Dehydroascorbate reductases and glutathione set a threshold for high light-induced ascorbate accumulation.

Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis triple mutant lacking all three DHAR genes (herein called dhar) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated dhar Arabidopsis plants as well as a quadruple mutant line combining dhar with an additional vtc2 mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a non-negligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Since GSH itself can reduce DHA non-enzymatically, we used the pad2 mutant that contains approximately 30% of the wild-type GSH level. The pad2 mutant accumulated ascorbate at a wild-type level under high light; however, when the pad2 mutation was combined with dhar, there was near complete inhibition of high light-dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in dhar pad2 plants, and that both DHAR activity and GSH content set a threshold for high light-induced ascorbate accumulation.