title: Genome-wide survey and expression analysis of Chlamydomonas reinhardtii U-box E3 ubiquitin ligases (CrPUBs) reveal a functional lipid metabolism module.
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.
authors.affiliation: Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, 571101, China.
authors.forename: Qiulan
authors.initials: Q
authors.lastname: Luo
authors.affiliation: Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, 571101, China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, 571101, China.
authors.forename: Wenquan
authors.initials: W
authors.lastname: Wang
authors.affiliation: School of Science, Hainan Medical College, Haikou, 571101, China.
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Fei
authors.affiliation: Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, 571101, China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Deng
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D032461:Chromosomes, Plant / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics; D050356:Lipid Metabolism / Q000235:genetics*; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D054875:Ubiquitination / Q000235:genetics
chemical_list: D010940:Plant Proteins; D044767:Ubiquitin-Protein Ligases
references.citation: Curr Biol. 2000 Feb 24;10(4):R132-4
references.pmid: 10704423
references.citation: Biochem J. 2008 Aug 1;413(3):447-57
references.pmid: 18393940
references.citation: Plant Physiol. 2014 Jun 25;165(4):1647-1656
references.pmid: 24965176
references.citation: J Biol Chem. 2001 Aug 31;276(35):33111-20
references.pmid: 11435423
references.citation: Plant Physiol. 2003 Jun;132(2):861-9
references.pmid: 12805616
references.citation: Plant Physiol. 2008 Aug;147(4):2084-95
references.pmid: 18552232
references.citation: Plant Biol (Stuttg). 2011 Sep;13(5):725-30
references.pmid: 21815976
references.citation: PLoS One. 2011 Jan 28;6(1):e16219
references.pmid: 21297981
references.citation: J Microbiol Methods. 2009 Apr;77(1):41-7
references.pmid: 19162091
references.citation: Plant Physiol. 2012 Sep;160(1):556-68
references.pmid: 22829319
references.citation: Plant Cell. 2008 Jul;20(7):1899-914
references.pmid: 18664614
references.citation: Mol Cell Proteomics. 2012 Oct;11(10):973-88
references.pmid: 22787274
references.citation: J Proteome Res. 2014 Mar 7;13(3):1373-96
references.pmid: 24528286
references.citation: Plant J. 2006 May;46(4):649-57
references.pmid: 16640601
references.citation: PLoS One. 2011;6(6):e21321
references.pmid: 21738636
references.citation: Plant Cell. 2006 Apr;18(4):1084-98
references.pmid: 16531496
references.citation: Mol Biol Rep. 2012 May;39(5):5883-8
references.pmid: 22201023
references.citation: Plant J. 2013 May;74(3):511-23
references.pmid: 23398263
references.citation: Trends Plant Sci. 2001 Aug;6(8):354-8
references.pmid: 11495788
references.citation: Front Plant Sci. 2014 Feb 19;5:37
references.pmid: 24600457
references.citation: Annu Rev Plant Biol. 2004;55:555-90
references.pmid: 15377232
references.citation: Plant Signal Behav. 2012 Aug;7(8):898-900
references.pmid: 22827949
references.citation: Bioresour Technol. 2013 Sep;143:623-31
references.pmid: 23845710
references.citation: J Exp Bot. 2009;60(4):1109-21
references.pmid: 19196749
references.citation: Plant Cell. 2010 Oct;22(10):3474-88
references.pmid: 20971894
references.citation: Plant Physiol. 2005 Dec;139(4):1597-611
references.pmid: 16339806
references.citation: Science. 2007 Oct 12;318(5848):245-50
references.pmid: 17932292
references.citation: Plant Cell. 2012 Sep;24(9):3708-24
references.pmid: 23012436
references.citation: Plant Cell. 2004 Oct;16(10):2795-808
references.pmid: 15377756
references.citation: J Biol Chem. 2001 Jun 1;276(22):19617-23
references.pmid: 11274149
references.citation: Plant Reprod. 2014 Jun;27(2):59-68
references.pmid: 24573467
references.citation: Eur J Biochem. 1990 Aug 17;191(3):571-6
references.pmid: 2167845
references.citation: Plant Physiol. 2012 Sep;160(1):28-37
references.pmid: 22659522
references.citation: Plant Physiol. 2004 Jan;134(1):59-66
references.pmid: 14657406
references.citation: Mol Plant. 2008 Sep;1(5):800-15
references.pmid: 19825583
references.citation: J Cell Biol. 1975 Dec;67(3):587-605
references.pmid: 1202015
references.citation: Plant J. 2004 Nov;40(4):611-21
references.pmid: 15500475
references.citation: Biochim Biophys Acta. 2003 Sep 23;1651(1-2):50-9
references.pmid: 14499588
references.citation: Plant Physiol. 2010 Dec;154(4):1737-52
references.pmid: 20935180
references.citation: Nature. 2000 Feb 24;403(6772):913-6
references.pmid: 10706292
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D445-51
references.pmid: 23172288
references.citation: Plant J. 2009 Jul;59(1):39-51
references.pmid: 19309463
references.citation: Cell. 2001 Aug 10;106(3):343-54
references.pmid: 11509183
references.citation: Bioresour Technol. 2014;152:572-5
references.pmid: 24360518
references.citation: Plant J. 2009 Oct;60(1):168-80
references.pmid: 19508425
references.citation: Curr Microbiol. 2012 May;64(5):477-85
references.pmid: 22358185
references.citation: PLoS One. 2012;7(11):e49207
references.pmid: 23166612
references.citation: Plant Cell. 2014 Apr 18;26(4):1410-1435
references.pmid: 24748044
references.citation: Mol Biol Rep. 2010 Feb;37(2):653-60
references.pmid: 19288292
references.citation: Mol Biol Evol. 2013 Dec;30(12):2725-9
references.pmid: 24132122
references.citation: Syst Biol. 2003 Oct;52(5):696-704
references.pmid: 14530136
references.citation: Prikl Biokhim Mikrobiol. 2012 Mar-Apr;48(2):150-68
references.pmid: 22586908
references.citation: Mol Cells. 2011 Mar;31(3):201-8
references.pmid: 21347703
references.citation: Plant Cell. 2003 Oct;15(10):2408-20
references.pmid: 14507999
references.citation: Genes Dev. 2008 Apr 1;22(7):825-31
references.pmid: 18381887
references.citation: Plant J. 2007 Jan;49(2):228-37
references.pmid: 17241447
references.citation: J Cell Biol. 2009 Aug 24;186(4):601-13
references.pmid: 19704024
references.citation: Bioresour Technol. 2013 Oct;146:310-316
references.pmid: 23948268
references.citation: Plant Cell. 2006 Apr;18(4):1067-83
references.pmid: 16531490
references.citation: Proteomics. 2011 Nov;11(21):4266-73
references.pmid: 21928291
references.citation: Mol Plant. 2013 Sep;6(5):1388-404
references.pmid: 23307436
references.citation: Plant Cell. 1992 Dec;4(12):1575-88
references.pmid: 1281700
references.citation: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W585-7
references.pmid: 17517783
references.citation: Cell. 1999 Mar 5;96(5):635-44
references.pmid: 10089879
references.citation: PLoS One. 2010 Jul 14;5(7):e11579
references.pmid: 20644651
references.citation: Plant J. 2007 Oct;52(2):309-21
references.pmid: 17714429
references.citation: Plant Physiol. 2006 Dec;142(4):1664-82
references.pmid: 17041029
references.citation: Plant J. 2011 Jan;65(2):194-205
references.pmid: 21223385
references.citation: J Biol Chem. 2012 May 4;287(19):15811-25
references.pmid: 22403401
references.citation: Mol Plant. 2011 Nov;4(6):938-46
references.pmid: 21502661