High level of reactive oxygen species inhibits triacylglycerols accumulation in Chlamydomonas reinhardtii

Many microalgae accumulate large amounts of lipid droplets (LDs) following N deprivation. The triacylglycerols (TAGs) in these LDs can be used as a feedstock to produce biodiesel. To investigate the molecular mechanism underlying LD formation, we used a percoll gradient-based method to enrich for mutants with defects in LD formation and generated an insertional mutagenesis library containing >11,000 Chlamydomonas reinhardtii transformants. One of the mutants harbored a mutation in a gene encoding a glutathione peroxidase (GPX5) that catalyzes the formation of water from hydrogen peroxide (H2O2) or organic hydroperoxide. The gpx5 mutant had a 35% reduction in the number of LDs and a 50% reduction in TAG content compared with the parental strain CC4348 at 24 h of N deprivation. The full-length of GPX5 or truncate GPX5 without the putative signal peptide or the putative signal peptide of GPX5 fused YFP localized in the cytoplasm, but not in chloroplasts. Complementation with full-length GPX5 rescued the mutant phenotypes, and the expression of GPX5 in a complemented strain L27 was increased 2–3 fold after 24 h of N deprivation. Moreover, the gpx5 mutant showed increased sensitivity to the singlet oxygen (1O2) stress generated by the photosensitizer Rose Bengal (RB). The ROS concentration of the gpx5 was about 1.5 times that of CC4348 during N deprivation. Artificial increasing of ROS concentration in cells abolished the formation of LDs in Chlamydomonas. These data suggest that a high level of ROS attenuates LD formation.