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Molecular Regulation of Carotenoid Biosynthesis in Tomato Fruits. New Biotechnological Strategies Lucio D'andrea

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Molecular Regulation of Carotenoid Biosynthesis in Tomato Fruits. New Biotechnological Strategies Lucio D'andrea ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en molecular regulation of carotenoid biosynthesis in tomato fruit new biotechnological strategies Lucio D’Andrea 2016 Universitat Autónoma de Barcelona Facultat de Biociències Doctorat de Biologia i Biotecnologia Vegetal 2016 Molecular regulation of carotenoid biosynthesis in tomato fruits New biotechnological strategies Memoria presentada por Lucio D’Andrea para optar al título de doctor por la Universitat Autónoma de Barcelona Lucio D’Andrea Candidato a doctor Manuel Rodriguez-Concepción Briardo Llorente Director Co-director Joan Barceló Coll Tutor “Procuremos inventar pasiones nuevas, o reproducir las viejas con pareja intensidad” Del libro Rayuela de Julio Cortázar “Caminando en línea recta no puede uno llegar muy lejos” Del libro El Principito de Antoine de Saint-Exupéry. Esta tesis se la dedico A mi abuela Nelly y a mi madre. A la primera por las raíces y a la segunda por las alas. Table of contents Agradecimientos i Resumen iii Summary v Index of Figures vii Index of Tables xi Introduction 1 Section I: The tomato fruit 3 o Stages of tomato fruit development 4 . System I or Maturation 4 . System II or Ripening 5 o Metabolic changes during ripening 5 o The chloroplast to chromoplast transition 7 o Ethylene production and perception 9 o Transcriptional regulators of tomato ripening 10 Section II: Carotenoid biosynthesis 13 o The MEP pathway 14 o Carotenoid biosynthesis 17 . GGPP to phytoene: phytoene synthase (PSY) 17 . From phytoene to lycopene 19 . From lycopene to cyclic carotenes 20 . Biosynthesis of xanthophylls 21 Xanthophylls cycle enzymes 22 Neoxanthin synthase 22 o Biosynthesis of apocarotenids 23 Section III: Molecular regulation of carotenoid biosynthesis 24 o The coordination between the MEP pathway and carotenoid biosynthetic pathway 24 o Transcriptional regulation 26 . Developmental regulation 26 . Light signaling 28 o Post-transcriptional regulation 30 . Modulation of enzyme levels and activity 30 . Metabolite channeling of multi-enzyme complexes 32 Section IV: Economic and nutritional value of tomato fruit and Carotenoids 34 o Nutritional Quality 34 o Genetic engineering of carotenoid production in tomato fruit 36 Objectives 43 Results 45 Chapter I: A role for shade signaling on the regulation of carotenoid biosynthesis during tomato fruit ripening. 47 Background and rationale: light, PSY and carotenoids 49 The ripening-induced tomato PIF1a is a true PIF1 homologue 50 PIF1a represses PSY1 expression by binding to a PBE box in its promoter 55 Tomato fruit chlorophyll reduces the R/FR ratio of sunlight as it penetrates the fruit flesh 59 Fruit pigmentation-dependent changes in the R/FR ratio specifically influence PSY1 expression 61 Changes in the R/FR ratio of the light sensed in pericarp cells likely adjust carotenoid biosynthesis to the actual progress of ripening 64 Chapter II: A role for the Clp protease complex during tomato fruit ripening. 69 Background and rationale: the Clp protease complex in plants 71 Genes encoding Clp protease subunits are induced during tomato fruit ripening 74 Silencing of the tomato ClpR1 gene during fruit ripening affects carotenoid accumulation profile 78 Transgenic E8:amiR1 fruit show an orange color when ripe due to an enrichment in β-carotene, the main pro-vitamin A carotenoid 83 E8:amiR1 fruits do not fully differentiate typical chromplasts 89 Higher levels of DXS protein (but not transcripts) in transgenic amiR1 fruits are consistent with a reduction in Clp protease activity 91 Clp-defective amiR1 fruits have a similar chromoplast proteome to control fruits at the R stage 92 Clp protease-dependent protein turnover plays an important role during tomato fruit ripening. 95 The rate-limiting enzymes of the MEP and carotenoid pathways might be targets of the Clp protease in tomato fruit chromoplasts 103 Discussion 107 Section I: A role for shade signaling on the regulation of carotenoid biosynthesis during tomato fruit ripening. 109 . The self-shading model: recycling of a PIF-based mechanism to monitor tomato fruit ripening 109 . Carotenoids and seed-dispersion: an evolutionary perspective 112 Section II: A role for the Clp protease complex during tomato fruit ripening 114 A role for the Clp protease in carotenoid biosynthesis 114 A role for the Clp protease in chromoplast ultrastructure 119 Section III: All roads lead to Rome: PSY as a central regulator of carotenogenesis in tomato fruit. 122 Conclusions 125 Material and Methods 129 Plant material and growth conditions 131 o Plant material 131 o Plant growth conditions 132 Nucleic acids thechniques 133 o PCR. Cloning and colony screening 133 o Gateway cloning 133 . Virus induced gene silencing (VIGS) cloning 133 . Artifitial microRNA (amiRNA) cloning 134 o Bacteria transformation by heat shock 136 o Plasmidic DNA extraction 137 o Gel Purification 137 o RNA extraction 137 o cDNA synthesis 138 o Gene expression analysis 138 o Chromatin inmuno-precipitation (ChIP) coupled to qPCR 140 Protein techniques 141 o Protein extraction 141 o SDS-PAGE 141 o Western blot 142 . Densitometry 142 Metabolite techniques 143 o Plastidial Isoprenoids analysis by HPLC 143 Plant molecular biology techniques o Seed sterilization and sowing 143 o Plant transformation 143 . MicroTom (MT) stable transformation 144 . Transient transformation 144 o Selection of transgenic plant 145 . Genomic DNA extraction 146 . Genotyping PCR 146 o Cycloheximide (CHX) experiment 146 Microscopy and imaging 147 o Laser confocal microscopy 147 o Transmition electronic microscopy (TEM) 147 o Raman imaging 147 o Photography 148 Biophotonics 148 System biology techniques 148 o Plastid isolation 148 o Protein solubilization from isolated plastids 149 o Proteomic analysis. 149 . Sample preparation 149 . TMT labeling 149 . High pH reverse phase (hpRP) fractionation 150 . Nano-scale reverse phase chromatography and tandem mass spectrometry (nanoLC-MS/MS) 151 . Data processing, protein identification and data analysis 152 Statistical analysis 153 Bioinformatic Analysis 153 o Gene expression analysis from microarray and RNA-seq data 153 o MapMan 153 o Sequence aligment and phylogenetic trees 154 References 155 Annexes 181 Publications 195 Agradecimientos Probablemente esta sea la parte que más me costó escribir. No porque no supiese a quién agradecer, sino más bien porque es tanta la gente, que es muy probable que la memoria me juegue una mala pasada. Por tanto, prefiero asumir que será inevitable dicho olvido y agradecer a todas aquellas personas que formaron parte de mi vida durante estos cuatro años. Empiezo. Quiero agradecer: Al Ministerio de Educación, Cultura y Deporte del Gobierno de España por haberme concedido la beca de Formación de Personal Universitario y con ello la posibilidad de realizar esta tesis doctoral. A mi director, Manuel, porque esta tesis no habría sido posible sino fuese porque cuatro años atrás, cuando estaba a punto de dejar de intentarlo, me tendió su mano. Sin duda, uno de los momentos más gratos de mi vida fue leer aquel e-mail, diciendo que tendría mi segunda oportunidad para realizar la tesis doctoral. Agradecerle también por su dirección continua, por su predisposición, por el apoyo constante, por las discusiones científicas y, fundamentalmente, por enseñarme a pensar. Más aún, por la libertad y la confianza en el laboratorio. Y sobre todo, gracias por enseñarnos que es mejor ser líder que jefe. A mi co-director y amigo, Briardo. Me considero un afortunado, por haber tenido al mejor ejemplo de amor a la ciencia, tenacidad y perseverancia. Gracias por el apoyo, tanto personal como científico durante estos años. Gracias por enseñarme que cuando se persigue, se consigue. Pero de lo que más agradecido estoy es de haber podido aprender de vos todos estos años; sin dudas sos y serás uno de mis pilares fundadores en esto de ser científico. A la Dra Li, Li y al Dr. Theodore Thannhauser de la Universidad de Cornell, por haberme dado la oportunidad de conocer, no solo nuevas técnicas, sino también, nuevas maneras de hacer ciencia. A mi familia. Que cuando hace 6 años dije que me venía a Europa todo fueron palabras de aliento. Porque siempre me brindaron todo el apoyo para que mis proyectos se cumplan. A mis padres, mis hermanas, mis cuñados, y en especial, a mis sobrinos, Emilia, Tiago y Juani, que sus sonrisas han hecho que esos días no tan buenos sean maravillosos. Al 2.01 TODO. A los viejos, Águila, Jordi, David, Mike, Cata, Pablo, Esther; a los nuevos Neto, Vicky, Miguel, Sofía; y a las que estuvieron durante todos estos años Miriam y Rosa. Por las risas, por los llantos (pero sobre todo por las risas). Por todas las fú, por “las casas por la ventana”, “los i tenis” y los asados en Segur. Por aguantarme las locuras y dejarme aguantar las de todos ustedes. Sin dudas todo este tiempo ha hecho que seamos más que un laboratorio, una gran familia. Al personal investigador del CRAG. Especialmente a mis compañeros (algunos ya viejas glorias). Esos con los que hemos andado juntos algunos años. Agradecerle especialmente a Mariana, Pat, Luis, Walter, Elena, Norma, Pep, Sil, Chiqui y Carmen. A los servicios científicos y personal de administración del CRAG, por hacer todo lo posible para que las cosas nos sean fáciles y rápidas. Especialmente a Pilar, porque fueron varios años de luchar codo a codo, contra aún no tenemos muy claro qué; pero parece que le hemos ganado. A las troupes que me acogieron estos años en España.
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