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Kessler, Félix
Nom
Kessler, Félix
Affiliation principale
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Professeur.e ordinaire
Email
felix.kessler@unine.ch
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Voici les éléments 1 - 10 sur 115
- PublicationAccès libreCharacterization of domesticated clove ("Syzygium aromaticum") and comparison with related species(2024)
; ; Ivanov NicolaiLe giroflier, Syzygium aromaticum, est un arbre à épices appartenant à la famille des Myrtacées. Les boutons floraux encore fermés et séchés (clous de girofle), l'huile essentielle et l'oléorésine produits à partir du giroflier sont riche en eugénol et utilisés commercialement dans un large éventail d'industries. Malgré son importance économique et le potentiel thérapeutique prometteur de l'eugénol, peu de données omiques étaient disponibles pour la recherche fondamentale et appliquée sur le giroflier. Un génome de référence est un outil important pour l'avancement de la recherche en biologie végétale et des programmes d'amélioration des cultures. Dans le cadre de cette thèse, le génome du giroflier a été séquencé, et assemblé à l'échelle chromosomique, puis analysé en utilisant des approches de génomique comparative et multi-omiques dans le but d’étudier l'évolution du génome du giroflier et la base génétique de la biosynthèse de l'eugénol. Les assemblages des génomes de Syzygium malaccense (430 Mb), Syzygium aqueum (392 Mb), Syzygium jambos (426 Mb), et Syzygium syzygioides (431 Mb) ont également été construits pour être comparés aux génomes du giroflier (370 Mb), de Syzygium grande (405 Mb) ainsi qu’au génome d’une autre espèce de Myrtacées, Eucalyptus grandis (690 Mb), afin d'acquérir de nouvelles connaissances sur l'évolution des génomes des espèces de Syzygium. En analysant l'assemblage du génome du giroflier et en intégrant les données transcriptomiques et métabolomiques produites à partir des feuilles et des bourgeons floraux, les principales familles de gènes impliquées dans la biosynthèse des précurseurs de la lignine et des phénylpropènes volatils (eugénol, isoeugénol, chavicol) ont été identifiées ainsi que les gènes potentiellement impliqués dans la biosynthèse de l'eugénol. Sur la base de cette étude multiomiques, nous avons émis l'hypothèse que l'acétate d'eugénol pourrait jouer un rôle clé dans la forte accumulation d'eugénol dans les feuilles et les bourgeons floraux du giroflier. En outre, un nombre inférieur de gènes codant pour une possible eugénol synthase — une enzyme clé dans la biosynthèse de l'eugénol — ont été identifiés dans les génomes des 4 espèces de Syzygium nouvellement assemblés (1 à 3) que dans le génome du giroflier (15), suggérant que cette variation du nombre de copie peut être impliquée dans la teneur élevée en eugénol des organes de ce dernier. Les analyses de synténie ont révélé une bonne conservation de l'organisation chromosomique des 6 espèces de Syzygium étudiées et des réarrangements intrachromosomiques sur 7 des 11 chromosomes entre les espèces de Syzygium et E. grandis. Ces nouveaux génomes de Syzygium sont une contribution précieuse aux ressources génomiques de la famille des Myrtacées, et en particulier pour le genre Syzygium, le plus riche en espèces d’arbre au monde. De plus, la disponibilité d’un génome de référence pour le giroflier ouvre de nouvelles perpectives pour la recherche appliquée visant à optimiser la productivité des girofliers. ABSTRACT Clove, Syzygium aromaticum, is a valuable spice crop tree belonging to the Myrtaceae family. Its dried, unopened flower buds, essential oil, and oleoresin-rich in eugenol, are used commercially in a broad range of industries. Despite its economic importance and the promising therapeutic potential of eugenol, little -omics information was available for fundamental and applied research on clove. A reference genome is an important tool for the advancement of plant biology research and crop improvement programs. In this thesis, the clove genome was sequenced, assembled at the chromosome level and subsequently analysed using comparative genomics and multi-omics approaches to investigate clove genome evolution and the genetic basis of eugenol biosynthesis. In addition, the genome assemblies of Syzygium malaccense (430 Mb), Syzygium aqueum (392 Mb), Syzygium jambos (426 Mb), and Syzygium syzygioides (431 Mb) were constructed and compared to the genome assemblies of clove (370 Mb), Syzygium grande (405 Mb) and their Myrtaceae relative Eucalyptus grandis (690 Mb) to provide further genomic insights into the evolution of Syzygium genomes. By analysing the clove genome assembly and sets of transcriptomic and metabolomic data from leaves and buds, the key gene families involved in the biosynthesis of the precursor of lignin and phenylpropene volatiles (eugenol, isoeugenol, chavicol) were identified as well as genes possibly involved in the biosynthesis of eugenol. On the basis of this multi -omics study, we hypothesized that eugenol acetate may play a key role in the high accumulation of eugenol in clove leaves and buds. Fewer genes were found in the newly assembled Syzygium genomes (1 to 3) than in clove (15) to encode for putative eugenol synthase—a key enzyme in the eugenol biosynthetic pathway—suggesting that this copy number variation may be involved in the high content in eugenol of clove. Synteny analyses revealed a high conservation of the chromosomal organization of the 6 Syzygium species studied and intrachromosomal rearrangements on 7 of the 11 chromosomes, between the Syzygium species and E. grandis. This new set of Syzygium genomes is a valuable contribution to genomics resources for the Myrtaceae family, and especially to the Syzygium genus, the world’s most species-rich tree genus. Moreover, the availability of the clove reference genome opens new perspectives for applied research aiming to optimize clove production. - PublicationAccès libreThe atypical kinase ABC1K1 / PGR6 allocates geranylgeranyldiphosphate to the carotenoid biosynthesis pathway(2023)
; Les espèces fruitières cultivées jouent un rôle important dans la vie sur Terre. En effet, les fruits fournissent des nutriments et des vitamines essentiels qui ne sont pas synthétisés par l'homme mais qui sont nécessaires à sa santé. La synthèse de ces nutriments essentiels a lieu au cours du processus complexe de maturation et de mûrissement des fruits, orchestré par des gènes régulateurs, des facteurs de transcription et des hormones. Au cours de ce processus, de nombreux événements biologiques et physiologiques entraînent des changements dans les fruits et sont indispensables pour la couleur, la saveur, l'arôme et la qualité des fruits. Dans le fruit de la tomate, nombre de ces événements biologiques se produisent dans un plaste coloré spécialisé dans la biosynthèse et la séquestration des caroténoïdes, le chromoplaste. La couleur rouge caractéristique des fruits de la tomate est due à la synthèse et à l'accumulation de lycopène dans un compartiment lipoprotéique sous-organellaire des chloroplastes et des chromoplastes appelé plastoglobule (PG). Le noyau hydrophobe des plastoglobules des chloroplastes et des chromoplastes sert de réservoir et de compartiment de biosynthèse des esters de phytyle, des lipides prényliques et des caroténoïdes. Dans le premier chapitre intitulé "Plastoglobules : A hub of lipid metabolism in the chloroplast" (Shanmugabalaji et al., 2022), nous avons passé en revue les fonctions et le métabolisme des plastoglobules dans différents types de plastes. Dans le deuxième chapitre "Chapter II : Les plastoglobules du chromoplaste recrutent la voie de biosynthèse des caroténoïdes et contribuent à l'accumulation des caroténoïdes pendant la maturation du fruit de la tomate " (Zita et al., 2022), nous avons utilisé une approche protéomique pour étudier le remodelage du protéome des plastoglobules pendant la transition chloroplaste-chromoplaste dans le fruit de la tomate. Cette étude protéomique a révélé que le plastoglobule de la tomate contient environ 30 protéines et des membres de différentes familles d'enzymes dont les fibrillines (FBNs), l'activité du complexe BC1 kinase (ABC1Ks), la tocophérol cyclase (VTE1), la NAD(P)H-ubiquinone oxydoréductase C1 (NDC1). L'étude a révélé la présence de la voie complète de biosynthèse des caroténoïdes dans les plastoglobules du chromoplaste : phytoène synthase 1 (PSY1), phytoène désaturase (PDS), zeta carotène désaturase (ZDS), caroténoïde isomérase (CRTISO), et lycopène beta cyclase (LYC-B). Les résultats montrent que le plastoglobule devient une plateforme pour la biosynthèse des caroténoïdes pendant la transition chloroplaste-chromoplaste. En outre, les lipidomes du chloroplaste et du plastoglobule du chromoplaste subissent un remodelage au cours des étapes de maturation progressive du fruit. En particulier, le β-carotène et le lycopène sont fortement enrichis dans les plastoglobules du chromoplaste par rapport aux plastoglobules du chloroplaste. Dans l'ensemble, ce chapitre démontre que le plastoglobule joue un rôle central dans le processus de maturation. Dans le troisième chapitre "A quantitative method to measure geranylgeranyl diphosphate (GGPP) and geranylgeranyl monophosphate (GGP) in tomato (Solanum lycopersicum) fruit" (Zita et al., 2023), une méthode de quantification du GGPP à partir de tissus de fruits de tomates a été mise au point en utilisant la chromatographie liquide à ultra-haute performance couplée à la spectrométrie de masse en tandem (UHPLC-MS/MS). La méthode a été validée en utilisant un type sauvage (WT) et des matrices mutantes défectueuses dans la synthèse du GGPP. En outre, ce chapitre souligne l'importance de la préparation des échantillons pour préserver le GGPP et limiter sa conversion en GGP (produit hydrolysé). Cette méthode est essentielle pour la suite de l'analyse dans ma thèse. Enfin, dans le quatrième chapitre de ma thèse "An atypical kinase ABC1K1 allocates geranylgeranyl diphosphate to the carotenoid biosynthesis pathway in plastoglobules of chromoplasts", j'étudie le rôle de SlABC1K1 dans le métabolisme des caroténoïdes du fruit de la tomate. Le mutant CRISPR-Cas9 de SlABC1K1 (slabc1k1) présente le phénotype photosynthétique PGR6 dans les feuilles qui avait été observé précédemment chez Arabidopsis. De manière surprenante, les fruits de slabc1k1 présentaient un phénotype orange. Le phénotype slabc1k1 est lié à une accumulation réduite de lycopène et de son précurseur, le phytoène. Sur la base de nos résultats, je propose deux scénarios possibles pour la fonction de SlABC1K1 dans les plastoglobules des fruits de tomate. SlABC1K1 peut agir comme un régulateur clé pour l'allocation du géranylgéranyl diphosphate (GGPP) à la voie des caroténoïdes ou comme un co-régulateur des enzymes de biosynthèse des caroténoïdes dans les plastoglobules. La découverte que SlABC1K1 est nécessaire pour l'allocation du GGPP au plastoglobule est le principal résultat de ma thèse. ABSTRACT Crop fruit species play an important role in life on Earth. Indeed, fruits provide essential nutrients and vitamins which are not synthesized by humans but are necessary for their health. The synthesis of these essential nutrients occurs during the complex process of fruit maturation and ripening, orchestrated by regulatory genes, transcription factors, and hormones. During this process, many biological, and physiological events lead to fruit changes and are indispensable for the fruit color, flavor, aroma and quality. In tomato fruit, many of these biological events occur in a colored plastid specialized in carotenoid biosynthesis and sequestration, the chromoplast. The hallmark red color of tomato fruit is due to the synthesis and accumulation of lycopene in a lipoprotein sub-organellar compartment of chloroplasts and chromoplasts called plastoglobule (PG). The hydrophobic core of chloroplast and chromoplast plastoglobules serves as a reservoir and biosynthetic compartment of phytyl esters, prenyl lipids and carotenoids. In the first chapter entitled “Plastoglobules: A hub of lipid metabolism in the chloroplast” (Shanmugabalaji et al., 2022) we reviewed plastoglobule functions and metabolism in different plastid types. In the second chapter “Chapter II: Chromoplast plastoglobules recruit the carotenoid biosynthetic pathway and contribute to carotenoid accumulation during tomato fruit maturation” (Zita et al., 2022), we used a proteomic approach to investigate plastoglobule proteome remodeling during the chloroplast to chromoplast transition in tomato fruit. This proteomic study revealed that the tomato plastoglobule contains around 30 proteins and members of different enzyme families including fibrillins (FBNs), the activity of BC1 complex kinase (ABC1Ks), tocopherol cyclase (VTE1), AD(P)Hubiquinone oxidoreductase C1 (NDC1). The study revealed the presence of the complete carotenoid biosynthesis pathway in chromoplast plastoglobules: phytoene synthase 1 (PSY1), phytoene desaturase (PDS), zeta carotene desaturase (ZDS), carotenoid isomerase (CRTISO), and lycopene beta cyclase (LYC-B). The results show that the plastoglobule becomes a platform for carotenoid biosynthesis during the chloroplast to chromoplast transition. In addition, the lipidomes of chloroplast and chromoplast plastoglobule undergo remodeling during progressive fruit ripening stages. Specifically, β-carotene and lycopene were highly enriched in chromoplast plastoglobules compared to chloroplast plastoglobules. Overall, the chapter demonstrates that the plastoglobule plays a central role in the ripening process. The third chapter “A quantitative method to measure geranylgeranyl diphosphate (GGPP) and geranylgeranyl monophosphate (GGP) in tomato (Solanum lycopersicum) fruit” (Zita et al., 2023), a method to quantify GGPP from tomato fruit tissue was developed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method has been validated by using a wild-type (WT) and mutant matrices defective in GGPP synthesis. In addition, this chapter highlight the importance of sample preparation to preserve the GGPP and limited its conversion to GGP (hydrolyzed product). This method is essential for further analysis in my thesis. Finally, in the fourth chapter of my thesis “An atypical kinase ABC1K1 allocates geranylgeranyl diphosphate to the carotenoid biosynthesis pathway in plastoglobules of chromoplasts”, I investigate the role of SlABC1K1 in tomato fruit carotenoid metabolism. The CRISPR-Cas9 mutant of SlABC1K1 (slabc1k1) showed the photosynthetic PGR6 phenotype in leaves that had previously been observed in Arabidopsis. Surprisingly, slabc1k1 fruit had an orange phenotype. The slabc1k1 phenotype was linked to reduced lycopene accumulation as well as that of its phytoene precursor. Based on our results, I propose two possible scenarios for the function of SlABC1K1 in tomato fruit plastoglobules. SlABC1K1 may acts as a key regulator for geranylgeranyl diphosphate (GGPP) allocation to the carotenoid pathway or as a co-regulator of carotenoid biosynthesis enzymes in plastoglobules. The discovery that SlABC1K1 is required for the GGPP allocation to plastoglobule is the principal result of my thesis. - PublicationAccès libreStudy of the physiological and molecular functions of ABC1K1 protein during early development of "Arabidopsis thaliana"Photosynthesis is a key bioenergetic mechanism allowing photosynthetic organisms such as plants or algae to convert sunlight energy into chemical energy to produce sugar, while releasing molecular oxygen. This process takes place in a specific cellular organelle called chloroplast. Inside the chloroplast, the components of the photosynthetic machinery required to perform the photochemical part of photosynthesis, are inserted in a highly dynamic structure, the thylakoid membrane. Plastoglobules, small lipoprotein particles (lipid droplets) associated with the thylakoid membrane, are essential for chloroplast lipid metabolism, thylakoid formation and photoprotection. These structures contain a large diversity of neutral lipids some of which have strong antioxidant properties, such as tocopherols, carotenoids or plastoquinone. Most of the plastoglobule proteins are involved in lipids metabolisms. Among them, the ABC1K proteins are involved in the regulation of neutral lipid metabolism and contribute to the maintenance of photosynthetic activity by plastoquinone homestasis. My PhD consists in the study of the physiological and molecular functions of ABC1K1 protein in the early development of Arabidopsis thaliana. In particular, we studied its role in early chloroplast biogenesis under stress-enhancing red and high light conditions. We have discovered a new signaling mechanism in which ABC1K1 promotes the degradation of EX1, a singlet oxygen trigger (1O2), though the FTSH2 protease, particularly active under red light conditions. The accumulation of EX1 in abc1k1 and ftsh2 mutant led to the arrest of chloroplast biogenesis and a greening defect. Mutation of EX1 by CRISPR/Cas9 in the abc1k1 background partially alleviated the greening defect observed in the abc1k1 mutant (Chapter 2.1). During my PhD, we also observed that abc1k1 displayed a variegated phenotype under high light conditions, similarly to the ftsh2 mutant. This result was integrated in the publication called “Plastoquinone homoeostasis by Arabidopsis proton gradient regulation 6 is essential for photosynthetic efficiency” published in the scientific journal “Communication Biology” in 2019 (Chapter 2.2). Finally, we have shown that the dark re-oxidation of the photoactive plastoquinone pool is impaired in the abc1k1 mutant suggesting a defect in the plastoquinone mobility. This defect is restored in the abc1k1/abc1k3 double mutant. This result was integrated in the publication “Mutation of the Atypical Kinase ABC1K3 Partially Rescues the PROTON GRADIENT REGULATION 6 Phenotype in Arabidopsis thaliana” published in the scientific journal “Frontiers in Plant Science” in 2020 (Chapter 2.3). This thesis provides new insight into the role of ABC1K1 and ABC1K3 proteins in theregulation of early chloroplast biogenesis and photosynthesis under stressful light conditions.
- PublicationAccès libreEngineering Light Harvesting Complex IITo sustain the life on Earth solar energy has to be converted into chemical energy, this is possible thanks to the process called photosynthesis. Through a set of interconnected pigment-binding proteins, collectively called “light harvesting complex” (LHC), photon energy is collected and funneled towards the reaction centers (RCs) of the two photosystems. The RCs feed a series of redox reactions that ultimately allow the production of reducing power (NADPH) and ATP, used for the synthesis of organic molecules. Despite having a highly conserved core structure, the light harvesting complex II (LHCII) is capable to acclimate to a wide range of environmental conditions. LHCII can functionally associate to both photosystems thus allowing a fine tuning of the electron transport chain, or act as dissipators of excess light energy protecting the photosystems from photodamage. This dynamic regulation is crucial for the adaptation of plants to different light conditions. LHCII is mostly composed of homo and hetero trimers of three isoforms: LHCB1, LHCB2 and LHCB3. The first two, through the phosphorylation of a threonine present in the N-terminal domain, are crucial for the regulation of the dynamics of the LHCII network. LHCB2 phosphorylation plays a central role in LHCII association to photosystem I and is thus regarded as the regulatory isoform of LHCII. The role of LHCB1 phosphorylation is less obvious; however, it has an impact in vivo allowing a partial adaptive response also in absence of LHCB2. Thanks to the CRISPR/Cas9 technique, we produced multiple mutants for the clustered genes coding for LHCB1 and LHCB2, thus allowing the production of complete null mutants for these two LHCII isoforms. These mutant lines constitute an ideal platform to study the impact of targeted modifications on the LHCII network via the production of complemented lines. LHCB1 is the most abundant isoform of LHCII and, consequently, a multiple mutation of the five genes encoding this protein results in a pale phenotype, reduced PSII antenna cross-section, altered thylakoid structure along with lower Photosystem I over Photosystem II reaction center ratio. Interestingly, the loss of one of these two major isoforms results in compensatory effects at the phosphorylation level of the remaining. Loss of LHCB1 results in a de-phosphorylation of the remaining LHCB2, while loss of LHCB2 results in an over-phosphorylation of LHCB1. The complete knock out plants for both LHCB1 and LHCB2 were tested under prolonged fluctuating light, moderate temperature stress and their combination. This revealed an increased susceptibility to only the combined stress for the complete LHCB1 knock out, visible as a clear growth delay, combined with a decrease in the photosynthetic efficiency. Surprisingly, the loss of LHCB2, which impairs the antenna re-allocation between the two photosystems, did not result in any major defect under the combined stress condition. Modification of the threonine of the phosphorylation site to alanine (non phosphorylable) or aspartate (constitutive negative charge "phospho-mimic») for both LHCB1 and LHCB2 reveals the impact of such irreversible modification on photosynthetic acclimation and on the dynamics of the photosynthetic complexes. We demonstrated that the complete removal of LHCB2 protein or the substitution of its phosphorylation site by alanine or aspartate largely, result in a physiologically overlapping phenotype with sharp reduction of state transitions and decreased LHCII-PSI-LHCI supercomplex formation. These defects result in slower acclimation to fluctuating light. Our results show that only with the phospho-threonine group LHCB2 can fully accomplish state transitions and that the negative charge of the aspartate substitution has no impact on short-term photosynthetic acclimation. Disentangling the defined role of each antenna isoform, LHCB1 and LHCB2, could shed light in short and long term acclimatory processes. Leading to a better comprehension on how each isoform contributes to LHCII network organization and results in an optimal balance between light capture and photoprotection. The multiple null lines produced during this project are a milestone along this path and open future perspectives towards the design of innovative LHCII complementation studies.
- PublicationMétadonnées seulementCharacterization of a Plastoglobule-Localized SOUL4 Heme-Binding Protein in Arabidopsis thaliana(2020-1-31)Heme plays an active role in primary plant metabolic pathways as well as in stress signaling. In this study, we characterized the predicted heme-binding protein SOUL4. Proteomics evidence suggests that SOUL4 is a component of Arabidopsis plastoglobules (PGs, chloroplast lipid droplets). SOUL4 contains heme-binding motifs and the recombinant protein is shown here to bind heme in vitro. Fluorescence-tagged SOUL4 colocalized with the specific PG marker Fibrillin1A (FBN1A) in transiently transformed Nicotiana benthamiana leaves. In addition, SOUL4 cofractionated with another PG marker Fibrillin2 (FBN2) in sucrose gradient ultracentrifugation experiments. In vitro kinase experiments revealed that SOUL4 is phosphorylated by a yet unknown chloroplast protein kinase. Our data demonstrate that SOUL4 is a bona fide PG protein and may function in heme-buffering in the chloroplast.
- PublicationAccès libreHow chloroplasts protect themselves from unfolded proteins(2019-10-15)
; A genetic screen has identified the first signaling component of the unfolded protein response in chloroplasts. - PublicationAccès librePlastoquinone homoeostasis by Arabidopsis proton gradient regulation 6 is essential for photosynthetic efficiency(2019-6-20)
; ; ; ; ; - PublicationAccès libreThe novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery(2017)
Toc159 joue un rôle essentiel dans la biogenèse du chloroplaste. Les bases de données de phosphoprotéomique montrent que le domaine A de Toc159 est fortement phosphorylé. La protéine cytosolique caséine kinase II phosphoryle le domaine A in vitro. Toutefois d’autres kinases ayant la même fonction ont aussi été prédites. Tandis que la phosphorylation contrôle l’assemblage et l’activité des complexes d’importation de protéines dans les chloroplastes et les mitochondries, aucune kinase organite-spécifique n’a été identifiée jusqu’à présent. Par co-purification avec Toc159, nous avons découvert une protéine kinase dans la membrane externe du chloroplaste (KOC1 « Kinase at the Outer Chloroplast membrane 1 »). KOC1 est une protéine intégrale de membrane orientée vers le cytosol et associée de manière stable avec le complexe TOC. KOC1 phosphoryle le domaine A chez les membres de la famille Toc159 in vitro. Dans les chloroplastes des mutants koc1, l’efficience de l’importation des protéines a été réduite. Par ailleurs, les plantules koc1 ont un taux de survie réduit quand elles sont déplacées de l’obscurité à la lumière, quand une importation rapide des pré-protéines est nécessaire pour une biogenèse de chloroplastes complète. Nos résultats indiquent que KOC1 est un composant de la machinerie d’importation TOC en phosphorylant les récepteurs, en soutenant l’importation de pré-protéines et en contribuant à une biogenèse de chloroplastes efficiente., The chloroplast constitutes the site of photosynthesis and is an essential organelle in plant cells. An endosymbiotic event was at the origin of the chloroplast, an ancestral eukaryotic cell engulfing a photosynthetic cyanobacterium. During evolution, the majority of ancestral chloroplast genes were lost or transferred to the nucleus. The protein products of the successfully transferred genes are now synthesized by cytosolic ribosomes and imported into the chloroplast. The chloroplast destined proteins (preproteins) acquired an additional sequence that encodes a cleavable N-terminal targeting peptide (transit peptides). The transit peptide is recognized by the chloroplast import machinery, which initiates import. The import machinery consists of translocon complexes at the outer (TOC) and inner membrane of the chloroplast (TIC). The import of hundreds of different chloroplast proteins depends on TOC and TIC complexes. The TOC complex core contains three proteins, the GTPase receptors: Toc159, Toc34 and the channel Toc75, together they recognize and transfer the pre-proteins across the outer membrane of the chloroplast. Both Toc34 and Toc159 are exposed at the surface of the chloroplast, consistent with a receptor function, and have homologous GTP-binding domains (G-domain). In addition to the G-domain, Toc159 has a N-terminal A- (acidic) domain that extends into the cytosol and controls receptor specificity and a C-terminal membrane anchoring M-domain. Toc75 belongs to the OMP85 family that serves to integrate proteins into the outer membrane of gram negative bacteria, in chloroplasts it has evolved to provide a protein translocation channel across the outer membrane.
Toc159 plays an essential role in chloroplast biogenesis. Phosphoproteomics databases show that Toc159 is highly phosphorylated at the A domain. Cytosolic casein kinase II phosphorylates the A-domain in vitro, however other A-domain kinases have been predicted.
While phosphorylation controls assembly and activity of protein import complexes in both mitochondria and chloroplasts no organelle-specific kinases have been identified so far. By co-purification with Toc159, we discovered "Kinase at the Outer Chloroplast membrane 1" (KOC1). KOC1 is an integral membrane protein facing the cytosol and stably associating with TOC. KOC1 phosphorylated the A-domain of Toc159 family members in vitro. In mutant koc1 chloroplasts preprotein import efficiency was diminished. Moreover, koc1 seedlings had reduced survival rates when moved from the dark to the light when protein import is required to rapidly complete chloroplast biogenesis. Our data indicate that KOC1 is a functional component of the TOC machinery phosphorylating import receptors, supporting preprotein import and contributing to efficient chloroplast biogenesis. - PublicationAccès libreThe role of alpha-tocopherol in the protection of tomato plants against abiotic stress(2017)
- PublicationAccès libreChloroplast lipid droplet type II NAD(P)H oxidoreductase, NDC1, is essential for vitamin E and K1 metabolism(2017)
; Les cellules végétales possèdent dans leurs différents tissus des organelles spécialisées appartenant à la famille des plastes. Le chloroplaste est le principal membre de cette famille et il est responsable de la photosynthèse dans les plantes. La majorité des plastes contiennent des particules lipoprotéiques ("gouttelettes lipidiques") appelées plastoglobules.
Peu est connu au sujet des plastoglobules qui ont été pendant une longue période imaginés comme des gouttelettes de stockage passif. En effet, le cœur hydrophobe des plastoglobules chloroplastiques contiennent des lipides neutres comme les prénylquinones (plastoquinone, plastochromanol-8, phylloquinone, tocophérol), les caroténoïdes, les triacylglycérols, les phytyl esters et d'autres lipides inconnus.
Les plastoglobules sont aussi composés de protéines et certaines d'entre-elles participent à des réactions métaboliques qui se déroulent dans les plastoglobules.
Pendant mon doctorat, j’ai démontré que NDC1 (NADP(H) déshydrogénase C1 (At5g08740), prédite comme une NAD(P)H-dépendante réductase de quinones, est physiquement associé aux gouttelettes lipidiques des chloroplastes.
Grâce à la génétique inverse et une approche in vitro il a été démontré que NDC1 contrôle l'état redox du réservoir de plastoquinone en injectant des électrons dans le plastoquinone à l’intérieur des plastoglobules. Cet effet sur l'état redox des plastoquinones facilitent l'accumulation du plastochromanol. Nous pouvons supposer que NDC1 puisse jouer un rôle en tant que réducteur des intermédiaires quinones qui précèdent la cyclisation par VTE1.
De manière surprenante, NDC1 est requis pour la dernière étape de méthylation lors de la biosynthèse de la phylloquinone (Vitamine K1). En effet, les mutants ndc1 accumulent le précurseur non-méthylé, le 2-phythyl-1,4-naphtoquinone ce qui montre que NDC1 est une enzyme indispensable de cette voie de biosynthèse.
L’ensemble des découvertes permettent d'affirmer que les plastoglobules ne sont pas un simple lieu de stockage de lipides mais ils possèdent un rôle dans les métabolismes biosynthétique et énergétique., Plant cells in different tissues contain specialized organelles belonging to the family of plastids. The chloroplast is the most prominent family member and responsible for photosynthesis in leaves. Most plastid types contain lipoprotein particles ("lipid droplets") termed plastoglobules. Little is known about plastoglobules that were long regarded as passive storage droplets. Indeed, the hydrophobic core of chloroplast plastoglobules contains neutral lipids such as the prenylquinones, carotenoids, triacylglycerols, phytyl esters and others unknown. Plastoquinone, plastochromanol-8, phylloquinone and tocopherol are prenylquinone molecules stored partly in the plastoglobule but functioning in the chloroplast thylakoids. Plastoglobules also carry proteins and some of these have been demonstrated to participate in metabolic reactions taking place at plastoglobules.
During my PhD work I demonstrated that NDC1 (NAD(P)H dehydrogenase C1 (At5g08740)), a candidate plastoglobule protein and predicted NAD(P)H-dependent quinone reductase is physically associated with the lipid droplets. A combined reverse genetic and in vitro approach demonstrated that NDC1 controls the overall REDOX state of the total plastoquinone reservoir. NDC1 does so by reducing the plastoquinone reservoir of plastoglobules. These findings provided evidence that plastoglobules are not simply a lipid storage site but have a role in energy metabolism. Besides its effects on the plastoquinone REDOX state NDC1 also facilitates plastochromanol accumulation and, surprisingly, is required for the last methylation step in phylloquinone (Vitamin K1) biosynthesis. The ndc1 mutant accumulates the non-methylated precursor, the 2-phythyl-1,4-naphtoquinone but up to now we have been unable to determine the precise mechanism. In conclusion, I have shown that NDC1 is a unique electron input device affecting the REDOX state of the overall plastoquinone pool. But more than that NDC1 is a key player at the intersection of a variety of prenylquinone metabolic pathways. By mutant analysis, I identified that NDC1 is the second enzyme that is implicated in the tocopherol redox cycle. Presumably NDC1 plays a role as reducer of quinone intermediates foregoing the cyclization by VTE1. It has been also demonstrated that high light stress triggers far-ranging changes in prenylquinone composition studied in mutants and overexpressing lines of VTE1 and NDC1 enzymes. The discovery that NDC1 is a new component of phylloquinone biosynthesis pathway is the single most important result of my thesis.