Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation

BackgroundNitrogen limitation can induce neutral lipid accumulation in microalgae, as well as inhibiting their growth. Therefore, to obtain cultures with both high biomass and high lipid contents, and explore the lipid accumulation mechanisms, we implemented nitrogen deprivation in a model diatom Phaeodactylum tricornutum at late exponential phase.ResultsNeutral lipid contents per cell subsequently increased 2.4-fold, both the number and total volume of oil bodies increased markedly, and cell density rose slightly. Transcriptional profile analyzed by RNA-Seq showed that expression levels of 1213 genes (including key carbon fixation, TCA cycle, glycerolipid metabolism and nitrogen assimilation genes) increased, with a false discovery rate cut-off of 0.001, under N deprivation. However, most light harvesting complex genes were down-regulated, extensive degradation of chloroplast membranes was observed under an electron microscope, and photosynthetic efficiency declined. Further identification of lipid classes showed that levels of MGDG and DGDG, the main lipid components of chloroplast membranes, dramatically decreased and triacylglycerol (TAG) levels significantly rose, indicating that intracellular membrane remodeling substantially contributed to the neutral lipid accumulation.ConclusionsOur findings shed light on the molecular mechanisms of neutral lipid accumulation and the key genes involved in lipid metabolism in diatoms. They also provide indications of possible strategies for improving microalgal biodiesel production.

[1]  H. Ohta,et al.  Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation. , 2009, The Plant journal : for cell and molecular biology.

[2]  J. Scott Brockenbrough,et al.  Computational Genomics: Theory and Application , 2005 .

[3]  G. Lepage,et al.  Improved recovery of fatty acid through direct transesterification without prior extraction or purification. , 1984, Journal of lipid research.

[4]  C. Ratledge,et al.  The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi. , 1999, Microbiology.

[5]  Wei Chen,et al.  A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. , 2009, Journal of microbiological methods.

[6]  P. Hedley,et al.  Early response mechanisms of perennial ryegrass (Lolium perenne) to phosphorus deficiency , 2010, Annals of botany.

[7]  L. McAlister-Henn,et al.  Sources of NADPH in Yeast Vary with Carbon Source* , 2005, Journal of Biological Chemistry.

[8]  M. Osteras,et al.  Increased pyruvate orthophosphate dikinase activity results in an alternative gluconeogenic pathway in Rhizobium (Sinorhizobium) meliloti. , 1997, Microbiology.

[9]  G. Price,et al.  Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation. , 2011, Bioresource technology.

[10]  W. Plaxton,et al.  In vitro phosphorylation of phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum. , 2002, Plant & cell physiology.

[11]  Todd W. Lane,et al.  Triacylglycerol accumulation and profiling in the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Baccilariophyceae) during starvation , 2009, Journal of Applied Phycology.

[12]  Jean-Michel Robert,et al.  Diatom cultivation and biotechnologically relevant products. Part I: Cultivation at various scales , 2003, Applied Microbiology and Biotechnology.

[13]  F. Morel,et al.  The Role of the C4 Pathway in Carbon Accumulation and Fixation in a Marine Diatom1 , 2004, Plant Physiology.

[14]  Michael A. Borowitzka,et al.  Light and nitrogen deficiency effects on the growth and composition ofPhaeodactylum tricornutum , 1993 .

[15]  J. Reinfelder,et al.  The Role of the C 4 Pathway in Carbon Accumulation and Fixation in a Marine Diatom 1 , 2004 .

[16]  E. Ojeda Land.  Changes in the fatty acid composition of Phaeodactylum tricornutum and Dunaliella tertiolecta during growth and under phosphorus deficiency , 2022 .

[17]  J. Raven,et al.  Carbon acquisition by diatoms , 2007, Photosynthesis Research.

[18]  Leszek Rychlewski,et al.  The Phaeodactylum genome reveals the evolutionary history of diatom genomes , 2008, Nature.

[19]  Ying Zhang,et al.  Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. , 2007, Microbiology.

[20]  P. Falkowski,et al.  Biogeochemical Controls and Feedbacks on Ocean Primary Production , 1998, Science.

[21]  L. Tretter,et al.  Alpha-ketoglutarate dehydrogenase: a target and generator of oxidative stress , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[22]  T. Mock,et al.  Photosynthetic energy conversion under extreme conditions--I: important role of lipids as structural modulators and energy sink under N-limited growth in Antarctic sea ice diatoms. , 2002, Phytochemistry.

[23]  Shin-Han Shiu,et al.  Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism1[W][OA] , 2010, Plant Physiology.

[24]  D. Taylor,et al.  Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content. , 2008, Plant biotechnology journal.

[25]  E. Grima,et al.  Gram-scale purification of eicosapentaenoic acid (EPA, 20:5n-3) from wetPhaeodactylum tricornutum UTEX 640 biomass , 1996, Journal of Applied Phycology.

[26]  S. Fowler,et al.  Nile red: a selective fluorescent stain for intracellular lipid droplets , 1985, The Journal of cell biology.

[27]  Anders S Carlsson,et al.  Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis1 , 2004, Plant Physiology.

[28]  Yuelu Jiang,et al.  Photosynthetic performance, lipid production and biomass composition in response to nitrogen limitation in marine microalgae. , 2012, Plant physiology and biochemistry : PPB.

[29]  F. Morel,et al.  Expression and Inhibition of the Carboxylating and Decarboxylating Enzymes in the Photosynthetic C4 Pathway of Marine Diatoms1 , 2007, Plant Physiology.

[30]  Ursula Goodenough,et al.  Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and Starchless Chlamydomonas reinhardtii , 2009, Eukaryotic Cell.

[31]  K. Niyogi,et al.  PHOTOPROTECTION REVISITED: Genetic and Molecular Approaches. , 1999, Annual review of plant physiology and plant molecular biology.

[32]  A. Grossman,et al.  MACRONUTRIENT UTILIZATION BY PHOTOSYNTHETIC EUKARYOTES AND THE FABRIC OF INTERACTIONS. , 2001, Annual review of plant physiology and plant molecular biology.

[33]  A. Kaplan,et al.  A Model for Carbohydrate Metabolism in the Diatom Phaeodactylum tricornutum Deduced from Comparative Whole Genome Analysis , 2008, PloS one.

[34]  Xuemin Wang,et al.  Differential Degradation of Extraplastidic and Plastidic Lipids during Freezing and Post-freezing Recovery in Arabidopsis thaliana* , 2008, Journal of Biological Chemistry.

[35]  G. Schmidt,et al.  Chlororespiration: an adaptation to nitrogen deficiency in Chlamydomonas reinhardtii. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Milton Sommerfeld,et al.  Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol. , 2010, Metabolic engineering.

[37]  C. Demandre,et al.  Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography , 1985 .

[38]  A. Zarka,et al.  Cloning and molecular characterization of a novel acyl‐CoA:diacylglycerol acyltransferase 1‐like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum , 2011, The FEBS journal.

[39]  R. Carlson,et al.  Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum , 2012, Biotechnology for Biofuels.

[40]  G Peltier,et al.  Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress. , 2000, The Plant journal : for cell and molecular biology.

[41]  T. Larson,et al.  CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) 1 , 1996 .

[42]  Y. Chisti.  Biodiesel from microalgae. , 2007, Biotechnology advances.

[43]  S. Kopriva,et al.  The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants1[W] , 2011, Plant Physiology.

[44]  K. V. van Wijk,et al.  Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes1[W] , 2006, Plant Physiology.

[45]  C. Bowler,et al.  Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions 1 , 2007 .

[46]  F. Morel,et al.  INAUGURAL ARTICLE by a Recently Elected Academy Member:Efficiency of the CO2-concentrating mechanism of diatoms , 2011 .

[47]  D. Campbell,et al.  Chlorophyll Fluorescence Analysis of Cyanobacterial Photosynthesis and Acclimation , 1998, Microbiology and Molecular Biology Reviews.