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

Primers used for real-time RT-PCR and amplifying the genes around the insertion site of mu2.

<p>Primers used for real-time RT-PCR and amplifying the genes around the insertion site of mu2.</p

The random DNA sequences binding to FEMU2 3C2H2 ZF.

<p>(A) List of the 37 of random binding oligonucleotide sequences selected by FEMU2 3C2H2 ZF. Among them, 20 contain the TGGT core sequence, accounting for 65% of the total sequence; 13 contain the TGGG core sequence, accounting for 35% of the total sequence; and 30 contain TGGG/T the core sequence, accounting for 81% of the total sequences. (B) Frequency of flanking nucleotide selection. The consensus-binding sequences (G/T)TTGG(G/T)(G/T)T are also given.</p

Analysis of <i>C. reinhardtii</i> mu2.

<p>(A) Analysis of DNA insertion in <i>C. reinhardtii</i> mu2 by DNA gel blot. After the total DNA of <i>C. reinhardtii</i> mu2 and <i>C. reinhardtii</i> 2A38 were digested with <i>Eco</i>RI or <i>Hind</i>III and verified using agarose gel electrophoresis and gel blot analysis, the mutant gene was hybridized with a 100-bp P³²-labeled <i>ble</i> CDS DNA fragment. (B) The growth curve of <i>C. reinhardtii</i> mu2 and parent strain <i>C. reinhardtii</i> 2A38. -Fe indicates the algae was cultured in 0 µM Fe TAP medium; 0.5, 18, and 200 mean that the algae were cultured in 0.5, 18, and 200 µM Fe TAP medium, respectively. (C) The chlorophyll content of <i>C. reinhardtii</i> mu2 and parent strain <i>C. reinhardtii</i> 2A38. + indicates that the algae was cultured in 18 µM Fe TAP medium; - indicates that the algae was cultured in 0 µM Fe TAP medium. (D) Primarily photochemical efficiency of photosystem II (Fv/Fm) assay of <i>C. reinhardtii</i> mu2 and parent strain <i>C. reinhardtii</i> 2A38 cultivated in –Zn, –Fe, –Mn, and –Cu TAP medium after 4 days. (E) Quantity analysis of ARS activity of <i>C. reinhardtii</i> mu2 and parent strain <i>C. reinhardtii</i> 2A38 cultivated in –Zn, –Fe, –Mn, and –Cu TAP medium after 4 days. T-test was conducted on the experiments using SPSS statistical software. Significance was set to *<i>P</i><0.05, ** <i>P</i><0.01.</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

Comparison of the mRNA abundance of Fe-regulated genes.

<p>Strains IR7, mu2, 2A38, and Maa7- were grown for 4 days in Fe-deficient TAP medium. 2A38, <i>C. reinhardtii</i>. 2A38, CC425 with <i>fox1</i> promoter::<i>ars</i> expression cassette inserted in genome; Maa7-, transgenic algae with pMaa7IR/XIR transformed to <i>C. reinhardtii</i> 2A38; mu2, mutant with the <i>femu2</i> gene deleted from the genome of <i>C. reinhardtii</i> 2A38; IR7, pMaa7IR/Femu2IR transgenic algae.</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

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