Illumina transcriptomic sequencing identified 1971 metabolites. The results showed that flavonoids, amino acids and their derivatives, and phenolic acids accounted for nearly half of the total metabolites. The major active substances exhibited significant variations across different developmental stages. The summer months (June to August) represented the most active growth and metabolic phase. Active compounds, represented by flavonoids such as tiliroside, scopoletin, naringenin, dihydrokaempferol, apigenin, luteolin, quercetin, kaempferol, and rutin, are secondary metabolites with potential medicinal value in <i>T. miqueliana</i> leaves. There were significant differences in differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) across developmental stages. The synthesis of key secondary metabolites is co-regulated by endogenous hormones, enzyme activities, and differentially expressed candidate genes. This study provides new insights for determining the appropriate harvesting time for <i>T. miqueliana</i> leaves and the metabolic regulation of secondary metabolites.