Metabolomic Profiling of Cassava Leaves Highlights Flavonoid Dynamics and Antioxidant Potential Across Developmental Stages

Cassava leaves represent an abundant agricultural byproduct rich in bioactive flavonoids, yet the dynamic changes in flavonoid composition across cassava varieties and developmental stages remain poorly understood. Here, UPLC-MS/MS was used to profile flavonoid metabolites in three cassava varieties with contrasting leaf colors-South China 9 (SC9), Manihot esculenta 'Variegata' (MEV), and purple-leaf cassava (PC)-across young (Y), tender (T), and mature (M) leaf stages. A total of 296 flavonoid metabolites were identified, revealing pronounced variety- and stage-dependent accumulation patterns. Purple-leaf cassava exhibited the highest flavonoid accumulation and antioxidant capacity, while SC9 and MEV showed peak antioxidant activity at the young leaf stage. Differential metabolite analysis identified a core set of 16 differential flavonoids, including Dihydroquercetin, Aromadendrin, Naringenin, Apiin, and Pelargonidin-3-O-glucoside. Correlation analysis further revealed that seven flavonoids, such as apiin, tricin-7-O-(2''-feruloyl)glucoside, eriodictyol-5,3'-di-O-glucoside, amentoflavone-7''-O-glucoside, sophoricoside, and chrysoeriol-7-O-(6''-feruloyl)glucoside-were identified as the primary contributors to antioxidant activity. Further expression analysis of key upstream biosynthetic genes corresponding to the differential metabolites revealed that the transcript levels of MeF3H, MeF3'5'H and MeLAR closely mirrored the flavonoid accumulation patterns. This indicates that these genes play critical roles in regulating flavonoid biosynthesis and can serve as potential targets for genetic improvement of cassava nutritional quality.