Ascorbate deficiency does not limit non-photochemical quenching in Chlamydomonas reinhardtii

Ascorbate (vitamin C) plays essential roles in development, signaling, hormone biosynthesis, regulation of gene expression, stress resistance and photoprotection. In vascular plants, violaxanthin de-epoxidase (VDE) requires ascorbate (Asc) as reductant, thereby it is required for the energy-dependent component of non-photochemical quenching (NPQ). In order 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 the VTC2 gene, essential for Asc biosynthesis. The Crvtc2-1 knockout mutant was viable and, depending on the growth conditions, it contained 10 to 20% Asc relative to its wild type. When Chlamydomonas 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 VDE found in Chlamydomonas does not require Asc as a reductant. The rapidly induced, energy-dependent NPQ component, characteristic of photoautotrophic Chlamydomonas 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 Chlamydomonas than in vascular plants.\n\nOne-sentence summaryIn Chlamydomonas -in contrast to seed plants-, ascorbate is not required for violaxanthin deepoxidation and energy-dependent non-photochemical quenching but it mitigates photoinhibitory quenching.