MibHLH35L regulates AsA homeostasis via MiMDHAR4/MiAPX6 to attenuate oxidative stress and delay mango senescence

Postharvest deterioration of mango fruit is closely linked to ethylene-induced oxidative stress and disrupted ascorbate homeostasis. However, the molecular regulatory mechanisms underlying ethylene-mediated ascorbic acid (AsA) metabolism remain unclear. This study identified an ethylene-responsive module— MibHLH35L-MiMDHAR4-MiAPX6— that regulates ascorbate homeostasis and ripening in mango. Ethylene treatment accelerated fruit senescence, as characterized by increased ethylene production, respiration rate, and the accumulation of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), coupled with a rapid decline in AsA content. Conversely, 1-methylcyclopropene (1-MCP) delayed senescence by sustaining higher AsA levels and reducing reactive oxygen species (ROS) accumulation. Transcriptomic and RT-qPCR analyses showed that ethylene strongly suppressed the expression of MibHLH35L, MiMDHAR4, and MiAPX6, while 1-MCP treatment induced MibHLH35L and rebalanced the expression of its downstream targets. Using yeast one-hybrid, EMSA, and dual-luciferase assays, we demonstrated that MibHLH35L directly binds to E-box motifs in the promoters of MiMDHAR4 and MiAPX6 to activate their transcription. Functional validation through transient overexpression and VIGS in mango, as well as stable genetic transformation in tomato, confirmed that MibHLH35L reduces peroxide accumulation, delays ripening, and increases AsA content by upregulating MiMDHAR4 and MiAPX6. In summary, MibHLH35L serves as a key transcriptional activator of MiMDHAR4 and MiAPX6, promoting AsA regeneration and ROS scavenging to maintain AsA levels and inhibit fruit senescence. These findings suggest that ETH/1-MCP treatments are associated with changes in MibHLH35L-mediated redox regulation, offering important insights into the transcriptional regulation of AsA metabolism in horticultural crops.