Constitutive zeaxanthin accumulation affects photosynthetic activity and non-photochemical quenching in Chlorella...

Carotenoids play crucial roles in light harvesting and photoprotection in photosynthetic organisms, although their functions in non-model microalgae remain poorly understood. Here, we investigated the effects of constitutive zeaxanthin accumulation in the green microalga Chlorella vulgaris. A mutant strain, named fm53 (fluorescence mutant 53), was isolated by UV mutagenesis and characterized by constitutive zeaxanthin accumulation together with the absence of other β-β xanthophylls. Whole-genome sequencing identified 36 single-nucleotide variants, including a mutation in the 5' untranslated region of the zeaxanthin epoxidase (ZEP) gene, associated with the absence of detectable ZEP protein accumulation. Compared with the wild type, fm53 accumulated higher levels of zeaxanthin, showed altered Photosystem II supercomplex organization, reduced maximum photosynthetic efficiency, and strongly decreased light-dependent non-photochemical quenching (NPQ). Fluorescence lifetime analysis revealed constitutive zeaxanthin-dependent quenching mechanisms in the mutant. Despite these alterations, biomass productivity remained similar to the wild type under most growth conditions tested. These results provide new insights into the roles of zeaxanthin and the xanthophyll cycle in regulating photosynthesis and photoprotection in C. vulgaris. Moreover, the constitutive accumulation of zeaxanthin highlights the potential of the fm53 strain as a source of high-value carotenoids.