The expression patterns of CrPUB genes as determined by semi-quantitative PCR analyses.

<p>Microalgae cells were grown in complete (HSM) and N-starvation (HSM-N) media for 2, 4 and 6 days. Semi-quantitative PCR analyses were performed. The 18S rRNA gene was used as an internal control.</p

Lipid contents in CrPUB gene RNAi transgenic lines.

<p>(A)The lipid contents in the RNAi lines containing the constructs harboring the fragments amplified using primer set A as the dsRNA. (B) The lipid content in RNAi lines containing the constructs harboring the fragments amplified using primers set B as the dsRNA. The CrPUB gene RNAi lines were cultured in HSM for 12 days. The cells were resuspended in 200 μL of Nile red staining solution, followed by FD. The lipid content (ng/10⁶ cells) was calculated using the equation mentioned previously. Both the wild type CC425 and empty-vector transformants Maa7-RNAi were presented as controls. Significant differences were assessed using ANOVA. The p-values indicate the level of significance for differences between RNAi lines and wild type (*P<0.05, **P<0.01). The data are expressed as the means±SD of 3 replicates. CrPUB5 and CrPUB14 gene silencing induced lipid accumulation, and CrPUB11, CrPUB23 and CrPUB28 gene silencing showed the opposite effect.</p

List of the 30 U-box genes identified in <i>C</i>. <i>reinhardtii</i> and their sequence characteristics.

<p>^aF and R represent the forward and reverse directions on the chromosome, respectively.</p><p>^bWoLF PSORT. N, nucleus; C, chloroplast; c, cytoplasm; V, vacuole; E, endoplasmic reticulum; M, mitochondria; n.a., not available.</p><p>In total, 30 CrPUB proteins were obtained by BLASTP search using the <i>C</i>. <i>reinhardtii</i> V5.5 proteome database and PUB proteins from <i>Arabidopsis thaliana</i> and <i>Oryza sativa</i> as queries. The 30 CrPUB genes were named based on their chromosome position. The molecular weights and pIs of the 30 CrPUB proteins were predicted using ExPASy. The CrPUB sub-cellular locations were predicted using the WOLF PSORT program.</p><p>List of the 30 U-box genes identified in <i>C</i>. <i>reinhardtii</i> and their sequence characteristics.</p

Phylogenetic relationship between <i>C</i>. <i>reinhardtii</i> and <i>Arabidopsis</i> U-box proteins.

<p>The amino acid sequences of U-box proteins from the two proteomes were used for analysis. The unrooted tree was inferred using Mega 6.0 software and the neighbor-joining method with 1000 bootstrap replicates. The CrPUB proteins are indicated in pink font; only the <i>Arabidopsis thaliana</i> and <i>C</i>. <i>reinhardtii</i> homologous branches show the bootstrap values as percentages.</p

Table_1_Flagella-Associated WDR-Containing Protein CrFAP89 Regulates Growth and Lipid Accumulation in Chlamydomonas reinhardtii.docx

<p>WD40-repeat (WDR) domain-containing proteins are subunits of multi-protein E3 ligase complexes regulating various cellular and developmental activities in eukaryotes. Chlamydomonas reinhardtii serves as a model organism to study lipid metabolism in microalgae. Under nutrition deficient conditions, C. reinhardtii accumulates lipids for survival. The proteins in C. reinhardtii flagella have diverse functions, such as controlling the motility and cell cycle, and environment sensing. Here, we characterized the function of CrFAP89, a flagella-associated WDR-containing protein, which was identified from C. reinhardtii nitrogen deficiency transcriptome analysis. Quantitative real time-PCR showed that the transcription levels of CrFAP89 were significantly enhanced upon nutrient deprivation, including nitrogen, sulfur, or iron starvation, which is considered an effective condition to promote triacylglycerol (TAG) accumulation in microalgae. Under sulfur starvation, the expression of CrFAP89 was 32.2-fold higher than the control. Furthermore, two lines of RNAi mutants of CrFAP89 were generated by transformation, with gene silencing of 24.9 and 16.4%, respectively. Inhibiting the expression of the CrFAP89 gene drastically increased cell density by 112–125% and resulted in larger cells, that more tolerant to nutrition starvation. However, the content of neutral lipids declined by 12.8–19.6%. The fatty acid content in the transgenic algae decreased by 12.4 and 13.3%, mostly decreasing the content of C16:0, C16:4, C18, and C20:1 fatty acids, while the C16:1 fatty acid in the CrFAP89 RNAi lines increased by 238.5 to 318.5%. Suppressed expression of TAG biosynthesis-related genes, such as CrDGAT1 and CrDGTTs, were detected in CrFAP89 gene silencing cells, with a reduction of 16–78%. Overall our results suggest that down-regulating of the expression of CrFAP89 in C. reinhardtii, resulting in an increase of cell growth and a decrease of fatty acid synthesis with the most significant decrease occurring in C16:0, C16:4, C18, and C20:1 fatty acid. CrFAP89 might be a regulator for lipid accumulation in C. reinhardtii.</p

Genome-Wide Survey and Expression Analysis of <i>Chlamydomonas reinhardtii</i> U-box E3 Ubiquitin Ligases (CrPUBs) Reveal a Functional Lipid Metabolism Module

<div><p>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 <i>Arabidopsis</i> and 77 PUB proteins have been identified in <i>Oryza</i>. However, detailed studies on U-box genes in the model microalgae <i>Chlamydomonas reinhardtii</i> are lacking. Here, we present a comprehensive analysis of the genes encoding U-box family proteins in <i>C</i>. <i>reinhardtii</i>. Following BLASTP analysis, 30 full-length U-box genes were identified in the <i>C</i>. <i>reinhardtii</i> 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.</p></div

Results of qPCR analysis of the CrPUB genes.

<p>A. N starvation up-regulated the mRNA expression of 15 CrPUB genes as shown by real-time PCR analysis. B. The transcription of 5 CrPUB genes was repressed under N starvation. C. CrPUB9, CrPUB 26 and CrPUB30 were all expressed under both N starvation and normal conditions; however, no relationship between mRNA abundances and N starvation was found. <i>C</i>. <i>reinhardtii</i> CC124 were pre-cultured in HSM to the mid-logarithmic phase, followed by centrifugation and resuspension in HSM and HSM-N with continued culturing for 0, 2, 4, 6 days. The cells were collected, and the RNA samples were isolated. The gene transcript levels were determined using real-time quantitative PCR. All expression values were normalized to the value of the 18S rRNA gene. The relative amounts were calibrated based on the number of transcripts of the corresponding genes in cells maintained in HSM-N for 0 days. The data are shown as the means (±SD, n = 3). Significance is indicated as *P<0.05, **P<0.01. The hollow circles represent CrPUB gene mRNA abundances under N starvation, and the solid circles represent CrPUB gene mRNA abundances under normal conditions.</p

Known PUB E3 ubiquitin ligases and targets of the Ub/26S proteasome pathway involved in plant growth and development.

<p>Known PUB E3 ubiquitin ligases and targets of the Ub/26S proteasome pathway involved in plant growth and development.</p

Multiple alignment of U-box domains from CrPUB proteins.

<p>The U-box domains in CrPUB proteins were predicted using PROSITE and MEME programs. Their sequences were aligned using ClustalX 2.1, and the alignments were edited using the GeneDoc 2.7 sequence editor. Black, gray and light gray shading indicates the identities and similarities among these sequences as 100%, 80%, and 60%, respectively.</p

Microscopic observations of CrPUB gene RNAi transgenic algae.

<p>(A) Fluorescence microscopy for the detection of nonpolar lipid accumulation using Nile red staining of CrPUB RNAi lines containing the constructs harboring the fragments amplified using primer set A as the dsRNA. (B) Fluorescence microscopy for the detection of nonpolar lipid accumulation using Nile red staining of CrPUB RNAi lines containing the constructs harboring the fragments amplified using primer set B as the dsRNA. After culturing in HSM for 12 days, the cells of the RNAi lines were stained with Nile red and imaged using a Nikon 80i fluorescence microscope. Yellow fluorescence signals indicate lipid drops, while the red background indicates chlorophyll autofluorescence. Scale bars = 5 μm. Each picture represents a bright field image (left) and a fluorescent image (right).</p