Both major xanthophyll cycles present in nature promote nonphotochemical quenching in a model diatom.

Xanthophyll cycling contributes to photoprotection by regulating nonphotochemical quenching (NPQ), a form of excess energy dissipation through heat. While most photosynthetic eukaryotes (including land plants) use the violaxanthin cycle, some algae like diatoms and haptophytes rely on the diadinoxanthin cycle for photoprotection. These algae also contain minor amounts of violaxanthin cycle pigments, thought to serve only as the precursors of other major xanthophylls. Both cycles are catalyzed by the enzymes violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP). Here, we characterized the role of VDE and of the ZEP paralogs ZEP2 and ZEP3 in the model diatom Phaeodactylum tricornutum. We generated knockout lines for each gene and treated exponentially growing mutants and wild type with periodic high-light stress. As knockouts of VDE and ZEP3 were significantly impaired in the diadinoxanthin cycle, we concluded that VDE and ZEP3 are the main regulators of the diadinoxanthin cycle in this diatom. Strikingly, under light stress, ZEP2 knockouts mainly accumulated pigments of the violaxanthin cycle instead of the diadinoxanthin cycle, but still displayed the same NPQ capacity as the wild type. We conclude that both major xanthophyll cycles present in nature can contribute to NPQ with comparable efficiency within the same diatom species, offering perspective on the evolution of xanthophyll-mediated photoprotection.