Mechanism of ClFLS1 regulated amentoflavone biosynthesis in mediating drought and cold stress tolerance in Cunninghamia lanceolata

Amentoflavone, a natural biflavonoid with anti-inflammatory, antitumor, vasodilatory, hypoglycemic, and antioxidant properties, is widely used in healthcare. Cunninghamia lanceolata (Lamb.) Hook ( C. lanceolata ), is a fast-growing timber species endemic of China, contains abundant flavonoid compounds, including 3–5 % amentoflavone, though its biosynthesis remains unclear. Transcriptomic analysis of the high-amentoflavone C. lanceolata identified a flavonol biosynthase gene ClFLS1 , which as a candidate regulator of amentoflavone biosynthesis. We cloned ClFLS1 , analyzed its expression and subcellular localization (root-dominant, cytoplasm-specific), and generated ClFLS1 -overexpressing ( ClFLS1-OX ) Arabidopsis thaliana ( A. thaliana ) via Agrobacterium -mediated transformation method. ClFLS1-OX lines exhibited elevated amentoflavone levels compared to wild-type (Col-0) and atfls1 mutants, with further increases under drought/cold stress. ClFLS1 accelerated bolting, flowering, and leaf/root proliferation, indicating dual roles in growth and metabolism. This study elucidates ClFLS1 ’s regulatory mechanisms in amentoflavone biosynthesis and stress adaptation, offering strategies for engineering medicinal timber species.