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Table of contents
CHAPTER I STATE OF ART
I. JOURNEY TO THE CENTRE OF PLANT DEFENCE
A. THROUGH THE LOOKING-GLASS: OPEN YOUR EYES, DON’T BE PLANT BLIND!
B. THE DEFENCE IN OUR PLANTS: FROM MARTIAL STRATEGIES TO THE COST OF WAR
B.1. Defence starts before the establishment of defensive mechanisms
B.1.a. Various enemies imply various defences
B.1.b. How to realise you are under attack?
B.1.c. Signalling pathways to trigger defences
B.2. Various defence mechanisms to respond to various threats
B.2.a. Indirect defences: asking for help?
B.2.b. Direct defences: physical mechanisms to build a reinforced armour
B.2.c. Direct defences: chemical mechanisms to concoct poison pills
B.2.c.1. Defensive proteins
B.2.c.2. Specialised metabolites
B.3. The cost of defence: growing or fighting?
B.3.a. Defence is a resource-consuming process
B.3.b. The multiple theories of trade-off between growth and defence
C. FEED ME KILL ME: AGRONOMICAL INTEREST AND DEFENCE OF FICUS CARICA
C.1. The fig tree, a plant of agronomical interest
C.2. Ficus defence mechanisms
C.2.a. Tough mineralised leaves and specialised tissues
C.2.b. Ficus defensive proteins and metabolites
D. SMELLS LIKE TOXIC FURANOCOUMARINS
D.1. Furanocouma-what?
D.1.a. Furanocoumarins constitute one of the four classes of coumarins
D.1.b. Distribution of furanocoumarins in higher plants
D.1.c. Repartition and variation of furanocoumarins within plants
D.2. Defensive properties of furanocoumarins
D.2.a. Toxic properties and P450 inhibition
D.2.b. Phototoxic properties and phytophotodermatitis
D.2.c. From defensive properties to interesting bioactivities
D.3. Consequences of these defensive properties for the plants themselves
D.3.a. Emergence of linear and angular furanocoumarins
D.3.b. Defensive properties and spatio-temporal variation within plants
D.4. The furanocoumarins in the Moraceae family
D.4.a. Furanocoumarins described in various Ficus species
D.4.b. Repartition of furanocoumarins in Ficus carica
E. THE FURANOCOUMARIN PATHWAY MUST GO ON
E.1. The different steps of the furanocoumarin biosynthesis pathway
E.2. Evolutionary perspectives and concluding remarks
II. ENDLESS P450S MOST BEAUTIFUL
A. INTRODUCTION: ALL YOU NEED IS A P450
B. A BRIEF HISTORY OF P450S: RESEARCHES AND DISCOVERIES OVER TIME
C. ONE CLASSIFICATION TO NAME THEM ALL
D. IF YOU PLEASE DRAW ME A FUNCTIONAL P450: FROM STRUCTURE TO ACTIVITY
D.1. P450s: from the primary to the secondary and tertiary structures
D.1.a. Discovery of the P450 fold
D.1.b. The “P450 fold”: overall architecture
D.1.c. Structurally conserved regions
D.1.d. Substrate Recognition Sites
D.1.d.1. Discovery and description of the SRSs
D.1.d.2. SRSs, a hot spot for site-directed mutagenesis
D.2. Catalytic activity of P450s
D.2.a. P450 redox partner systems
D.2.b. Catalytic cycle of a typical class II P450
D.3. Atypical P450s and unusual P450-mediated reactions
E. HIGHWAY TO PHYTOCHEMISTRY: P450S’ IMPORTANCE IN THE PLANT KINGDOM
E.1. In plants, P450s constitute one of the largest gene superfamily
E.2. The diversification of P450s is at the heart of plant chemical diversity
F. ON THE ORIGIN OF P450 GENES: EVOLUTIVE STORY OF P450S IN LAND PLANTS
F.1. Early emergence of all plant P450 clans
F.2. Subsequent diversification of plant P450 families and subfamilies
F.3. The diversification of P450s families: different functions, different patterns
F.3.a. Plant-specific P450s and the conquest of land
F.3.b. P450s involved in plant specialised metabolism
F.3.b.1. Evolutionary patterns
F.3.b.2. Two examples of lineage-specific evolution
F.6. Summary of plant P450 evolution
III. OBJECTIVE AND APPROACH OF THE PHD
CHAPTER II WHOLE NEW GENES
A. INTRODUCTION AND STRATEGY: IN SEARCH OF THE LOST GENE
B. CANDIDATE GENES AND HOW TO FIND THEM
B.1. APPROACH: WHERE TO SEARCH, WHAT TO SEARCH?
B.2. IN SILICO SCREENING OF THE F. CARICA RNA-SEQ LIBRARY
C. I’LL MAKE AN ENZYME OUT OF YOU: CLONING AND EXPRESSION OF THE P450S
C.1. AMPLIFICATION, CLONING AND SEQUENCING OF THE P450 CANDIDATES
C.2. HETEROLOGOUS EXPRESSION OF THE P450 CANDIDATES
D. CONVERT THIS AND I’LL LOVE YOU: ENZYME ASSAYS AND CHARACTERISATION
D.1. ENZYME ASSAYS AND FUNCTIONAL SCREENING
D.1.a. CYP76F112, a marmesin synthase
D.1.b. CYP82J18, a P450 that hydroxylates auraptene
D.1.c. CYP81BN4, a P450 that hydroxylates cnidilin
D.2. FUNCTIONAL CHARACTERISATION OF CYP76F112, CYP82J18 AND CYP81BN4
D.2.a. CYP76F112: an enzyme with a strong specificity and affinity
D.2.a.1. Substrate specificity
D.2.a.2. Optimal enzymatic conditions
D.2.a.3. Kinetic parameters: affinity, catalytic constant and catalytic efficiency
D.2.b. The cases of CYP82J18 and CYP81BN4
E. DISCUSSION: ANOTHER P450 IN THE PATHWAY
E.1. AN APPROACH THAT PROVED ITSELF EFFECTIVE
E.2. PHYSIOLOGICAL OR PROMISCUOUS ACTIVITIES?
E.2.a. CYP76F112: a physiological marmesin synthase activity?
E.2.b. CYP81BN4 and CYP82J18: promiscuous activities?
E.3. NEW P450 FAMILIES INVOLVED IN THE FURANOCOUMARIN PATHWAY
E.3.a. The CYP76 family: from terpenoids to furanocoumarins?
E.3.a.1. General overview of the CYP76 family
E.3.a.2. Focus on the CYP76F subfamily and the marmesin synthase
E.3.b. The cases of CYP81BN4 and CYP82J18
E.4. CONCLUSION AND FUTURE PERSPECTIVES
CHAPTER III ONCE UPON A P450
A. BACK TO THE PAST: INTRODUCTION AND STRATEGY
A.1. OBJECTIVE AND STRATEGY
A.2. PREREQUISITE: HOW DO GENES EVOLVE AND DIVERSIFY?
A.2.a. Gene duplication
A.2.b. The fate of duplicated genes
A.2.c. All duplicates are not created equal
B. DATA MINING: THE P450S COMING OUT OF THE NITROGEN FIXING CLADE
B.1. APPROACH: FROM FICUS TO THE NITROGEN FIXING CLADE
B.2. CONSTITUTION OF THE DATASETS
C. INFERRING PHYLOGENIES: THE REALM OF THE ELDER GENES
C.1. EXPANSION AND DIVERSIFICATION OF CYP76FS
C.1.a. Gene-family phylogeny of the CYP76Fs in the Nitrogen Fixing Clade
C.1.b. Evolution of CYP76Fs’ SRSs
C.2. EVOLUTIONARY PATTERNS OF CYP81BNS ACROSS THE NITROGEN FIXING CLADE
C.3. CONSERVATION OF THE CYP82J SUBFAMILY ACROSS THE NITROGEN FIXING CLADE
D. DISCUSSION: THE STORY O’ MY P450S
D.1. EVOLUTIVE STORY OF CYP76F112, THE F. CARICA MARMESIN SYNTHASE
D.1.a. Evolution of the CYP76 family and emergence of CYP76F112
D.1.b. Clustering of CYP76Fs
D.1.c. Multiple origin of the furanocoumarin pathway in higher plants
D.1.d. The CYP76Fs: a marmesin synthase activity specific of the Ficus genus?
D.2. EVOLUTIVE STORY OF CYP81BN4
D.3. EVOLUTIVE STORY OF CYP82J18
D.4. CONCLUSION AND FUTURE PERSPECTIVES
CHAPTER IV IN THE ACTIVE SITE OF THE MARMESIN SYNTHASES
A. RISE OF THE MARMESIN SYNTHASES: INTRODUCTION AND STRATEGY
B. THE MOLECULAR SHAPE OF YOUR P450: 3D MODELLING AND DOCKING
B.1. HOMOLOGY MODELLING OF CYP76F111 AND CYP76F
B.1.a. Approach: the homology modelling technique
B.1.b. Building the 3D models of CYP76F111 and CYP76F112
B.2. DOCKING EXPERIMENTS WITHIN CYP76F111 AND CYP76F112
B.2.a. Approach: the docking technique
B.2.b. Docking of the heme within CYP76F111 and CYP76F112
B.2.c. Docking of the DMS within CYP76F111 and CYP76F112
B.2.c.1. Approach: definition of the grid receptor and the flexible residues
B.2.c.2. Docking of the DMS within CYP76F112
B.2.c.3. Docking of the DMS within CYP76F111
C. FINDING KEY AMINO ACIDS INFLUENCING THE DOCKING OF THE DMS
C.1. COMPARISON OF THE DOCKING SITES OF CYP76F111 AND CYP76F112
C.2. INFLUENCE OF THE VARIABLE AMINO ACIDS DURING THE DOCKING OF THE DMS
C.2.a. Preliminary results: identification of 4 key amino acids
C.2.b. Simultaneous modification of the 4 amino acids A, B, C and D
C.2.c. Individual modifications of A, B, C and D
C.2.d. Simultaneous modifications of A and B
D. SITE-DIRECTED MUTAGENESIS: 4 AMINO ACIDS, AND NOTHING ELSE MATTERS?
D.1. THE CHOICE OF THE MUTANTS
D.2. SYNTHESIS AND EXPRESSION OF THE MUTANTS
D.3. FUNCTIONAL CHARACTERISATION: INFLUENCE OF THE 4 AMINO ACIDS
E. GOTTA DOCK THEM ALL: ADDITIONAL DOCKINGS WITH THE F112-LIKE
F. DISCUSSION: A SINGLE AMINO ACID IS MISSING, AND ALL BEGINS ANEW
F.1. CYP76F111 AND CYP76F112: RELIABLE MODELLING AND ACCURATE DOCKING
F.1.a. Overall architecture of CYP76F111 and CYP76F112
F.1.b. Confronting the in-silico dockings with the in-vitro experiments
F.2. THE IMPACT OF THE RESIDUES A, B, C AND D ON THE MARMESIN SYNTHASE ACTIVITY
F.2.a. Key residues from the SRSs
F.2.b. The residues A and B might stabilise the DMS in the active site
F.2.c. The residue D might stabilise the DMS in the active site
F.2.d. The residue C contribute to shape the substrate-docking site
F.2.e. Summary of the influence of the residues A, B, C and D
F.2.f. The residue C: a hotspot position?
F.2.g. The limits of the models and the importance of the access channel
F.3. RECENT AMINO ACIDS FOR A RECENT MARMESIN SYNTHASE ACTIVITY
CHAPTER V THE COST OF FURANOCOUMARINS
A. INTRODUCTION: DO TOMATOES DREAM OF TOXIC FURANOCOUMARINS?
B. BRICK BY BRICK: THE GOLDENBRAID MULTI-GENIC CONSTRUCTIONS
B.1. APPROACH: WHICH PLASMID TO CONSTRUCT, AND HOW?
B.1.a. Overall strategy
B.1.b. Presentation of the GoldenBraid cloning system
B.1.c Using the GoldenBraid technology, or how to construct the desired plasmid
B.2. CONSTRUCTION OF THE PLASMID
C. THE TOMATOES OF EVIL: TOMATO TRANSFORMATION AND REGENERATION
C.1. APPROACH AND CHOICE OF THE NEGATIVE CONTROL
C.2. GENERATION OF THE TRANSGENIC TOMATOES
C.2.a. Tomato transformation and regeneration
C.2.b. Confirmation of the transformations
D. DISCUSSION: GET A BETTER PLASMID, DON’T GIVE UP THE TRANSFORMATIONS
D.1. SUMMARY OF THE PRELIMINARY RESULTS
D.2. LIMITS, HYPOTHESES AND RECOMMENDATIONS FOR A FUTURE CONTINUATION
D.2.a. A matter of size?
D.2.b. Reordering the transcriptional units
D.2.c. Avoiding the repetitive use of identical promoters and terminators
D.2.d. Using inducible instead of 35S promoters
D.2.e. Additional TUs to prevent autotoxicity?
CHAPTER VI GENERAL CONCLUSION AND PERSPECTIVES
A. INTO THE UNKNOWN STEPS OF THE FURANOCOUMARIN PATHWAY
A.1. CYP76F112: A RECENT MARMESIN SYNTHASE THAT OPENS MANY PROSPECTS
A.1.a. The marmesin synthase activity
A.1.b. The furanocoumarin pathway, a case of convergent evolution
A.2. CYP81BN4 AND CYP82J18: PROMISCUOUS AND NON-SPECIES-SPECIFIC ENZYMES?
A.3. NEW PROSPECTS TO PURSUE THE ELUCIDATION OF THE FURANOCOUMARIN PATHWAY
A.3.a. A complete genome for Ficus carica
A.3.b. Finding the ancestral substrate of the Ficus CYP76Fs
A.3.c. Other Ficus CYP76Fs potentially involved in the furanocoumarin pathway
A.3.d. Other P450 families potentially involved in the furanocoumarin pathway
A.3.e. The marmesin synthases in other plant families
A.3.f. Other enzymes families: Ficus methyltransferases and dioxygenases
A.3.g. Application and study of plant biosynthesis pathways
B. TOO MUCH FURANOCOUMARINS WILL COST YOU
CHAPTER VII MATERIALS AND METHODS
A. MATERIALS
A.1. PLANT MATERIAL
A.1.a. Ficus carica
A.1.b. Solanum lycopersicum
A.2. BACTERIAL STRAIN
A.2.a. Escherichia coli MC1022
A.2.b. Escherichia coli ccdB Survival™
A.2.c. Agrobacterium tumefaciens EHA105
A.3. YEAST STRAIN: SACCHAROMYCES CEREVISIÆ WAT21
A.4. VECTORS
A.4.a. pCR™8/GW/TOPO™
A.4.b. pYeDP60 and pYeDP60_GW®
A.4.c. GoldenBraid commercial vectors
A.4.c.1. The pUPD vectors: pUPD, pUPD-35S and pUPD-tNOS
A.4.c.2. The α-level vectors: pDGB1_α1 and pDGB1_α2
A.4.c.3. The Ω-level vectors: pDGB1_Ω1 and pDGB1_Ω2
A.4.d. Recombinant GoldenBraid vectors
A.4.d.1. pDGB1_Ω1 [PsDiox+PsPT1]
A.4.d.2. pUPD-CYP71AJ3
A.4.e. pSoup
A.4.f. plCSL11024 vector
A.5. CULTURE MEDIA
A.5.a. Bacteria culture medium: LB medium and associated antibiotics
A.5.b. Yeast culture media
A.5.c. Tomato in vitro culture media
A.6. BIOINFORMATIC TOOLS
A.6.a. Databases
A.6.a.1. Ficus carica RNA-seq library
A.6.a.2. Public online databases
A.6.b. Software
A.6.b.1. Software used for molecular biology and basic sequence analyses
A.6.b.2. Software used for phylogenetic analyses
A.6.b.3. Software used for modelling and docking
A.6.c. Online tools
B. METHODS
B.1. COMMON MOLECULAR BIOLOGY AND MICROBIOLOGY METHODS
B.1.a. Plant tissue grinding
B.1.b. Extraction and purification of plant RNA
B.1.c. Synthesis of complementary DNA
B.1.d. Extraction of plant genomic DNA
B.1.e. Amplification of DNA fragments by PCR
B.1.e.1. PrimeSTAR® Max DNA Polymerase
B.1.e.2. SapphireAmp® Fast PCR Master Mix 2X
B.1.f. DNA extraction from agarose gel
B.1.g. DNA digestion using restriction enzymes
B.1.h. Cloning techniques
B.1.h.1. Cloning of a PCR-amplified fragment in pCR™8/GW/TOPO™
B.1.h.2. Recombination into the pYeDP60_GW® vector
B.1.i. Preparation of electrocompetent bacteria
B.1.i.1. Preparation of electrocompetent Escherichia coli
B.1.i.2. Preparation of electrocompetent Agrobacterium tumefaciens EHA105
B.1.j. Transformation of competent bacteria
B.1.k. Isolation of plasmid DNA from bacteria
B.1.l. Spectrophotometry quantification
B.1.m. Sequencing
B.1.n. Synthesis of the CYP76F mutants
B.2. METHODS LINKED TO P450 HETEROLOGOUS EXPRESSION, ASSAY AND CHARACTERISATION
B.2.a. Yeast transformation
B.2.a.1. Preparation of competent S. cerevisiae WAT21
B.2.a.2. Transformation of S. cerevisiae WAT21
B.2.b. Isolation of plasmid DNA from yeast
B.2.c. Heterologous expression of P450s in S. cerevisiae
B.2.c.1. Yeast culture and P450 expression
B.2.c.2. Preparation of yeast microsomes
B.2.d. Western-Blotting: confirmation of the presence of the P450s of interest
B.2.d.1. Polyacrylamide gel electrophoresis in denaturing conditions
B.2.d.2. Transfer of the proteins to a polyvinylidene difluoride membrane
B.2.d.3. Immunodetection
B.2.e. Quantification of functional P450s with the differential CO spectrum method
B.2.f. Enzymatic assay and functional characterisation
B.2.f.1. Functional screening
B.2.f.2. Determination of the optimal conditions: temperature and pH
B.2.f.3. Determination of the kinetic parameters
B.3. METABOLIC ANALYSES
B.3.a. Extraction of phenolic compounds from plant grinded sample
B.3.b. UHPLC-MS analyses
B.3.c. Orbitrap-IDX analyses
B.4. THE GOLDENBRAID CLONING TECHNIQUE
B.4.a. Domestication of the genes of interest
B.4.a.1. Domestication of CYP76F112
B.4.a.2. Domestication of the KanaR gene
B.4.b. Cloning into the pUPD vector
B.4.c. Assembly of simple transcriptional units
B.4.d. Repeated assembly of multiple transcriptional units
B.4.d.1. Assembly of two transcriptional units: CYP76F112 and CYP71AJ3
B.4.d.2. Assembly of four transcriptional units: PsDiox, PsPT1, CYP76F112 and CYP71AJ3
B.4.d.3. Assembly of 5 transcriptional units to construct the final 5-TUs plasmid
B.5. TOMATO STABLE TRANSFORMATION AND REGENERATION
B.5.a. Preparation of the tomatoes to be transformed
B.5.a.1. Sterilisation of the tomato seeds
B.5.a.2. Germination of the sterile tomato seeds
B.5.a.3. Preparation of the cotyledons
B.5.b. Preparation of the Agrobacterium suspension to transform the tomatoes
B.5.b.1. Co-transformation of the Agrobacterium
B.5.b.2. Preparation of the Agrobacterium suspension
B.5.c. Transfection of the cotyledon fragments
B.5.d. Regeneration and selection of the transgenic cotyledons
B.5.e. Rooting of the transgenic plantlets
B.5.f. Transfer into the soil and growth of fully developed tomato plants
B.6. BIOINFORMATIC ANALYSES
B.6.a. Identification of P450 candidates from the F. carica RNA-seq library
B.6.b. Phylogenetic analyses
B.6.b.1. Constitution of the CYP76F, CYP81BN and CYP82J initial datasets
B.6.b.2. Sequence alignment
B.6.b.3. Phylogenetic analyses
B.6.c. Modelling and docking analyses
B.6.c.1. Homology modelling of the CYP76Fs
B.6.c.2. Docking experiments
RÉSUMÉ DÉTAILLÉ EN FRANÇAIS
A. ETAT DE L’ART ET OBJECTIF DE LA THESE
A.1. Défense des plantes et furocoumarines
A.2. Les cytochromes P450s
A.3. Objectifs de la thèse
B. IDENTIFICATION DE GENES IMPLIQUES DANS LA VOIE DE BIOSYNTHESE DES FUROCOUMARINES
B.1. APPROCHE, CHOIX DE LA PLANTE MODÈLE ET DES FAMILLES ENZYMATIQUES D’INTÉRÊT
B.2. IDENTIFICATION, CLONAGE ET EXPRESSION HÉTÉROLOGUE DES P450 CANDIDATS
B.3. CRIBLAGE FONCTIONNEL ET CARACTÉRISATION ENZYMATIQUE
B.4. DISCUSSION
C. ANALYSE PHYLOGENETIQUE DE CYP76F112, CYP82J18 ET CYP81BN4
C.1. APPROCHE, CONSTITUTION DES JEUX DE DONNÉES ET CONSTRUCTION DES ARBRES
C.2. CYP76F112 : ANALYSE PHYLOGÉNÉTIQUE ET DISCUSSION
C.3. LES CAS DE CYP81BN4 ET CYP82J18
D. EMERGENCE DE L’ACTIVITE MARMESINE SYNTHASE
D.1. APPROCHE
D.2. MODÉLISATION ET EXPÉRIENCE DE DOCKING MOLÉCULAIRE
D.3. MUTAGENÈSE DIRIGÉE, INFLUENCE DES ACIDES AMINÉS ET DISCUSSION
D.4. PERSPECTIVES ÉVOLUTIVES ET APPARITION DE L’ACTIVITÉ MARMÉSINE SYNTHASE
E. RECONSTITUTION DE LA VOIE DES FUROCOUMARINES DANS LA TOMATE
E.1. APPROCHE GLOBALE
E.2. CONSTRUCTION D’UN PLASMIDE MULTIGÉNIQUE
E.3. GÉNÉRATION DE TOMATES TRANSGÉNIQUES
E.4.DISCUSSION
F. CONCLUSION GENERALE ET PERSPECTIVES
REFERENCES
