Short-term elevated rhizosphere CO₂ enhances antioxidant capacity in facility melon seedlings by promoting flavonoids accumulation

Melon ( Cucumis melo ) is one of major commercial crop in facility cultivation. However, continuous cropping, excessive organic fertilizer application, heightened microbial activity, and plastic film mulching often lead to rhizosphere CO 2 accumulation during production. Previous studies have demonstrated that short-term elevated rhizosphere CO 2 significantly promote melon growth. While the carbon secondary assimilation products and key secondary metabolic mechanisms that promote the growth of melon seedlings under ER-CO 2 unclear. Therefore, we investigated carbon assimilation, allocation, and secondary metabolism of melon seedlings under elevated rhizosphere CO 2 through physiological, 13 C-isotope labeling and metabolic flux analyses. Results indicated that δ 13 C values and 13 C assimilation rates in roots and leaves initially increased (peaking at 48 h) and then declined. Non-targeted 13 C metabolic flux and metabolite content analyses demonstrated that differential metabolites of melon seedlings were significantly enriched in tricarboxylic acid (TCA) cycle and phenylpropanoid metabolism. qRT-PCR confirmed short-term Elevated Rhizosphere CO₂ (ER-CO 2 ) up-regulated CmPEPC, CmMDH, CmPEPCK and CmCS while down-regulated CmIDH and CmACO in the TCA cycle. Concurrently, negative feedback mechanism was activated to up-regulate CmPEPCK expression, thereby facilitating carbon partitioning, which ultimately up-regulated the genes of flavonoid pathway in phenylpropanoid metabolism ( CmSDH, CmPAL and CmCHS ) and promoted the accumulation of flavonoids. Flavonoid metabolites contributed to the favorable cellular environment for the growth of melon seedlings by enhancing the endogenous antioxidant defense system through both direct and indirect pathways while maintaining ROS accumulation of signaling levels. This study provides a theoretical foundation for optimizing the regulation of adverse rhizosphere conditions in facility-grown oriental melons. • Elucidating TCA cycle and phenylpropanoid pathway as key for carbon partitioning in melon under elevated rhizosphere CO 2 . • Identifying flavonoids as the primary end product of carbon redistribution in melon under elevated rhizosphere CO 2 . • Elucidating melon alleviated ROS accumulation by accumulating flavonoids under elevated rhizosphere CO 2 .