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• This is the first study focused on the dynamic responses of ascorbate metabolism to long-term continuous light. • The increase of lettuce shoot biomass under continuous light dependent on specific leaf weight rather than leaf area. • Increased AsA and DHA level under continuous light was mainly attributed to greater GalLDH and APX activity respectively. • Ascorbate-glutathione cycle pays little contribution to ascorbate accumulation under continuous light. To understand the dynamic changes of lettuce growth, ascorbate pool and metabolism under long-term red and blue continuous light (CL, 24/0 h, 200 μmol·m−2·s−1), growth parameters, ascorbate pool size, as well as activities and gene expression levels of key enzymes involved in the ascorbate metabolism in lettuce leaves were investigated every 3 days during 15 days. In comparison with normal photoperiod (Control, 16/8 h, 200 μmol·m−2·s−1), lettuce plants gained greater shoot fresh weight and dry matter under CL, which depended more on specific leaf weight than leaf area. Both reduced ascorbate (AsA) and dehydroascorbate (DHA) contents were remarkably enhanced by CL and keep a relatively stable level during the experiment. The consistent greater contents of AsA and DHA were respectively attributed to the up-regulation of L-galactono-1,4-lactone dehydrogenase and ascorbate peroxidase activity rather than their gene expression. Although glutathione contents and glutathione reductase activities were elevated by CL, their contribution to AsA regeneration was limited on account of similar activity and expression level of dehydroascorbate reductase with control. In addition, H 2 O 2 content was also raised obviously under CL compared with control, but it was maintained at a relative safety and stable level from day 6. Fluorescence imaging results also showed that CL did not induce oxidative damage on lettuce leaves compared with control. It is concluded that lettuce plants grown under red and blue continuous light for 15 days obtained greater shoot biomass and ascorbate pool size without leaf injury, and 9 days was the most cost-effective duration for yield improvement from an energy standpoint. Ascorbate accumulation under continuous light was primarily and rapidly regulated by the enzymes involved in AsA biosynthesis and oxidation at enzymatic activity level rather than gene expression level. [ABSTRACT FROM AUTHOR]