Ascorbate (Asc; vitamin C) plays essential roles in development, signaling, hormone biosynthesis, regulation of gene expression, stress resistance, and photoprotection. In vascular plants, violaxanthin de-epoxidase requires Asc as a reductant; thereby, Asc is required for the energy-dependent component of nonphotochemical quenching (NPQ). To assess the role of Asc in NPQ in green algae, which are known to contain low amounts of Asc, we searched for an insertional Chlamydomonas reinhardtii mutant affected in theVTC2 gene encoding GDP-L-Gal phosphorylase, which catalyzes the first committed step in the biosynthesis of Asc. The Crvtc2-1 knockout mutant was viable and, depending on the growth conditions, contained 10% to 20% Asc relative to its wild type. When C. reinhardtii was grown photomixotrophically at moderate light, the zeaxanthin-dependent component of NPQ emerged upon strong red illumination both in the Crvtc2-1 mutant and in its wild type. Deepoxidation was unaffected by Asc deficiency, demonstrating that the Chlorophycean violaxanthin de-epoxidase found in C. reinhardtii does not require Asc as a reductant. The rapidly induced, energy-dependent NPQ component characteristic of photoautotrophic C. reinhardtii cultures grown at high light was not limited by Asc deficiency either. On the other hand, a reactive oxygen species-induced photoinhibitory NPQ component was greatly enhanced upon Asc deficiency, both under photomixotrophic and photoautotrophic conditions. These results demonstrate that Asc has distinct roles in NPQ formation in C. reinhardtii as compared to vascular plants.

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