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Kessler, Félix
Nom
Kessler, Félix
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Professeur.e ordinaire
Email
felix.kessler@unine.ch
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Voici les éléments 1 - 8 sur 8
- 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.
- 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. - PublicationMétadonnées seulementAtToc90, a new GTP-binding component of the Arabidopsis chloroplast protein import machinery(2004)
- PublicationMétadonnées seulementEssential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane(2002)
; - PublicationMétadonnées seulementProtein translocon at the Arabidopsis outer chloroplast membrane(2001)
- PublicationAccès libreThe Novel Chloroplast Outer Membrane Kinase KOC1 Is a Required Component of the Plastid Protein Import Machinery
; - PublicationAccès libreRole of plastoglobules in metabolite repair in the tocopherol redox cycle
; ; Besagni, CélinePlants are exposed to ever changing light environments and continuously forced to adapt. Excessive light intensity leads to the production of reactive oxygen species that can have deleterious effects on photosystems and thylakoid membranes. To limit damage, plants increase the production of membrane soluble antioxidants such as tocopherols. Here, untargeted lipidomics after high light treatment showed that among hundreds of lipid compounds alpha-tocopherol is the most strongly induced, underscoring its importance as an antioxidant. As part of the antioxidant mechanism, a-tocopherol undergoes a redox cycle involving oxidative opening of the chromanol ring. The only enzyme currently known to participate in the cycle is tocopherol cyclase (VTE1, At4g32770), that re-introduces the chromanol ring of a-tocopherol. By mutant analysis, we identified the NAD(P)H-dependent quinone oxidoreductase (NDC1, At5g08740) as a second enzyme implicated in this cycle. NDC1 presumably acts through the reduction of quinone intermediates preceding cyclization by VTE1. Exposure to high light also triggered far-ranging changes in prenylquinone composition that we dissect herein using null mutants and lines overexpressing the VTE1 and NDC1 enzymes. - PublicationAccès libreNucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency
; Jarvis, PaulThe atToc33 protein is one of several pre-protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis-related pre-proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild-type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159-deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.