The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii.

ABSTRACT

Microalgae-based biodiesel production has many advantages over crude oil extraction and refinement, thus attracting more and more concern. Protein ubiquitination is a crucial mechanism in eukaryotes to regulate physiological responses and cell development, which is highly related to algal biodiesel production. Cullins as the molecular base of cullin-RING E3 ubiquitin ligases (CRLs), which are the largest known class of ubiquitin ligases, control the life activities of eukaryotic cells. Here, three cullins (CrCULs) in the green microalgae Chlamydomonas reinhardtii were identified and characterized. To investigate the roles of CrCULs in lipid metabolism, the gene expression profiles of CrCULs under nutrition starvation were examined. Except for down-regulation under nitrogen starvation, the CrCUL3 gene was induced by sulfur and iron starvation. CrCUL2 seemed insensitive to nitrogen and sulfur starvation because it only had changes after treatment for eight days. CrCUL4 exhibited an expression peak after nitrogen starvation for two days but this declined with time. All CrCULs expressions significantly increased under iron deficiency at two and four days but decreased thereafter. The silencing of CrCUL2 and CrCUL4 expression using RNAi (RNA interference) resulted in biomass decline and lipids increase but an increase of 20% and 28% in lipid content after growth for 10 days, respectively. In CrCUL2 and CrCUL4 RNAi lines, the content of fatty acids, especially C160 and C180, notably increased as well. However, the lipid content and fatty acids of the CrCUL3 RNAi strain slightly changed. Moreover, the subcellular localization of CrCUL4 showed a nuclear distribution pattern. These results suggest CrCUL2 and CrCUL4 are regulators for lipid accumulation in C. reinhardtii. This study may offer an important complement of lipid biosynthesis in microalgae.