Dehydroascorbate Reductases and Glutathione Set a Threshold for High-Light–Induced Ascorbate Accumulation

Abstract

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 (<i>Arabidopsis thaliana</i>) triple mutant lacking all three <i>DHAR</i> genes (herein called ∆<i>dhar</i>) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated ∆<i>dhar</i> Arabidopsis plants as well as a quadruple mutant line combining ∆<i>dhar</i> with an additional <i>vtc2</i> mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the <i>pad2</i> mutant that contains ∼30% of the wild-type GSH level. The <i>pad2</i> mutant accumulated ascorbate at a wild-type level under high light; however, when the <i>pad2</i> mutation was combined with ∆<i>dhar</i>, 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 ∆<i>dhar pad2</i> plants, and that both DHAR activity and GSH content set a threshold for high-light-induced ascorbate accumulation.