Remodeling of Membrane Lipids in Iron-starved Chlamydomonas*

Background: Iron starvation triggers lipid droplet and triacylglycerol (TAG) accumulation in Chlamydomonas reinhardtii. Results: The overall lipid profile shows a decrease in the absolute content of monogalactosyldiacylglycerol (MGDG) and an increase in saturated and monounsaturated fatty acids. Conclusion: Iron starvation has an early and distinct effect on membrane lipids, before onset of chlorosis. Significance: Iron deficiency affects distribution of lipid type as well as fatty acid profile. Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24–48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

[1]  Christoph Benning,et al.  Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas reinhardtii: Discovery of the Betaine Lipid Synthase BTA1Cr , 2005, Eukaryotic Cell.

[2]  B. Fox,et al.  Stearoyl-acyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[3]  C. Pérez,et al.  Effect of iron chlorosis on mineral nutrition and lipid composition of thylakoid biomembrane in Prunus persica (L.) Bastch. , 1993, Plant and Soil.

[4]  Ian K. Blaby,et al.  Three Acyltransferases and Nitrogen-responsive Regulator Are Implicated in Nitrogen Starvation-induced Triacylglycerol Accumulation in Chlamydomonas* , 2012, The Journal of Biological Chemistry.

[5]  C. Benning,et al.  A Cytochrome b5-Containing Plastid-Located Fatty Acid Desaturase from Chlamydomonas reinhardtii , 2012, Eukaryotic Cell.

[6]  E. Heinz,et al.  The suitability of lipase from Rhizopus arrhizus delemar for analysis of fatty acid distribution in dihexosyl diglycerides, phospholipids and plant sulfolipids. , 1973, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[7]  A. Busch,et al.  Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo-oxidative stress in response to iron availability in Chlamydomonas reinhardtii. , 2008, The Plant journal : for cell and molecular biology.

[8]  J. Folch,et al.  A simple method for the isolation and purification of total lipides from animal tissues. , 1957, The Journal of biological chemistry.

[9]  Y. Li-Beisson,et al.  Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves , 2011, BMC biotechnology.

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

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

[12]  E. Cahoon,et al.  Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169. , 2012, Phytochemistry.

[13]  Meng Chen,et al.  Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta. , 2011, Bioresource technology.

[14]  Wenxu Zhou,et al.  The Metabolome of Chlamydomonas reinhardtii following Induction of Anaerobic H2 Production by Sulfur Depletion* , 2009, The Journal of Biological Chemistry.

[15]  H. Ohta,et al.  Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis. , 2013, The Plant journal : for cell and molecular biology.

[16]  T. S. Moore,et al.  Membrane lipid biosynthesis in Chlamydomonas reinhardtii. In vitro biosynthesis of diacylglyceryltrimethylhomoserine. , 2001, Plant physiology.

[17]  T. S. Moore,et al.  Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Partial characterization of CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase , 2003 .

[18]  Scott I. Hsieh,et al.  Fe Sparing and Fe Recycling Contribute to Increased Superoxide Dismutase Capacity in Iron-Starved Chlamydomonas reinhardtii[W] , 2012, Plant Cell.

[19]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[20]  P. Weers,et al.  Growth and reproduction of Daphnia galeata in response to changes in fatty acids, phosphorus, and nitrogen in Chlamydomonas reinhardtii , 1997 .

[21]  E. H. Harris.  The Chlamydomonas sourcebook , 2009 .

[22]  J. Ohlrogge,et al.  DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development[W][OA] , 2009, The Plant Cell Online.

[23]  S. Merchant,et al.  Adaptation to Fe‐deficiency requires remodeling of the photosynthetic apparatus , 2002, The EMBO journal.

[24]  Cassandra Johnny,et al.  A Galactoglycerolipid Lipase Is Required for Triacylglycerol Accumulation and Survival Following Nitrogen Deprivation in Chlamydomonas reinhardtii[C][W] , 2012, Plant Cell.

[25]  Y. Li-Beisson,et al.  Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: With focus on proteins involved in lipid metabolism , 2011, Proteomics.

[26]  S. Merchant,et al.  FEA1, FEA2, and FRE1, Encoding Two Homologous Secreted Proteins and a Candidate Ferrireductase, Are Expressed Coordinately with FOX1 and FTR1 in Iron-Deficient Chlamydomonas reinhardtii , 2007, Eukaryotic Cell.

[27]  S. Merchant,et al.  TAG, you're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation. , 2012, Current opinion in biotechnology.

[28]  O. Kruse,et al.  Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii. , 2012, Journal of biotechnology.

[29]  Q. Hu,et al.  Phospholipid:Diacylglycerol Acyltransferase Is a Multifunctional Enzyme Involved in Membrane Lipid Turnover and Degradation While Synthesizing Triacylglycerol in the Unicellular Green Microalga Chlamydomonas reinhardtii[C][W] , 2012, Plant Cell.

[30]  K. Gable,et al.  Metabolic Response to Iron Deficiency in Saccharomyces cerevisiae* , 2010, The Journal of Biological Chemistry.

[31]  K. Niyogi,et al.  Trophic status of Chlamydomonas reinhardtii influences the impact of iron deficiency on photosynthesis , 2010, Photosynthesis Research.

[32]  N. Terry.  Limiting Factors in Photosynthesis: I. USE OF IRON STRESS TO CONTROL PHOTOCHEMICAL CAPACITY IN VIVO. , 1980, Plant physiology.

[33]  C. Benning,et al.  RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in Chlamydomonas reinhardtii , 2009, Eukaryotic Cell.

[34]  N. Terry.  Limiting factors in photosynthesis. , 1980 .

[35]  A. Abadı́a,et al.  Iron deficiency in pea leaves: Effect on lipid composition and synthesis , 1988 .

[36]  C. Benning,et al.  Rapid Triacylglycerol Turnover in Chlamydomonas reinhardtii Requires a Lipase with Broad Substrate Specificity , 2012, Eukaryotic Cell.

[37]  Yajun Li,et al.  The effects of nutritional restriction on neutral lipid accumulation in Chlamydomonas and Chlorella , 2011 .

[38]  C. Stocking.  Iron deficiency and the structure and physiology of maize chloroplasts. , 1975, Plant physiology.

[39]  W. Huber,et al.  which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets , 2011 .

[40]  Scott I. Hsieh,et al.  A Ferroxidase Encoded by FOX1 Contributes to Iron Assimilation under Conditions of Poor Iron Nutrition in Chlamydomonas , 2008, Eukaryotic Cell.

[41]  M. Pellegrini,et al.  A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii. , 2011, The Plant journal : for cell and molecular biology.

[42]  S. E. Taylor,et al.  Changes in Thylakoid Galactolipids and Proteins during Iron Nutrition-Mediated Chloroplast Development. , 1985, Plant Physiology.

[43]  Scott I. Hsieh,et al.  Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage[W][OA] , 2012, Plant Cell.

[44]  Scott I. Hsieh,et al.  The Proteome of Copper, Iron, Zinc, and Manganese Micronutrient Deficiency in Chlamydomonas reinhardtii* , 2012, Molecular & Cellular Proteomics.

[45]  Changcheng Xu,et al.  A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii , 2011, FEBS letters.

[46]  Wenyu Yang,et al.  Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase. , 2004, The Biochemical journal.

[47]  A. Busch,et al.  Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii , 2007, Proteomics.

[48]  John Shanklin,et al.  DESATURATION AND RELATED MODIFICATIONS OF FATTY ACIDS1. , 1998, Annual review of plant physiology and plant molecular biology.

[49]  E. Heinz,et al.  Desaturation of oleoyl groups in envelope membranes from spinach chloroplasts. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[50]  S. Merchant,et al.  Copper-responsive gene expression during adaptation to copper deficiency. , 1998, Methods in enzymology.

[51]  J. Schloss.  A Chlamydomonas gene encodes a G protein β subunit-like polypeptide , 1990, Molecular and General Genetics MGG.

[52]  S. Eberhard,et al.  A single mutation that causes phosphatidylglycerol deficiency impairs synthesis of photosystem II cores in Chlamydomonas reinhardtii. , 2004, European journal of biochemistry.

[53]  C. Benning,et al.  The Sulfolipids 2′-O-Acyl-Sulfoquinovosyldiacylglycerol and Sulfoquinovosyldiacylglycerol Are Absent from a Chlamydomonas reinhardtii Mutant Deleted in SQD11 , 2003, Plant Physiology.

[54]  C. Benning,et al.  Lipid metabolism in microalgae distinguishes itself. , 2013, Current opinion in biotechnology.

[55]  I. Graham,et al.  A Cytosolic Acyltransferase Contributes to Triacylglycerol Synthesis in Sucrose-Rescued Arabidopsis Seed Oil Catabolism Mutants1[W][OA] , 2012, Plant Physiology.

[56]  P. Covello,et al.  Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. , 2001, Plant physiology.

[57]  J. Browse,et al.  A new class of Arabidopsis mutants with reduced hexadecatrienoic acid fatty acid levels. , 1998, Plant physiology.