高产油小球藻基因组测序和蛋白质组学研究
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摘要
Chlorella protothecoides既可以自养生长也可以异养生长，异养细胞油脂含量达到50%，同时细胞密度可提高20倍以上。C. protothecoides的高产油特性使其适宜于制备生物柴油。为建立C. protothecoides分子研究平台并探索进一步提高微藻产油率的方法，本研究在优化微藻培养生产生物柴油的基础上，进行C.protothecoides基因组测序并对自养和异养藻的全蛋白做差异表达分析。
全基因组测序采用Solexa Paird-End、Solexa Mate-Pair和454三种方法，过滤后序列的总覆盖度为120x。最终拼装得到406个Scaffold，N50为24，全长22.9Mb。显示三个主要的特点：第一，基因组小，基因组比近缘藻Chlorella variabilisNC64A小一倍，基因数目7,039，在已测序绿藻中最少。第二，发现一类在小球藻属及近缘藻中独立进化而来的编码己糖转运蛋白的基因，C. protothecoides中有三个，可能参与异养细胞对糖信号的感应及葡萄糖的吸收。第三，C. protothecoides是唯一缺少编码硝酸盐转运蛋白基因的真核绿藻，但编码氨基酸转运蛋白的基因很丰富，显示出对不同氮源的偏好性。
对自养和异养C. protothecoides的全细胞蛋白做质谱鉴定，共鉴定到1,931个蛋白，占总预测蛋白的27.4%，其中异养细胞上调蛋白206个，下调288个。对差异表达蛋白的功能分析显示出代谢通路的变化，糖酵解、TCA循环和脂肪合成过程均增强，光合作用和碳固定显著降低。首先，说明异养细胞的能量来源从光合磷酸化转变为氧化磷酸化；其次，糖酵解的酶成为异养细胞含量最高的蛋白，较高的糖酵解能力为脂肪酸的合成提供前体物质，而脂肪合成的酶上调为导致油脂积累的直接原因。另外，发现异养状态下脱落酸（ABA）信号转导蛋白表达上调，说明ABA在异养细胞中起到很重要的调控作用。
本研究建立了C. protothecoides的基因组和蛋白质组数据库，为分子研究奠定了基础。同时，揭示出C. protothecoides基因组的特点并阐述异养细胞代谢途径的变化及脂肪积累的机理，分析得到一些可能的调控蛋白及信号，为进一步研究微藻产油的机制提供了新的思路。
Chlorella protothecoides can grow in both autotrophic and heterotrophic conditions.In heterotrophic cells, the oil content reached to50%, the cell density was increasedmore than20-fold. The high oil yield of C. protothecoides makes it an importantresource for biodeisel production. In order to eatablish the platform for molecularresearch and explore methods to improve oil production of microalgae, we sequencedthe genome of C. protothecoides and investigated the proteomic changes betweenautotrophic and heterotrophic cells.
The genome of C. protothecoides was sequenced by Solexa Paird-End, Solexa MatePair and Roche454method, the coverage of the clean data was120x. After assembly406scaffolds (>100bp) were obtained, and the N50was24. The size of the genome is22.9Mb, and7.039genes are predicted. By comparative analysis of the genome, threemain characteristics were found. Firstly, the C. protothecoides has a small genome insize, and about half of what its close relative Chlorella variabilis NC64A has.Furthermore, it encodes the fewest number of genes among all sequenced green algae,and the decrease of total gene is partly because the decrease of genes with the similarfunction. Secondly, a family of hexose/H+cotransporter that restricts to Chlorella wasfound, and there are three such genes in C. protothecoides, which may participated inthe transform of glucose across the plasma membrane. Thirdly, C. protothecoides is theonly eukaryotic green algae that lack nitrate transporter, but represented a biaseddistribution of amino acid transporters, indicating that C. protothecoides prefer to useamino acid as nitrogen source.
The proteins of autotrophic and heterotrophic C. protothecoides were identified byLC/MS/MS.1,931proteins were identified which accounted for27.4%of the totalpredicted proteins. Compared with autotrophic cells,206proteins were up regulatedand288proteins were down regulated. The functional analysis of the differentlyexpressed proteins revealed the changes of the metabolic pathways. The process ofglycolysis, TCA cycle and the synthesis of oil are all enhanced, meanwhile, thephotosynthesis and carbon fixation were inhibited. The results illustrated the transitionof energy biosythesis from photophosphorylation to oxidative phosphorylation in heterotrophic cells. The enzymes in glycolysis are the most abundant proteins inheterotrphic cells. High glycolytic capacity ensured the cell to meet the demand ofprecursors for fatty acid synthesis, and the increase of the fatty acid synthesis enzymesdirectly lead to oil accumulation. The proteins of PP2C and SnRK2in abscisic acid(ABA) signal transduction were also upregulated in heterotrophic cells. ABA may playan important role in heterotrophic growth and oil accumulation.
This study established the molecular research platform of C. protothecoides throughthe completement of genomic sequencing and proteomic research. Meantime, the studyrevealed the characteristics of C. protothecoides genome, and the changes of metabolicpathways in autotrophic and heterotrophic cells were illustrated. The results indicatedthe mechanism of oil accumulation in heterotrophic cells. Moreover, the possiblesignal transduction and regulatory proteins were found. All these provide new ideas forfurther research in mechanism of microalgal oil production.
全基因组测序采用Solexa Paird-End、Solexa Mate-Pair和454三种方法，过滤后序列的总覆盖度为120x。最终拼装得到406个Scaffold，N50为24，全长22.9Mb。显示三个主要的特点：第一，基因组小，基因组比近缘藻Chlorella variabilisNC64A小一倍，基因数目7,039，在已测序绿藻中最少。第二，发现一类在小球藻属及近缘藻中独立进化而来的编码己糖转运蛋白的基因，C. protothecoides中有三个，可能参与异养细胞对糖信号的感应及葡萄糖的吸收。第三，C. protothecoides是唯一缺少编码硝酸盐转运蛋白基因的真核绿藻，但编码氨基酸转运蛋白的基因很丰富，显示出对不同氮源的偏好性。
对自养和异养C. protothecoides的全细胞蛋白做质谱鉴定，共鉴定到1,931个蛋白，占总预测蛋白的27.4%，其中异养细胞上调蛋白206个，下调288个。对差异表达蛋白的功能分析显示出代谢通路的变化，糖酵解、TCA循环和脂肪合成过程均增强，光合作用和碳固定显著降低。首先，说明异养细胞的能量来源从光合磷酸化转变为氧化磷酸化；其次，糖酵解的酶成为异养细胞含量最高的蛋白，较高的糖酵解能力为脂肪酸的合成提供前体物质，而脂肪合成的酶上调为导致油脂积累的直接原因。另外，发现异养状态下脱落酸（ABA）信号转导蛋白表达上调，说明ABA在异养细胞中起到很重要的调控作用。
本研究建立了C. protothecoides的基因组和蛋白质组数据库，为分子研究奠定了基础。同时，揭示出C. protothecoides基因组的特点并阐述异养细胞代谢途径的变化及脂肪积累的机理，分析得到一些可能的调控蛋白及信号，为进一步研究微藻产油的机制提供了新的思路。
Chlorella protothecoides can grow in both autotrophic and heterotrophic conditions.In heterotrophic cells, the oil content reached to50%, the cell density was increasedmore than20-fold. The high oil yield of C. protothecoides makes it an importantresource for biodeisel production. In order to eatablish the platform for molecularresearch and explore methods to improve oil production of microalgae, we sequencedthe genome of C. protothecoides and investigated the proteomic changes betweenautotrophic and heterotrophic cells.
The genome of C. protothecoides was sequenced by Solexa Paird-End, Solexa MatePair and Roche454method, the coverage of the clean data was120x. After assembly406scaffolds (>100bp) were obtained, and the N50was24. The size of the genome is22.9Mb, and7.039genes are predicted. By comparative analysis of the genome, threemain characteristics were found. Firstly, the C. protothecoides has a small genome insize, and about half of what its close relative Chlorella variabilis NC64A has.Furthermore, it encodes the fewest number of genes among all sequenced green algae,and the decrease of total gene is partly because the decrease of genes with the similarfunction. Secondly, a family of hexose/H+cotransporter that restricts to Chlorella wasfound, and there are three such genes in C. protothecoides, which may participated inthe transform of glucose across the plasma membrane. Thirdly, C. protothecoides is theonly eukaryotic green algae that lack nitrate transporter, but represented a biaseddistribution of amino acid transporters, indicating that C. protothecoides prefer to useamino acid as nitrogen source.
The proteins of autotrophic and heterotrophic C. protothecoides were identified byLC/MS/MS.1,931proteins were identified which accounted for27.4%of the totalpredicted proteins. Compared with autotrophic cells,206proteins were up regulatedand288proteins were down regulated. The functional analysis of the differentlyexpressed proteins revealed the changes of the metabolic pathways. The process ofglycolysis, TCA cycle and the synthesis of oil are all enhanced, meanwhile, thephotosynthesis and carbon fixation were inhibited. The results illustrated the transitionof energy biosythesis from photophosphorylation to oxidative phosphorylation in heterotrophic cells. The enzymes in glycolysis are the most abundant proteins inheterotrphic cells. High glycolytic capacity ensured the cell to meet the demand ofprecursors for fatty acid synthesis, and the increase of the fatty acid synthesis enzymesdirectly lead to oil accumulation. The proteins of PP2C and SnRK2in abscisic acid(ABA) signal transduction were also upregulated in heterotrophic cells. ABA may playan important role in heterotrophic growth and oil accumulation.
This study established the molecular research platform of C. protothecoides throughthe completement of genomic sequencing and proteomic research. Meantime, the studyrevealed the characteristics of C. protothecoides genome, and the changes of metabolicpathways in autotrophic and heterotrophic cells were illustrated. The results indicatedthe mechanism of oil accumulation in heterotrophic cells. Moreover, the possiblesignal transduction and regulatory proteins were found. All these provide new ideas forfurther research in mechanism of microalgal oil production.
引文
[1] Scott SA, Davey MP, Dennis JS et al. Biodiesel from algae: challenges and prospects. CurrOpin Biotechnol2010;21:277-86.
[2] Teresa M. Mata AAM, Nidia. S. Caetano. Microalgae for biodiesel production and otherapplications: A review. Renewable and Sustainable Energy Reviews2010;14:16.
[3] Li Y, Horsman M, Wu N et al. Biofuels from microalgae. Biotechnol Prog2008;24:815-20.
[4] Su Z, Kang R, Shi S et al. An economical device for carbon supplement in large-scalemicro-algae production. Bioprocess Biosyst Eng2008;31:641-5.
[5] Zhang QH, Wu X, Xue SZ et al. Hydrodynamic characteristics and microalgae cultivation in anovel flat plate photobioreactor. Biotechnol Prog2012.
[6] Breuer G, Lamers PP, Martens DE et al. The impact of nitrogen starvation on the dynamics oftriacylglycerol accumulation in nine microalgae strains. Bioresour Technol2012;124C:217-226.
[7] Berges JA, Charlebois DO, Mauzerall DC, Falkowski PG. Differential Effects of NitrogenLimitation on Photosynthetic Efficiency of Photosystems I and II in Microalgae. Plant Physiol1996;110:689-696.
[8] Miao X, Wu Q. High yield bio-oil production from fast pyrolysis by metabolic controlling ofChlorella protothecoides. J Biotechnol2004;110:85-93.
[9] Xiong W, Li X, Xiang J, Wu Q. High-density fermentation of microalga Chlorellaprotothecoides in bioreactor for microbio-diesel production. Appl Microbiol Biotechnol2008;78:29-36.
[10] Chen YH, Walker TH. Biomass and lipid production of heterotrophic microalgae Chlorellaprotothecoides by using biodiesel-derived crude glycerol. Biotechnol Lett2011;33:1973-83.
[11] O'Grady J, Morgan JA. Heterotrophic growth and lipid production of Chlorella protothecoideson glycerol. Bioprocess Biosyst Eng2011;34:121-5.
[12] Li X, Xu H, Wu Q. Large-scale biodiesel production from microalga Chlorella protothecoidesthrough heterotrophic cultivation in bioreactors. Biotechnol Bioeng2007;98:764-71.
[13] Xu H, Miao X, Wu Q. High quality biodiesel production from a microalga Chlorellaprotothecoides by heterotrophic growth in fermenters. J Biotechnol2006;126:499-507.
[14] Xiong W, Gao C, Yan D et al. Double CO(2) fixation in photosynthesis-fermentation modelenhances algal lipid synthesis for biodiesel production. Bioresour Technol2010;101:2287-93.
[15] Hortensteiner S, Chinner J, Matile P et al. Chlorophyll breakdown in Chlorella protothecoides:characterization of degreening and cloning of degreening-related genes. Plant Mol Biol2000;42:439-50.
[16] Xiong W, Liu L, Wu C et al.13C-tracer and gas chromatography-mass spectrometry analysesreveal metabolic flux distribution in the oleaginous microalga Chlorella protothecoides. PlantPhysiol2010;154:1001-11.
[17] Ball SG. Eukaryotic Microalgae Genomics. The Essence of Being a Plant. Plant Physiology2005;137:397–98.
[18] Merchant SS, Prochnik SE, Vallon O et al. The Chlamydomonas genome reveals the evolutionof key animal and plant functions. Science2007;318:245-50.
[19] Radakovits R, Jinkerson RE, Fuerstenberg SI et al. Draft genome sequence and genetictransformation of the oleaginous alga Nannochloropis gaditana. Nat Commun2012;3:686.
[20] Matsuzaki M, Misumi O, Shin IT et al. Genome sequence of the ultrasmall unicellular red algaCyanidioschyzon merolae10D. Nature2004;428:653-7.
[21] Gobler CJ, Berry DL, Dyhrman ST et al. Niche of harmful alga Aureococcus anophagefferensrevealed through ecogenomics. Proc Natl Acad Sci U S A2011;108:4352-7.
[22] Bowler C, Allen AE, Badger JH et al. The Phaeodactylum genome reveals the evolutionaryhistory of diatom genomes. Nature2008;456:239-44.
[23] Blanc G, Agarkova I, Grimwood J et al. The genome of the polar eukaryotic microalgaCoccomyxa subellipsoidea reveals traits of cold adaptation. Genome Biol2012;13:R39.
[24] Palenik B, Grimwood J, Aerts A et al. The tiny eukaryote Ostreococcus provides genomicinsights into the paradox of plankton speciation. Proc Natl Acad Sci U S A2007;104:7705-10.
[25] Worden AZ, Lee JH, Mock T et al. Green evolution and dynamic adaptations revealed bygenomes of the marine picoeukaryotes Micromonas. Science2009;324:268-72.
[26] Prochnik SE, Umen J, Nedelcu AM et al. Genomic analysis of organismal complexity in themulticellular green alga Volvox carteri. Science2010;329:223-6.
[27] Huang JC, Wang Y, Sandmann G, Chen F. Isolation and characterization of a carotenoidoxygenase gene from Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol2006;71:473-9.
[28] Li Y, Huang J, Sandmann G, Chen F. Glucose sensing and the mitochondrial alternativepathway are involved in the regulation of astaxanthin biosynthesis in the dark-grown Chlorellazofingiensis (Chlorophyceae). Planta2008;228:735-43.
[29] Morris HJ, Bermudez RC, Carrillo O, Alonso ME. Are the peptide sequences encrypted in foodChlorella protein a possible explanation for the immunostimulatory effects of microalgalsupplements? Med Hypotheses2008;70:896.
[30] Gazzarrini S, Kang M, Abenavoli A et al. Chlorella virus ATCV-1encodes a functionalpotassium channel of82amino acids. Biochem J2009;420:295-303.
[31] Fitzgerald LA, Graves MV, Li X et al. Sequence and annotation of the288-kb ATCV-1virusthat infects an endosymbiotic chlorella strain of the heliozoon Acanthocystis turfacea. Virology2007;362:350-61.
[32] Blanc G, Duncan G, Agarkova I et al. The Chlorella variabilis NC64A genome revealsadaptation to photosymbiosis, coevolution with viruses, and cryptic sex. Plant Cell2010;22:2943-55.
[33] Lee KH. Proteomics: a technology-driven and technology-limited discovery science. TrendsBiotechnol2001;19:217-22.
[34] Wang X, Li X, Deng X et al. A protein extraction method compatible with proteomic analysisfor the euhalophyte Salicornia europaea. Electrophoresis2007;28:3976-87.
[35] Faurobert M, Pelpoir E, Chaib J. Phenol extraction of proteins for proteomic studies ofrecalcitrant plant tissues. Methods Mol Biol2007;355:9-14.
[36] Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology forproteomics. Proteomics2004;4:3665-85.
[37] Roepstorff P. Mass spectrometry in protein studies from genome to function. Curr OpinBiotechnol1997;8:6-13.
[38] Domon B, Aebersold R. Mass spectrometry and protein analysis. Science2006;312:212-7.
[39] Hajduch M, Hearne LB, Miernyk JA et al. Systems analysis of seed filling in Arabidopsis:using general linear modeling to assess concordance of transcript and protein expression. PlantPhysiol2010;152:2078-87.
[40] Scigelova M, Makarov A. Orbitrap mass analyzer--overview and applications in proteomics.Proteomics2006;6Suppl2:16-21.
[41] Kalli A, Hess S. Effect of mass spectrometric parameters on peptide and protein identificationrates for shotgun proteomic experiments on an LTQ-orbitrap mass analyzer. Proteomics2012;12:21-31.
[42] Nesvizhskii AI, Vitek O, Aebersold R. Analysis and validation of proteomic data generated bytandem mass spectrometry. Nat Methods2007;4:787-97.
[43] Eng JK, Fischer B, Grossmann J, Maccoss MJ. A fast SEQUEST cross correlation algorithm. JProteome Res2008;7:4598-602.
[44] Forster B, Mathesius U, Pogson BJ. Comparative proteomics of high light stress in the modelalga Chlamydomonas reinhardtii. Proteomics2006;6:4309-20.
[45] Naumann B, Busch A, Allmer J et al. Comparative quantitative proteomics to investigate theremodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.Proteomics2007;7:3964-79.
[46] Schmidt M, Gessner G, Luff M et al. Proteomic analysis of the eyespot of Chlamydomonasreinhardtii provides novel insights into its components and tactic movements. Plant Cell2006;18:1908-30.
[47] Atteia A, Adrait A, Brugiere S et al. A proteomic survey of Chlamydomonas reinhardtiimitochondria sheds new light on the metabolic plasticity of the organelle and on the nature ofthe alpha-proteobacterial mitochondrial ancestor. Mol Biol Evol2009;26:1533-48.
[48] Le Bihan T, Martin SF, Chirnside ES et al. Shotgun proteomic analysis of the unicellular algaOstreococcus tauri. J Proteomics2011;74:2060-70.
[49] Lee do Y, Park JJ, Barupal DK, Fiehn O. System Response of Metabolic Networks inChlamydomonas reinhardtii to Total Available Ammonium. Mol Cell Proteomics2012;11:973-88.
[50] James GO, Hocart CH, Hillier W et al. Fatty acid profiling of Chlamydomonas reinhardtiiunder nitrogen deprivation. Bioresource Technology2011;102:3343-51.
[51] Wang ZT, Ullrich N, Joo S et al. Algal lipid bodies: stress induction, purification, andbiochemical characterization in wild-type and starchless Chlamydomonas reinhardtii. EukaryotCell2009;8:1856-68.
[52] Work VH, Radakovits R, Jinkerson RE et al. Increased lipid accumulation in theChlamydomonas reinhardtii sta7-10starchless isoamylase mutant and increased carbohydratesynthesis in complemented strains. Eukaryot Cell2010;9:1251-61.
[53] Zabawinski C, Van Den Koornhuyse N, D'Hulst C et al. Starchless mutants of Chlamydomonasreinhardtii lack the small subunit of a heterotetrameric ADP-glucose pyrophosphorylase. JBacteriol2001;183:1069-77.
[54] Guarnieri MT, Nag A, Smolinski SL et al. Examination of triacylglycerol biosyntheticpathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.PLoS One2011;6:e25851.
[55] Nguyen HM, Baudet M, Cuine S et al. Proteomic profiling of oil bodies isolated from theunicellular green microalga Chlamydomonas reinhardtii: with focus on proteins involved inlipid metabolism. Proteomics2011;11:4266-73.
[56] Yang L, Ding Y, Chen Y et al. The proteomics of lipid droplets: structure, dynamics, andfunctions of the organelle conserved from bacteria to humans. J Lipid Res2012;53:1245-53.
[57] Moellering ER, Benning C. RNA interference silencing of a major lipid droplet protein affectslipid droplet size in Chlamydomonas reinhardtii. Eukaryot Cell2010;9:97-106.
[58] Kwon YJ, Wang F, Liu CZ. Deep-bed solid state fermentation of sweet sorghum stalk toethanol by thermotolerant Issatchenkia orientalis IPE100. Bioresour Technol2011;102:11262-5.
[59] Liu R, Shen F. Impacts of main factors on bioethanol fermentation from stalk juice of sweetsorghum by immobilized Saccharomyces cerevisiae (CICC1308). Bioresour Technol2008;99:847-54.
[60] Cooley MB, Carychao D, Nguyen K et al. Effects of environmental stress on stability oftandem repeats in Escherichia coli O157:H7. Appl Environ Microbiol2010;76:3398-400.
[61] Hu Y, Zhang L, He S et al. Cold stress selectively unsilences tandem repeats in heterochromatinassociated with accumulation of H3K9ac. Plant Cell Environ2012.
[62] Hua Q, Yang C, Baba T et al. Responses of the central metabolism in Escherichia coli tophosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts. J Bacteriol2003;185:7053-67.
[63] Harper JW, Adams PD. Cyclin-dependent kinases. Chem Rev2001;101:2511-26.
[64] Ozcan S, Johnston M. Function and regulation of yeast hexose transporters. Microbiol Mol BiolRev1999;63:554-69.
[65] Caspari T, Will A, Opekarova M et al. Hexose/H+symporters in lower and higher plants. J ExpBiol1994;196:483-91.
[66] Sauer N, Tanner W. The hexose carrier from Chlorella. cDNA cloning of a eucaryoticH+-cotransporter. FEBS Lett1989;259:43-6.
[67] Wolf K, Tanner W, Sauer N. The Chlorella H+/hexose cotransporter gene. Curr Genet1991;19:215-9.
[68] Stadler R, Wolf K, Hilgarth C et al. Subcellular localization of the inducible Chlorella HUP1monosaccharide-H+symporter and cloning of a Co-induced galactose-H+symporter. PlantPhysiol1995;107:33-41.
[69] Saier MH, Jr., Beatty JT, Goffeau A et al. The major facilitator superfamily. J Mol MicrobiolBiotechnol1999;1:257-79.
[70] Sauer N, Caspari T, Klebl F, Tanner W. Functional expression of the Chlorella hexosetransporter in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A1990;87:7949-52.
[71] Will A, Caspari T, Tanner W. Km mutants of the Chlorella monosaccharide/H+cotransporterrandomly generated by PCR. Proc Natl Acad Sci U S A1994;91:10163-7.
[72] Stroud RM, Miercke LJ, O'Connell J et al. Glycerol facilitator GlpF and the associatedaquaporin family of channels. Curr Opin Struct Biol2003;13:424-31.
[73] Sweet G, Gandor C, Voegele R et al. Glycerol facilitator of Escherichia coli: cloning of glpFand identification of the glpF product. J Bacteriol1990;172:424-30.
[74] Sutherland FC, Lages F, Lucas C et al. Characteristics of Fps1-dependent and-independentglycerol transport in Saccharomyces cerevisiae. Journal of Bacteriology1997;179:7790-95.
[75] Glass AD, Britto DT, Kaiser BN et al. The regulation of nitrate and ammonium transportsystems in plants. J Exp Bot2002;53:855-64.
[76] Vidal EA, Tamayo KP, Gutierrez RA. Gene networks for nitrogen sensing, signaling, andresponse in Arabidopsis thaliana. Wiley Interdiscip Rev Syst Biol Med2010;2:683-93.
[77] Kanamori T, Kanou N, Atomi H, Imanaka T. Enzymatic characterization of a prokaryotic ureacarboxylase. J Bacteriol2004;186:2532-9.
[78] Glenn S. Maitz, Edward M. Haas, Castric PA. Purification and properties of theallophanatehydrolase from Chlamydomonasreinhardii. Biochimica et Biophysica Acta (BBA)-General Subjects1982;714:486-491.
[79] Forchhammer K. Global carbon/nitrogen control by PII signal transduction in cyanobacteria:from signals to targets. FEMS Microbiol Rev2004;28:319-33.
[80] Commichau FM, Forchhammer K, Stulke J. Regulatory links between carbon and nitrogenmetabolism. Curr Opin Microbiol2006;9:167-72.
[81] Ferris PJ, Goodenough UW. The mating-type locus of Chlamydomonas reinhardtii containshighly rearranged DNA sequences. Cell1994;76:1135-45.
[82] Goodenough U, Lin H, Lee JH. Sex determination in Chlamydomonas. Semin Cell Dev Biol2007;18:350-61.
[83] Malik SB, Pightling AW, Stefaniak LM et al. An expanded inventory of conserved meioticgenes provides evidence for sex in Trichomonas vaginalis. PLoS One2008;3:e2879.
[84] Plaxton WC. The Organization and Regulation of Plant Glycolysis. Annu Rev Plant PhysiolPlant Mol Biol1996;47:185-214.
[85] Zrenner R, Krause KP, Apel P, Sonnewald U. Reduction of the cytosolicfructose-1,6-bisphosphatase in transgenic potato plants limits photosynthetic sucrosebiosynthesis with no impact on plant growth and tuber yield. Plant J1996;9:671-81.
[86] Fischer K, Weber A. Transport of carbon in non-green plastids. Trends Plant Sci2002;7:345-51.
[87] Ohlrogge JB, Jaworski JG. Regulation of Fatty Acid Synthesis. Annu Rev Plant Physiol PlantMol Biol1997;48:109-136.
[88] Flugge UI, Hausler RE, Ludewig F, Gierth M. The role of transporters in supplying energy toplant plastids. J Exp Bot2011;62:2381-92.
[89] Olah J, Orosz F, Keseru GM et al. Triosephosphate isomerase deficiency: a neurodegenerativemisfolding disease. Biochem Soc Trans2002;30:30-8.
[90] Chen M, Thelen JJ. The plastid isoform of triose phosphate isomerase is required for thepostgerminative transition from heterotrophic to autotrophic growth in Arabidopsis. Plant Cell2010;22:77-90.
[91] Dorion S, Clendenning A, Jeukens J et al. A large decrease of cytosolic triosephosphateisomerase in transgenic potato roots affects the distribution of carbon in primary metabolism.Planta2012;236:1177-90.
[92] Valancin A, Srinivasan B, Rivoal J, Jolicoeur M. Analyzing the effect of decreasing cytosolictriosephosphate isomerase on Solanum tuberosum hairy root cells using a kinetic-metabolicmodel. Biotechnol Bioeng2012.
[93] Sweetlove LJ, Beard KF, Nunes-Nesi A et al. Not just a circle: flux modes in the plant TCAcycle. Trends Plant Sci2010;15:462-70.
[94] Schwender J, Shachar-Hill Y, Ohlrogge JB. Mitochondrial metabolism in developing embryosof Brassica napus. J Biol Chem2006;281:34040-7.
[95] Chai GH, Fu XH, Bai ZT et al. Research Progress on Plant endo-1,4-β-Glucanase Genes.Journal of Agricultural Science and Technology2009;11:17-22.
[96] Cosgrove DJ. Expansive growth of plant cell walls. Plant Physiol Biochem2000;38:109-24.
[97] MartíNez F, OrúS MI. Cell Cycle Alteration and β-1,4Endoglucanase in HeterotrophicChlorella vulgaris UAM101. Journal of Plant Physiology1991;138:17-23.
[98] Rioboo C, O'Connor JE, Prado R et al. Cell proliferation alterations in Chlorella cells understress conditions. Aquat Toxicol2009;94:229-37.
[99] SYRETT PJ, M.J.MERRETT, BOCKS SM. Enzymes of the Glyoxylate Cycle in Chlorellavulgaris. Journal of Experimental Botany1962;14:249-264.
[100] Graham IA, Denby KJ, Leaver CJ. Carbon Catabolite Repression Regulates Glyoxylate CycleGene Expression in Cucumber. Plant Cell1994;6:761-772.
[101] Eastmond PJ, Graham IA. Re-examining the role of the glyoxylate cycle in oilseeds. TrendsPlant Sci2001;6:72-8.
[102] Graham IA. Seed storage oil mobilization. Annu Rev Plant Biol2008;59:115-42.
[103] Eastmond PJ, Germain V, Lange PR et al. Postgerminative growth and lipid catabolism inoilseeds lacking the glyoxylate cycle. Proc Natl Acad Sci U S A2000;97:5669-74.
[104] Park SG, Ewalt KL, Kim S. Functional expansion of aminoacyl-tRNA synthetases and theirinteracting factors: new perspectives on housekeepers. Trends Biochem Sci2005;30:569-74.
[105] Han JM, Jeong SJ, Park MC et al. Leucyl-tRNA synthetase is an intracellular leucine sensor forthe mTORC1-signaling pathway. Cell2012;149:410-24.
[106] Davis MS, Solbiati J, Cronan JE, Jr. Overproduction of acetyl-CoA carboxylase activityincreases the rate of fatty acid biosynthesis in Escherichia coli. J Biol Chem2000;275:28593-8.
[107] Roesler K, Shintani D, Savage L et al. Targeting of the Arabidopsis homomericacetyl-coenzyme A carboxylase to plastids of rapeseeds. Plant Physiol1997;113:75-81.
[108] Kosa M, Ragauskas AJ. Lipids from heterotrophic microbes: advances in metabolism research.Trends Biotechnol2011;29:53-61.
[109] Ruddle P,2nd, Whetten R, Cardinal A et al. Effect of a novel mutation in aDelta9-stearoyl-ACP-desaturase on soybean seed oil composition. Theor Appl Genet2012.
[110] Kachroo A, Shanklin J, Whittle E et al. The Arabidopsis stearoyl-acyl carrier protein-desaturasefamily and the contribution of leaf isoforms to oleic acid synthesis. Plant Mol Biol2007;63:257-71.
[111] Vigeolas H, Waldeck P, Zank T, Geigenberger P. Increasing seed oil content in oil-seed rape(Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase underthe control of a seed-specific promoter. Plant Biotechnol J2007;5:431-41.
[112] Slocombe SP, Cornah J, Pinfield-Wells H et al. Oil accumulation in leaves directed bymodification of fatty acid breakdown and lipid synthesis pathways. Plant Biotechnol J2009;7:694-703.
[113] Machler F, Oberson A, Grub A, Nosberger J. Regulation of photosynthesis in nitrogen deficientwheat seedlings. Plant Physiol1988;87:46-9.
[114] Kane PM. Disassembly and reassembly of the yeast vacuolar H(+)-ATPase in vivo. J BiolChem1995;270:17025-32.
[115] Huss M, Vitavska O, Albertmelcher A et al. Vacuolar H(+)-ATPases: intra-and intermolecularinteractions. Eur J Cell Biol2011;90:688-95.
[116] Tarakhovskaya ER, Maslov YI, Shishova MF. Phytohormones in algae. Russian Journal ofPlant Physiology2007;54.
[117] Stirk W, rd g V, Staden JV, J ger K. Cytokinin-and auxin-like activity in Cyanophyta andmicroalgae. Journal of applied phycology2002;14:215-221.
[118] De-Bashan EL, Antoun H, Bashan Y. Involvement of indole-3-acetic-acid produced by themicroalgae growth-promoting bacterium Azospirillum spp. in growth promotion of Chlorellavulgaris. Journal of Phycology2008;44:938-947.
[119] Hirayama T, Umezawa T. The PP2C-SnRK2complex: the central regulator of an abscisic acidsignaling pathway. Plant Signal Behav2010;5:160-3.
[120] Arenas-Huertero F, Arroyo A, Zhou L et al. Analysis of Arabidopsis glucose insensitive mutants,gin5and gin6, reveals a central role of the plant hormone ABA in the regulation of plantvegetative development by sugar. Genes Dev2000;14:2085-96.
[121] Jadhav AS, Taylor DC, Giblin M et al. Hormonal regulation of oil accumulation in Brassicaseeds: metabolism and biological activity of ABA,7'-,8'-and9'-hydroxy ABA in microsporederived embryos of B. napus. Phytochemistry2008;69:2678-88.
[122] Yang Y, Yu X, Song L, An C. ABI4activates DGAT1expression in Arabidopsis seedlingsduring nitrogen deficiency. Plant Physiol2011;156:873-83.
[123] Lu CL, de Noyer SB, Hobbs DH et al. Expression pattern of diacylglycerol acyltransferase-1,an enzyme involved in triacylglycerol biosynthesis, in Arabidopsis thaliana. Plant Mol Biol2003;52:31-41.
[124] Penfield S, Li Y, Gilday AD et al. Arabidopsis ABA INSENSITIVE4regulates lipidmobilization in the embryo and reveals repression of seed germination by the endosperm. PlantCell2006;18:1887-99.
[125] Runswick MJ, Walker JE, Bisaccia F et al. Sequence of the bovine2-oxoglutarate/malatecarrier protein: structural relationship to other mitochondrial transport proteins. Biochemistry1990;29:11033-40.
[126] Weber A, Menzlaff E, Arbinger B et al. The2-oxoglutarate/malate translocator of chloroplastenvelope membranes: molecular cloning of a transporter containing a12-helix motif andexpression of the functional protein in yeast cells. Biochemistry1995;34:2621-7.
[127] Ortiz-Lopez A, Chang H, Bush DR. Amino acid transporters in plants. Biochim Biophys Acta2000;1465:275-80.
[128] Sauer N, Tanner W. Selection and characterization of chlorella mutants deficient in amino Acidtransport: further evidence for three independent systems. Plant Physiol1985;79:760-4.
[129] Murphy DJ, Vance J. Mechanisms of lipid-body formation. Trends Biochem Sci1999;24:109-15.
[2] Teresa M. Mata AAM, Nidia. S. Caetano. Microalgae for biodiesel production and otherapplications: A review. Renewable and Sustainable Energy Reviews2010;14:16.
[3] Li Y, Horsman M, Wu N et al. Biofuels from microalgae. Biotechnol Prog2008;24:815-20.
[4] Su Z, Kang R, Shi S et al. An economical device for carbon supplement in large-scalemicro-algae production. Bioprocess Biosyst Eng2008;31:641-5.
[5] Zhang QH, Wu X, Xue SZ et al. Hydrodynamic characteristics and microalgae cultivation in anovel flat plate photobioreactor. Biotechnol Prog2012.
[6] Breuer G, Lamers PP, Martens DE et al. The impact of nitrogen starvation on the dynamics oftriacylglycerol accumulation in nine microalgae strains. Bioresour Technol2012;124C:217-226.
[7] Berges JA, Charlebois DO, Mauzerall DC, Falkowski PG. Differential Effects of NitrogenLimitation on Photosynthetic Efficiency of Photosystems I and II in Microalgae. Plant Physiol1996;110:689-696.
[8] Miao X, Wu Q. High yield bio-oil production from fast pyrolysis by metabolic controlling ofChlorella protothecoides. J Biotechnol2004;110:85-93.
[9] Xiong W, Li X, Xiang J, Wu Q. High-density fermentation of microalga Chlorellaprotothecoides in bioreactor for microbio-diesel production. Appl Microbiol Biotechnol2008;78:29-36.
[10] Chen YH, Walker TH. Biomass and lipid production of heterotrophic microalgae Chlorellaprotothecoides by using biodiesel-derived crude glycerol. Biotechnol Lett2011;33:1973-83.
[11] O'Grady J, Morgan JA. Heterotrophic growth and lipid production of Chlorella protothecoideson glycerol. Bioprocess Biosyst Eng2011;34:121-5.
[12] Li X, Xu H, Wu Q. Large-scale biodiesel production from microalga Chlorella protothecoidesthrough heterotrophic cultivation in bioreactors. Biotechnol Bioeng2007;98:764-71.
[13] Xu H, Miao X, Wu Q. High quality biodiesel production from a microalga Chlorellaprotothecoides by heterotrophic growth in fermenters. J Biotechnol2006;126:499-507.
[14] Xiong W, Gao C, Yan D et al. Double CO(2) fixation in photosynthesis-fermentation modelenhances algal lipid synthesis for biodiesel production. Bioresour Technol2010;101:2287-93.
[15] Hortensteiner S, Chinner J, Matile P et al. Chlorophyll breakdown in Chlorella protothecoides:characterization of degreening and cloning of degreening-related genes. Plant Mol Biol2000;42:439-50.
[16] Xiong W, Liu L, Wu C et al.13C-tracer and gas chromatography-mass spectrometry analysesreveal metabolic flux distribution in the oleaginous microalga Chlorella protothecoides. PlantPhysiol2010;154:1001-11.
[17] Ball SG. Eukaryotic Microalgae Genomics. The Essence of Being a Plant. Plant Physiology2005;137:397–98.
[18] Merchant SS, Prochnik SE, Vallon O et al. The Chlamydomonas genome reveals the evolutionof key animal and plant functions. Science2007;318:245-50.
[19] Radakovits R, Jinkerson RE, Fuerstenberg SI et al. Draft genome sequence and genetictransformation of the oleaginous alga Nannochloropis gaditana. Nat Commun2012;3:686.
[20] Matsuzaki M, Misumi O, Shin IT et al. Genome sequence of the ultrasmall unicellular red algaCyanidioschyzon merolae10D. Nature2004;428:653-7.
[21] Gobler CJ, Berry DL, Dyhrman ST et al. Niche of harmful alga Aureococcus anophagefferensrevealed through ecogenomics. Proc Natl Acad Sci U S A2011;108:4352-7.
[22] Bowler C, Allen AE, Badger JH et al. The Phaeodactylum genome reveals the evolutionaryhistory of diatom genomes. Nature2008;456:239-44.
[23] Blanc G, Agarkova I, Grimwood J et al. The genome of the polar eukaryotic microalgaCoccomyxa subellipsoidea reveals traits of cold adaptation. Genome Biol2012;13:R39.
[24] Palenik B, Grimwood J, Aerts A et al. The tiny eukaryote Ostreococcus provides genomicinsights into the paradox of plankton speciation. Proc Natl Acad Sci U S A2007;104:7705-10.
[25] Worden AZ, Lee JH, Mock T et al. Green evolution and dynamic adaptations revealed bygenomes of the marine picoeukaryotes Micromonas. Science2009;324:268-72.
[26] Prochnik SE, Umen J, Nedelcu AM et al. Genomic analysis of organismal complexity in themulticellular green alga Volvox carteri. Science2010;329:223-6.
[27] Huang JC, Wang Y, Sandmann G, Chen F. Isolation and characterization of a carotenoidoxygenase gene from Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol2006;71:473-9.
[28] Li Y, Huang J, Sandmann G, Chen F. Glucose sensing and the mitochondrial alternativepathway are involved in the regulation of astaxanthin biosynthesis in the dark-grown Chlorellazofingiensis (Chlorophyceae). Planta2008;228:735-43.
[29] Morris HJ, Bermudez RC, Carrillo O, Alonso ME. Are the peptide sequences encrypted in foodChlorella protein a possible explanation for the immunostimulatory effects of microalgalsupplements? Med Hypotheses2008;70:896.
[30] Gazzarrini S, Kang M, Abenavoli A et al. Chlorella virus ATCV-1encodes a functionalpotassium channel of82amino acids. Biochem J2009;420:295-303.
[31] Fitzgerald LA, Graves MV, Li X et al. Sequence and annotation of the288-kb ATCV-1virusthat infects an endosymbiotic chlorella strain of the heliozoon Acanthocystis turfacea. Virology2007;362:350-61.
[32] Blanc G, Duncan G, Agarkova I et al. The Chlorella variabilis NC64A genome revealsadaptation to photosymbiosis, coevolution with viruses, and cryptic sex. Plant Cell2010;22:2943-55.
[33] Lee KH. Proteomics: a technology-driven and technology-limited discovery science. TrendsBiotechnol2001;19:217-22.
[34] Wang X, Li X, Deng X et al. A protein extraction method compatible with proteomic analysisfor the euhalophyte Salicornia europaea. Electrophoresis2007;28:3976-87.
[35] Faurobert M, Pelpoir E, Chaib J. Phenol extraction of proteins for proteomic studies ofrecalcitrant plant tissues. Methods Mol Biol2007;355:9-14.
[36] Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology forproteomics. Proteomics2004;4:3665-85.
[37] Roepstorff P. Mass spectrometry in protein studies from genome to function. Curr OpinBiotechnol1997;8:6-13.
[38] Domon B, Aebersold R. Mass spectrometry and protein analysis. Science2006;312:212-7.
[39] Hajduch M, Hearne LB, Miernyk JA et al. Systems analysis of seed filling in Arabidopsis:using general linear modeling to assess concordance of transcript and protein expression. PlantPhysiol2010;152:2078-87.
[40] Scigelova M, Makarov A. Orbitrap mass analyzer--overview and applications in proteomics.Proteomics2006;6Suppl2:16-21.
[41] Kalli A, Hess S. Effect of mass spectrometric parameters on peptide and protein identificationrates for shotgun proteomic experiments on an LTQ-orbitrap mass analyzer. Proteomics2012;12:21-31.
[42] Nesvizhskii AI, Vitek O, Aebersold R. Analysis and validation of proteomic data generated bytandem mass spectrometry. Nat Methods2007;4:787-97.
[43] Eng JK, Fischer B, Grossmann J, Maccoss MJ. A fast SEQUEST cross correlation algorithm. JProteome Res2008;7:4598-602.
[44] Forster B, Mathesius U, Pogson BJ. Comparative proteomics of high light stress in the modelalga Chlamydomonas reinhardtii. Proteomics2006;6:4309-20.
[45] Naumann B, Busch A, Allmer J et al. Comparative quantitative proteomics to investigate theremodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.Proteomics2007;7:3964-79.
[46] Schmidt M, Gessner G, Luff M et al. Proteomic analysis of the eyespot of Chlamydomonasreinhardtii provides novel insights into its components and tactic movements. Plant Cell2006;18:1908-30.
[47] Atteia A, Adrait A, Brugiere S et al. A proteomic survey of Chlamydomonas reinhardtiimitochondria sheds new light on the metabolic plasticity of the organelle and on the nature ofthe alpha-proteobacterial mitochondrial ancestor. Mol Biol Evol2009;26:1533-48.
[48] Le Bihan T, Martin SF, Chirnside ES et al. Shotgun proteomic analysis of the unicellular algaOstreococcus tauri. J Proteomics2011;74:2060-70.
[49] Lee do Y, Park JJ, Barupal DK, Fiehn O. System Response of Metabolic Networks inChlamydomonas reinhardtii to Total Available Ammonium. Mol Cell Proteomics2012;11:973-88.
[50] James GO, Hocart CH, Hillier W et al. Fatty acid profiling of Chlamydomonas reinhardtiiunder nitrogen deprivation. Bioresource Technology2011;102:3343-51.
[51] Wang ZT, Ullrich N, Joo S et al. Algal lipid bodies: stress induction, purification, andbiochemical characterization in wild-type and starchless Chlamydomonas reinhardtii. EukaryotCell2009;8:1856-68.
[52] Work VH, Radakovits R, Jinkerson RE et al. Increased lipid accumulation in theChlamydomonas reinhardtii sta7-10starchless isoamylase mutant and increased carbohydratesynthesis in complemented strains. Eukaryot Cell2010;9:1251-61.
[53] Zabawinski C, Van Den Koornhuyse N, D'Hulst C et al. Starchless mutants of Chlamydomonasreinhardtii lack the small subunit of a heterotetrameric ADP-glucose pyrophosphorylase. JBacteriol2001;183:1069-77.
[54] Guarnieri MT, Nag A, Smolinski SL et al. Examination of triacylglycerol biosyntheticpathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.PLoS One2011;6:e25851.
[55] Nguyen HM, Baudet M, Cuine S et al. Proteomic profiling of oil bodies isolated from theunicellular green microalga Chlamydomonas reinhardtii: with focus on proteins involved inlipid metabolism. Proteomics2011;11:4266-73.
[56] Yang L, Ding Y, Chen Y et al. The proteomics of lipid droplets: structure, dynamics, andfunctions of the organelle conserved from bacteria to humans. J Lipid Res2012;53:1245-53.
[57] Moellering ER, Benning C. RNA interference silencing of a major lipid droplet protein affectslipid droplet size in Chlamydomonas reinhardtii. Eukaryot Cell2010;9:97-106.
[58] Kwon YJ, Wang F, Liu CZ. Deep-bed solid state fermentation of sweet sorghum stalk toethanol by thermotolerant Issatchenkia orientalis IPE100. Bioresour Technol2011;102:11262-5.
[59] Liu R, Shen F. Impacts of main factors on bioethanol fermentation from stalk juice of sweetsorghum by immobilized Saccharomyces cerevisiae (CICC1308). Bioresour Technol2008;99:847-54.
[60] Cooley MB, Carychao D, Nguyen K et al. Effects of environmental stress on stability oftandem repeats in Escherichia coli O157:H7. Appl Environ Microbiol2010;76:3398-400.
[61] Hu Y, Zhang L, He S et al. Cold stress selectively unsilences tandem repeats in heterochromatinassociated with accumulation of H3K9ac. Plant Cell Environ2012.
[62] Hua Q, Yang C, Baba T et al. Responses of the central metabolism in Escherichia coli tophosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts. J Bacteriol2003;185:7053-67.
[63] Harper JW, Adams PD. Cyclin-dependent kinases. Chem Rev2001;101:2511-26.
[64] Ozcan S, Johnston M. Function and regulation of yeast hexose transporters. Microbiol Mol BiolRev1999;63:554-69.
[65] Caspari T, Will A, Opekarova M et al. Hexose/H+symporters in lower and higher plants. J ExpBiol1994;196:483-91.
[66] Sauer N, Tanner W. The hexose carrier from Chlorella. cDNA cloning of a eucaryoticH+-cotransporter. FEBS Lett1989;259:43-6.
[67] Wolf K, Tanner W, Sauer N. The Chlorella H+/hexose cotransporter gene. Curr Genet1991;19:215-9.
[68] Stadler R, Wolf K, Hilgarth C et al. Subcellular localization of the inducible Chlorella HUP1monosaccharide-H+symporter and cloning of a Co-induced galactose-H+symporter. PlantPhysiol1995;107:33-41.
[69] Saier MH, Jr., Beatty JT, Goffeau A et al. The major facilitator superfamily. J Mol MicrobiolBiotechnol1999;1:257-79.
[70] Sauer N, Caspari T, Klebl F, Tanner W. Functional expression of the Chlorella hexosetransporter in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A1990;87:7949-52.
[71] Will A, Caspari T, Tanner W. Km mutants of the Chlorella monosaccharide/H+cotransporterrandomly generated by PCR. Proc Natl Acad Sci U S A1994;91:10163-7.
[72] Stroud RM, Miercke LJ, O'Connell J et al. Glycerol facilitator GlpF and the associatedaquaporin family of channels. Curr Opin Struct Biol2003;13:424-31.
[73] Sweet G, Gandor C, Voegele R et al. Glycerol facilitator of Escherichia coli: cloning of glpFand identification of the glpF product. J Bacteriol1990;172:424-30.
[74] Sutherland FC, Lages F, Lucas C et al. Characteristics of Fps1-dependent and-independentglycerol transport in Saccharomyces cerevisiae. Journal of Bacteriology1997;179:7790-95.
[75] Glass AD, Britto DT, Kaiser BN et al. The regulation of nitrate and ammonium transportsystems in plants. J Exp Bot2002;53:855-64.
[76] Vidal EA, Tamayo KP, Gutierrez RA. Gene networks for nitrogen sensing, signaling, andresponse in Arabidopsis thaliana. Wiley Interdiscip Rev Syst Biol Med2010;2:683-93.
[77] Kanamori T, Kanou N, Atomi H, Imanaka T. Enzymatic characterization of a prokaryotic ureacarboxylase. J Bacteriol2004;186:2532-9.
[78] Glenn S. Maitz, Edward M. Haas, Castric PA. Purification and properties of theallophanatehydrolase from Chlamydomonasreinhardii. Biochimica et Biophysica Acta (BBA)-General Subjects1982;714:486-491.
[79] Forchhammer K. Global carbon/nitrogen control by PII signal transduction in cyanobacteria:from signals to targets. FEMS Microbiol Rev2004;28:319-33.
[80] Commichau FM, Forchhammer K, Stulke J. Regulatory links between carbon and nitrogenmetabolism. Curr Opin Microbiol2006;9:167-72.
[81] Ferris PJ, Goodenough UW. The mating-type locus of Chlamydomonas reinhardtii containshighly rearranged DNA sequences. Cell1994;76:1135-45.
[82] Goodenough U, Lin H, Lee JH. Sex determination in Chlamydomonas. Semin Cell Dev Biol2007;18:350-61.
[83] Malik SB, Pightling AW, Stefaniak LM et al. An expanded inventory of conserved meioticgenes provides evidence for sex in Trichomonas vaginalis. PLoS One2008;3:e2879.
[84] Plaxton WC. The Organization and Regulation of Plant Glycolysis. Annu Rev Plant PhysiolPlant Mol Biol1996;47:185-214.
[85] Zrenner R, Krause KP, Apel P, Sonnewald U. Reduction of the cytosolicfructose-1,6-bisphosphatase in transgenic potato plants limits photosynthetic sucrosebiosynthesis with no impact on plant growth and tuber yield. Plant J1996;9:671-81.
[86] Fischer K, Weber A. Transport of carbon in non-green plastids. Trends Plant Sci2002;7:345-51.
[87] Ohlrogge JB, Jaworski JG. Regulation of Fatty Acid Synthesis. Annu Rev Plant Physiol PlantMol Biol1997;48:109-136.
[88] Flugge UI, Hausler RE, Ludewig F, Gierth M. The role of transporters in supplying energy toplant plastids. J Exp Bot2011;62:2381-92.
[89] Olah J, Orosz F, Keseru GM et al. Triosephosphate isomerase deficiency: a neurodegenerativemisfolding disease. Biochem Soc Trans2002;30:30-8.
[90] Chen M, Thelen JJ. The plastid isoform of triose phosphate isomerase is required for thepostgerminative transition from heterotrophic to autotrophic growth in Arabidopsis. Plant Cell2010;22:77-90.
[91] Dorion S, Clendenning A, Jeukens J et al. A large decrease of cytosolic triosephosphateisomerase in transgenic potato roots affects the distribution of carbon in primary metabolism.Planta2012;236:1177-90.
[92] Valancin A, Srinivasan B, Rivoal J, Jolicoeur M. Analyzing the effect of decreasing cytosolictriosephosphate isomerase on Solanum tuberosum hairy root cells using a kinetic-metabolicmodel. Biotechnol Bioeng2012.
[93] Sweetlove LJ, Beard KF, Nunes-Nesi A et al. Not just a circle: flux modes in the plant TCAcycle. Trends Plant Sci2010;15:462-70.
[94] Schwender J, Shachar-Hill Y, Ohlrogge JB. Mitochondrial metabolism in developing embryosof Brassica napus. J Biol Chem2006;281:34040-7.
[95] Chai GH, Fu XH, Bai ZT et al. Research Progress on Plant endo-1,4-β-Glucanase Genes.Journal of Agricultural Science and Technology2009;11:17-22.
[96] Cosgrove DJ. Expansive growth of plant cell walls. Plant Physiol Biochem2000;38:109-24.
[97] MartíNez F, OrúS MI. Cell Cycle Alteration and β-1,4Endoglucanase in HeterotrophicChlorella vulgaris UAM101. Journal of Plant Physiology1991;138:17-23.
[98] Rioboo C, O'Connor JE, Prado R et al. Cell proliferation alterations in Chlorella cells understress conditions. Aquat Toxicol2009;94:229-37.
[99] SYRETT PJ, M.J.MERRETT, BOCKS SM. Enzymes of the Glyoxylate Cycle in Chlorellavulgaris. Journal of Experimental Botany1962;14:249-264.
[100] Graham IA, Denby KJ, Leaver CJ. Carbon Catabolite Repression Regulates Glyoxylate CycleGene Expression in Cucumber. Plant Cell1994;6:761-772.
[101] Eastmond PJ, Graham IA. Re-examining the role of the glyoxylate cycle in oilseeds. TrendsPlant Sci2001;6:72-8.
[102] Graham IA. Seed storage oil mobilization. Annu Rev Plant Biol2008;59:115-42.
[103] Eastmond PJ, Germain V, Lange PR et al. Postgerminative growth and lipid catabolism inoilseeds lacking the glyoxylate cycle. Proc Natl Acad Sci U S A2000;97:5669-74.
[104] Park SG, Ewalt KL, Kim S. Functional expansion of aminoacyl-tRNA synthetases and theirinteracting factors: new perspectives on housekeepers. Trends Biochem Sci2005;30:569-74.
[105] Han JM, Jeong SJ, Park MC et al. Leucyl-tRNA synthetase is an intracellular leucine sensor forthe mTORC1-signaling pathway. Cell2012;149:410-24.
[106] Davis MS, Solbiati J, Cronan JE, Jr. Overproduction of acetyl-CoA carboxylase activityincreases the rate of fatty acid biosynthesis in Escherichia coli. J Biol Chem2000;275:28593-8.
[107] Roesler K, Shintani D, Savage L et al. Targeting of the Arabidopsis homomericacetyl-coenzyme A carboxylase to plastids of rapeseeds. Plant Physiol1997;113:75-81.
[108] Kosa M, Ragauskas AJ. Lipids from heterotrophic microbes: advances in metabolism research.Trends Biotechnol2011;29:53-61.
[109] Ruddle P,2nd, Whetten R, Cardinal A et al. Effect of a novel mutation in aDelta9-stearoyl-ACP-desaturase on soybean seed oil composition. Theor Appl Genet2012.
[110] Kachroo A, Shanklin J, Whittle E et al. The Arabidopsis stearoyl-acyl carrier protein-desaturasefamily and the contribution of leaf isoforms to oleic acid synthesis. Plant Mol Biol2007;63:257-71.
[111] Vigeolas H, Waldeck P, Zank T, Geigenberger P. Increasing seed oil content in oil-seed rape(Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase underthe control of a seed-specific promoter. Plant Biotechnol J2007;5:431-41.
[112] Slocombe SP, Cornah J, Pinfield-Wells H et al. Oil accumulation in leaves directed bymodification of fatty acid breakdown and lipid synthesis pathways. Plant Biotechnol J2009;7:694-703.
[113] Machler F, Oberson A, Grub A, Nosberger J. Regulation of photosynthesis in nitrogen deficientwheat seedlings. Plant Physiol1988;87:46-9.
[114] Kane PM. Disassembly and reassembly of the yeast vacuolar H(+)-ATPase in vivo. J BiolChem1995;270:17025-32.
[115] Huss M, Vitavska O, Albertmelcher A et al. Vacuolar H(+)-ATPases: intra-and intermolecularinteractions. Eur J Cell Biol2011;90:688-95.
[116] Tarakhovskaya ER, Maslov YI, Shishova MF. Phytohormones in algae. Russian Journal ofPlant Physiology2007;54.
[117] Stirk W, rd g V, Staden JV, J ger K. Cytokinin-and auxin-like activity in Cyanophyta andmicroalgae. Journal of applied phycology2002;14:215-221.
[118] De-Bashan EL, Antoun H, Bashan Y. Involvement of indole-3-acetic-acid produced by themicroalgae growth-promoting bacterium Azospirillum spp. in growth promotion of Chlorellavulgaris. Journal of Phycology2008;44:938-947.
[119] Hirayama T, Umezawa T. The PP2C-SnRK2complex: the central regulator of an abscisic acidsignaling pathway. Plant Signal Behav2010;5:160-3.
[120] Arenas-Huertero F, Arroyo A, Zhou L et al. Analysis of Arabidopsis glucose insensitive mutants,gin5and gin6, reveals a central role of the plant hormone ABA in the regulation of plantvegetative development by sugar. Genes Dev2000;14:2085-96.
[121] Jadhav AS, Taylor DC, Giblin M et al. Hormonal regulation of oil accumulation in Brassicaseeds: metabolism and biological activity of ABA,7'-,8'-and9'-hydroxy ABA in microsporederived embryos of B. napus. Phytochemistry2008;69:2678-88.
[122] Yang Y, Yu X, Song L, An C. ABI4activates DGAT1expression in Arabidopsis seedlingsduring nitrogen deficiency. Plant Physiol2011;156:873-83.
[123] Lu CL, de Noyer SB, Hobbs DH et al. Expression pattern of diacylglycerol acyltransferase-1,an enzyme involved in triacylglycerol biosynthesis, in Arabidopsis thaliana. Plant Mol Biol2003;52:31-41.
[124] Penfield S, Li Y, Gilday AD et al. Arabidopsis ABA INSENSITIVE4regulates lipidmobilization in the embryo and reveals repression of seed germination by the endosperm. PlantCell2006;18:1887-99.
[125] Runswick MJ, Walker JE, Bisaccia F et al. Sequence of the bovine2-oxoglutarate/malatecarrier protein: structural relationship to other mitochondrial transport proteins. Biochemistry1990;29:11033-40.
[126] Weber A, Menzlaff E, Arbinger B et al. The2-oxoglutarate/malate translocator of chloroplastenvelope membranes: molecular cloning of a transporter containing a12-helix motif andexpression of the functional protein in yeast cells. Biochemistry1995;34:2621-7.
[127] Ortiz-Lopez A, Chang H, Bush DR. Amino acid transporters in plants. Biochim Biophys Acta2000;1465:275-80.
[128] Sauer N, Tanner W. Selection and characterization of chlorella mutants deficient in amino Acidtransport: further evidence for three independent systems. Plant Physiol1985;79:760-4.
[129] Murphy DJ, Vance J. Mechanisms of lipid-body formation. Trends Biochem Sci1999;24:109-15.