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Métabolisme Et Régulation Des Glycérolipides Dans Phaeodactylum Tricornutum Et Nannochloropsis Lina-Juana Dolch

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Métabolisme Et Régulation Des Glycérolipides Dans Phaeodactylum Tricornutum Et Nannochloropsis Lina-Juana Dolch Métabolisme et régulation des glycérolipides dans Phaeodactylum tricornutum et Nannochloropsis Lina-Juana Dolch To cite this version: Lina-Juana Dolch. Métabolisme et régulation des glycérolipides dans Phaeodactylum tricornu- tum et Nannochloropsis. Vegetal Biology. Université Grenoble Alpes, 2016. English. NNT : 2016GREAV026. tel-01656731 HAL Id: tel-01656731 https://tel.archives-ouvertes.fr/tel-01656731 Submitted on 6 Dec 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITÉ GRENOBLE ALPES Spécialité : Biologie végétale Arrêté ministériel : 7 août 2006 Présentée par Lina-Juana DOLCH Thèse dirigée par Eric MARECHAL préparée au sein du Laboratoire de Physiologie Cellulaire et Végétale dans l'École Doctorale de Chimie et Sciences du Vivant Glycerolipid metabolism and regulation in Phaeodactylum tricornutum and Nannochloropsis gaditana Thèse soutenue publiquement le 5 décembre 2016, devant le jury composé de : Dr. Giovanni FINAZZI Président Dr. Katrin PHILIPPAR Rapporteur Dr. Yonghua LI-BEISSON Rapporteur Dr. Olga SAYANOVA Examinateur Dr. Frédéric BEAUDOIN Examinateur Dr. Eric MARECHAL Directeur de thèse Wurzeln und Flügel Johann Wolfgang von Goethe RÉSUMÉ Phaeodactylum et Nannochloropsis sont des espèces photosynthétiques modèles pour le métabolisme des glycérolipides, se distinguant par un enrichissement en acides gras polyinsaturés à très longues chaînes (VLC-PUFA) et de grandes quantités en triacylglycérol (TAG). Les proportions des différents lipides sont influencées par des facteurs environnementaux. Nous avons caractérisé le remodelage lipidique chez Phaeodactylum en réponse à la carence en azote et en phosphate. Ces limitations en nutriments induisent une accumulation de TAG, exploitable comme biocarburant. Nous avons identifié de nouveaux composés induisant l'accumulation de TAG et étudié le rôle potentiel du monoxyde d’azote (NO•) dans la régulation du métabolisme lipidique. Nous avons montré qu’en fonction du site de production, le NO• était un signal émis lorsque les conditions de vie étaient critiques, déclenchant l'accumulation de TAG. Les VLC-PUFAs sont produits par des élongases et des désaturases localisées dans le RE. Nous avons identifié une nouvelle classe d’élongases d’acides gras saturés, agissant sur le 16:0, et appelées Δ0-ELO. Le knock out de Δ0-ELO1 de Nannochloropsis réduit le niveau du monogalactosyldiacylglycérol (MGDG), principal lipide des chloroplastes. Ce phénotype met en évidence le rôle de Δ0-ELO1 dans la «voie oméga» qui contrôle le trafic des VLC-PUFAs. Nous avons débuté une dissection de la «voie oméga» par des approches de génétique et des analyses du remodelage lipidique à basse température chez Nannochloropsis. Le diacylglycéryl hydroxyméthyltriméthyl-β-sérine (DGTS) apparaît comme le précurseur de base pour importer des VLC-PUFAs vers le chloroplaste, suivant une voie très régulée du DGTS au MGDG. De plus nous avons montré des fonctions possibles du MGDG et des VLC- PUFAs dans la photoprotection et la régulation de la fluidité membranaire latérale. Phaeodactylum and Nannochloropsis are photosynthetic model species for glycerolipid metabolism, standing out by an enrichment of very-long-chain polyunsaturated fatty acids (VLC-PUFAs) and high contents of neutral lipids such as triacylglycerol (TAG). Lipid profiles are influenced by environmental factors. We characterized the lipid remodelling occurring in Phaeodactylum in response to nitrogen and phosphate starvation. Nutrient limitations induce neutral lipid accumulation, which may be exploited as biofuels. We identified new triggers of TAG accumulation and investigated a potential role of nitric oxide (NO•) as second messenger in the regulation of neutral lipid levels. We conclude that in dependence of the production site, NO• serves as a signalling molecule for critical life conditions and thereby triggers TAG accumulation. VLC-PUFAs are produced by ER-located elongases and desaturases. We identified a novel class of elongases, called Δ0-ELOs, acting on saturated fatty acids, most importantly 16:0. Knock out of Δ0-ELO1 in Nannochloropsis resulted in reduced monogalactosyldiacylglycerol (MGDG) levels. MGDG is the major chloroplast lipid. This indicated a role of this initial elongase in fatty acid fate determination and thus in the elusive “omega pathway” for VLC- PUFA trafficking. We have started to investigate the “omega pathway” by reverse genetic approaches and analyses of low-temperature induced lipid remodelling in Nannochloropsis. Diacylglyceryl hydroxymethyltrimethyl-β-serine (DGTS) appears most likely at the base for the chloroplast import of VLC-PUFA, following a dynamically regulated DGTS-to-MGDG pathway. Additionally, we gave insights into possible functions of MGDG and VLC-PUFA in photoprotection and regulation of membrane fluidity. ABBREVIATIONS °C degree Celsius µE microEinstein 10N10P Enriched Seawater, Artificial Seawater with -1 -1 ESAW 466.7 mg.L NaNO3 and 30.94 mg.L NaH2PO4 AAE/AAS acyl-ACP synthetase ABAC9 ABC class A transporter 9 ACC acetyl-CoA carboxylase ACP acyl carrier protein ACS acyl-CoA synthetases AF alanine-phenylalanine dipeptide AMP adenosinmonophosphate ARA arachidonic acid asRNA antisense RNA At Arabidopsis thaliana ATP adenosintriphosphate ATPase adenosintriphosphate synthetase BLAST Basic Local Alignment Search Tool bp base-pair Bq Becquerel BSA bovine serum albumin CCAP Culture Collection of Algae and Protozoa CCM carbon concentration mechanism Pravosali-Guillard National CCMP Center for Culture of Marine Phytoplankton CDP cytidine diphosphate CDP-DAG cytidine diphosphate diacylglycerol CE collision energy CEF cyclic electron transfer Cer ceramide 3-[(3-Cholamidopropyl)dimethylammonio]-1- CHAPS propanesulfonate Chl chlorophyll CLSM confocal laser scanning microscopy CLSM Confocal laser scanning microscopy CoA coenzyme A CP26 Chloroplast protein 26 CPTIO 2-(4-Carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide Cq quantitation cycle Interdepartmental Biotechnology Centre of the CRIBI University of Padua Clustered Regularly Interspaced Short Palindromic CRISPR Repeats CRYP phytochrome Ctp chloroplast-like transit peptide Cyt cytochrome Cytb6f cytochrome b6f D5 desaturase 5 Da Dalton DAF-FM 4-amino-5-methylamino-2',7'-difluororescein diacetate DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea ddH2O double distilled water DGAT diacylglycerol acyltransferase DGCC diacylglyceryl-3-O-carboxyhydroxymethylcholine DGDG digalactosyldiacylglycerol DGTA diacylglyceryl hydroxymethyltrimethyl-β-alanine DGTS diacylglyceryl hydroxymethyltrimethyl-β-serine DHA docosahexaenoic acid DMP1 disintegration per minute DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DPH 1,6-diphenyl-hexa-1,3,5-triene dsRNA double stranded RNA DTT dithiothreitol ECR β-hydroxylacyl-CoA eonyl reductase EDTA ethylenediaminetetraacetic acid EGT endosymbiotic gene transfer ELO elongase eNOS endothelial EPA eicosapentaenoic acid ER eonyl-reductase ER endoplasmic reticulum ESAW Enriched Seawater, Artificial Seawater EST estimated sequence tag F steady state fluorescence in light-adapted cultures F0 steady state fluorescence in dark-adapted cultures FAME fatty acid methyl esters Fat1p fatty acid import protein 1 FATA/B acyl-ACP thioesterases FAX1 fatty acid export protein 1 FCP fucoxanthin chlorophyll a/c protein Fd ferredoxin FFA free fatty acid maximal fluorescence after a saturating light pulse on Fm dark-adapted cultures maximal fluorescence after a saturating light pulse on Fm’ light-adapted cultures FNR ferredoxin NADPH oxidoreductase FTSH1 ATP-dependent zinc metalloprotease Fv difference between F0 and Fm Fv/Fm maximum quantum efficiency of PSII g gram G3P glycerol-3-phosphate Gal galactose gas chromatography GC-FID coupled to ion flame detection gDNA genomic DNA GFP green fluorescence protein GLRXC2 glutaredoxin GPAT glycerol-sn-3-phosphate acyl-transferase GPAT glycerol-3-phosphate acyltransferase GPDH glycerol-3-phosphate dehydrogenase h hours HAD eonyl-dehydratase HCD β-hydroxylacyl-CoA dehydratase HCF136 photosynthesis system II assembly factor HGT horizontal gene transfer HII hexagonal-II phase HIS histidine HMM Pfam hidden Markov models HPLC high performance liquid chromatography IEM inner envelope membrane iNOS inducible k kilo KAR 3-ketoacyl-ACP reductase KASIII β-ketoacyl-ACP synthase kb kilobyte KCR β-ketoactyl-CoA reductase kd knock down KO knock out L litre LACS long acyl-CoA synthase LDL low-density lipoprotein LDs lipid droplets LEF linear electron flow LEU leucine LHC light harvesting complex LHCB light harvesting complex protein family B LHCF light harvesting complex protein family F LHCII light harvesting complex of photosystem II LHCR light harvesting complex protein family R LHCX1 light harvesting complex protein family X L-NAME Nicotinamide adenine dinucleotide phosphate L-NMMA NG-monomethyl L-arginine LPAT lysophosphatidate acyl-transferase LPCAT acyl-CoA: lysophosphatidylcholine acyltransferase LTQ ion-trap spectrometer LYS lysine m milli M molar m/z mass/charge Mb
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