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• EOP high light irradiation increased the ascorbate content of lettuce. • EOP high light intensity regulated the expression of genes and the activity of enzymes involved in ascorbate synthesis and cycling. • EOP high light intensity induced the production of ROS, and the content of ascorbate, as an important antioxidant, increased accordingly. • EOP high light intensity increased the carbohydrate content of lettuce. Increasing the L-ascorbate (AsA) content of vegetables is very important for improving plant quality in greenhouses and plant factories because AsA is an antioxidant that is essential for the proper function of plants and the human body. End-of-production (EOP) high light irradiation is an economical preharvest practice for improving product quality. Here, we studied the dynamic responses of the AsA pool and AsA metabolism in lettuce exposed to EOP high light irradiation (300, 400, and 500 μmol m−2·s−1) from red and blue LEDs over a 16-h time course. Compared to the control (200 μmol m−2·s−1), the content of AsA and total AsA (T-AsA) in lettuce leaves significantly increased after 8 h of high light irradiation, and the contents of AsA and T-AsA increased by 42.4 % and 36.9 %, respectively, under 500 μmol m−2·s−1 irradiation. Transcript levels of the AsA metabolism-related genes GME , GGP , GPP , GLDH , APX , MDHAR1 , MDHAR2 , DHAR1 , DHAR2 , and GR were upregulated under higher light. Likewise, the activities of AsA-related enzymes (GLDH, APX, DHAR, and GR) increased under 500 μmol m−2·s−1. The expression of genes (GGP , GPP , GLDH , MDHAR1 , MDHAR2 , DHAR1 , DHAR2 , and GR) was more sensitive to high light irradiation in AsA metabolism. Both high light intensity and long irradiation time increased the soluble sugar content. Moreover, high light intensity (500 μmol m−2·s−1) increased the content of H 2 O 2 , O 2 *−, and MDA in lettuce leaves, especially at 16 h. In conclusion, EOP high light increased AsA levels by upregulating the expression of genes and the activity of enzymes in AsA metabolism. The increase in soluble sugar availability and the signaling of reactive oxygen species also contributed to AsA accumulation. We therefore recommend 8 h of high light (500 μmol m−2·s−1) irradiation before harvest to increase the AsA content of lettuce. [ABSTRACT FROM AUTHOR]