An analytical view of the CrPUB gene family.

<p>A. An unrooted tree summarizing the evolutionary relationships among the 30 members of the CrPUB family. Multiple alignments of the 30 PUB protein sequences from <i>C</i>. <i>reinhardtii</i> were conducted using ClustalX 2.1. The phylogenetic tree was constructed using PhyML3.01 and the ML method with 1,000 bootstrap replicates. The numbers on each node are Shimodaira-Hasegawa-like test indices of statistical support provided by PhyML. Bar = 2.0 is a branch length that represents the number of amino acid substitutions per site. The tree shows the 3 phylogenetic subfamilies (numbered I to III and marked with different color backgrounds) with high predictive values. B. Intron/exon structure: The gene structures were drawn using the online tool GSDS. As shown in the legend, the exons and introns are indicated by green rectangles and thin lines, respectively. The untranslated regions (UTRs) are indicated by blue boxes. The sizes of exons and introns can be estimated using the scale shown at the bottom. C. Schematic representation of the conserved motifs in the 30 CrPUB proteins elucidated using SMART and PROSITE online. The different domains are indicated by different colored boxes denoted at the bottom right corner. The lengths of the proteins and motifs can be estimated using the scale shown at the bottom.</p

Effect of nitrogen starvation on lipid accumulation in <i>C</i>. <i>reinhardtii</i>.

<p>Microalgae cells were cultured in HSM and HSM-N for 4 days. The cell culture medium turned yellow when grown under N starvation. The nonpolar lipid in cells was stained using Nile red and imaged using a Nikon 80i fluorescence microscope. The lipid drops are shown as yellow dots under dark field. Red indicates chlorophyll autofluorescence. The upper panel shows cells grown in HSM, and the lower panel shows cells grown in HSM-N. From left to right: cell culture fluids, bright field images and fluorescent images. Scale bar = 5 μm.</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

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

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

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

Chromosomal locations of the 30 CrPUB genes.

<p>The locations of the 30 CrPUB genes were drafted using MapInspect software, and their duplication patterns were detected using a synteny plot in Plaza. The chromosome numbers and sizes are shown at the top of each chromosome. Each CrPUB gene on the chromosome is displayed on the left side according to the approximate physical location. The tandem gene duplications are indicated in red. The blue line indicates a gene cluster.</p