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The atypical kinase ABC1K1 / PGR6 allocates geranylgeranyldiphosphate to the carotenoid biosynthesis pathway
Auteur(s)
Editeur(s)
Date de parution
2023
Nombre de page
214
Résumé
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.
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.
Notes
Thesis committee:
Prof. Dr. Felix Kessler (Thesis Director) – University of Neuchâtel, Switzerland
Dr. Shanmugabalaji Ventakasalam (Thesis supervisor) – University of Neuchâtel, Switzerland
Prof. Dr. Theodoor Turlings – University of Neuchâtel, Switzerland
Prof. Dr. Christophe Rothan – University of Bordeaux, France
Defended on May 24th, 2023
No de thèse : 3064
Prof. Dr. Felix Kessler (Thesis Director) – University of Neuchâtel, Switzerland
Dr. Shanmugabalaji Ventakasalam (Thesis supervisor) – University of Neuchâtel, Switzerland
Prof. Dr. Theodoor Turlings – University of Neuchâtel, Switzerland
Prof. Dr. Christophe Rothan – University of Bordeaux, France
Defended on May 24th, 2023
No de thèse : 3064
Identifiants
Type de publication
doctoral thesis
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