Abstract
E3 ubiquitin ligases determine the substrate specificity of ubiquitination. Plant U-box (PUB) E3 ligases, with a typical 70-amino acid U-box domain, participate in plant developmental processes and environmental responses. Thus far, 64 PUB proteins have been identified in Arabidopsis and 77 PUB proteins have been identified in Oryza. However, detailed studies on U-box genes in the model microalgae Chlamydomonas reinhardtii are lacking. Here, we present a comprehensive analysis of the genes encoding U-box family proteins in C. reinhardtii. Following BLASTP analysis, 30 full-length U-box genes were identified in the C. reinhardtii genome sequence. Bioinformatics analyses of CrPUB genes were performed to characterize the phylogenetic relationships, chromosomal locations and gene structures of each member. The 30 identified CrPUB proteins are clustered into 3 distinct subfamilies, and the genes for these proteins are unevenly distributed among 14 chromosomes. Furthermore, the quantitative real-time RT-PCR or semi-quantitative RT-PCR analysis of 30 CrPUB mRNA abundances under nitrogen starvation showed that 18 CrPUB genes were induced by N starvation and that 7 genes were repressed in the N-poor environment. We selected five CrPUB genes exhibiting marked changes in expression under N-free conditions for further analysis in RNAi experiments and examined the oil content of these gene-silenced transgenic strains. The silencing of CrPUB5 and CrPUB14, which are typically down-regulated under N starvation, induced 9.8%-45.0% and 14.4%-61.8% lipid accumulation, respectively. In contrast, the silencing of CrPUB11, CrPUB23 and CrPUB28, which are markedly up-regulated under N-free conditions, decreased the lipid content by 5.5%-27.8%, 8.1%-27.3% and 6.6%-27.9%, respectively. These results provide a useful reference for the identification and functional analysis of this gene family and fundamental information for microalgae lipid metabolism research.
Highlights
Ubiquitin (Ub)-mediated protein degradation is a highly conserved process in eukaryotes
The lipid content in RNA interference transformants was examined. These results suggest an important role for CrPUB E3 ubiquitin ligases in lipid metabolism in C. reinhardtii
To identify the U-box proteins in C. reinhardtii, we first collected the known Plant U-box (PUB) gene sequences from Arabidopsis thaliana (AtPUB1-AtPUB64) and Oryza sativa (OsPUB1-OsPUB77) and performed a BLASTP search in the C. reinhardtii V5.5 proteome database using these sequences as queries
Summary
Ubiquitin (Ub)-mediated protein degradation is a highly conserved process in eukaryotes. Ub, which is a small 76-amino acid protein, forms a multi-Ub chain that serves as a degradation tag. The covalent attachment of Ub polymers to specific proteins involves the following threestep reaction: 1) E1 (Ub-activating enzyme) consumes ATP and actives Ub; 2) activated Ub is transferred to E2 (Ub-conjugating enzyme), forming the E2-Ub intermediate; and 3) E2 interacts with E3 (ubiquitin-protein ligases), which binds the substrate and delivers Ub to the target [1]. The E3 ligase-mediated recruitment of substrates is the key to the specificity of ubiquitination. E3 ligases are a diverse gene family in plants. Plant E3s participate in signaling pathways and control the cell cycle, morphogenesis, stress responses, self-incompatibility and pathogen defenses [2,3]. In Arabidopsis thaliana, more than 1,400 different E3s genes have been predicted through comprehensive genome analysis [4,5]
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