Integrated chemical composition identification and multi-omics analysis to reveal dynamic quality changes in mulberry leaves during growth

Mori Folium (also known as mulberry leaves), the dried leaves of Morus alba L., is a widely used traditional Chinese medicinal herb. With a growth cycle of approximately eight months, determining the optimal harvest period is essential to ensure the medicinal quality of mulberry leaves. Although traditional pharmacopoeias consider the superior quality of “post-frost mulberry leaves”, the underlying material basis and molecular regulatory mechanisms remain unclear. This study aims to systematically characterize the dynamic accumulation patterns of chemical constituents and elucidate the key components and regulatory networks responsible for the enhanced quality of “post-frost mulberry leaves”. Based on systematic inference of morphological, physiological, and compositional indicators, the growth of mulberry leaves may be tentatively divided into a vegetative growth phase and a secondary metabolite accumulation phase (S8–S9), although the precise timing of this transition could not be determined due to the lack of intermediate sampling points. A total of 76 compounds belonging to ten chemical classes were identified, with flavonoids being the predominant class and exhibiting clear stage-specific accumulation, notably from the S8 stage onward. Notably, key flavonoids, including astragalin and morachalcone A, were further significantly upregulated in the post‑frost stage (S9). Integrated transcriptomic and proteomic analyses identified four key pathways involved in flavonoid accumulation: “Phenylalanine, tyrosine and tryptophan biosynthesis”, “Phenylpropanoid biosynthesis”, “Flavonoid biosynthesis”, and “Flavone and flavonol biosynthesis”. Core regulatory factors, such as AST3 , TAT2 , PAL1 , 4CL2 , C4H , CHI , F3H , and UGT78D2 , were also identified. qRT-PCR validation confirmed that most flavonoid biosynthesis-related genes were significantly upregulated during stage S8-S9. This study provides a scientific foundation for the traditional use of “post-frost mulberry leaves”. The medicinal quality of mulberry leaves follows a decline-then-increase trend throughout the growth cycle. The significant accumulation of flavonoids under late-autumn and post-frost conditions constitutes the core material basis for the superior quality of “post-frost mulberry leaves” and can be considered as candidate quality markers, although their relevance requires future validation through direct correlation with pharmacological activities. This accumulation is synergistically regulated by a four-tiered pathway network: phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and flavonoid glycosylation.