Kessler, Félix

2011, Bischof, Sylvain, Baerenfaller, Katja, Wildhaber, Thomas, Troesch, Raphael, Vidi, Pierre-Alexandre, Roschitzki, Bernd, Hirsch-Hoffmann, Matthias, Hennig, Lars, Kessler, Félix, Gruissem, Wilhelm

2009, Meskauskiene, Rasa, Würsch, Marco, Laloi, Christophe, Vidi, Pierre-Alexandre, Coll, Núria S., Kessler, Félix, Baruah, Aiswarya, Kim, Chanhong, Apel, Klaus

The conditional flu mutant of Arabidopsis thaliana generates singlet oxygen (¹O₂) in plastids during a dark-to-light shift. Seedlings of flu bleach and die, whereas mature plants stop growing and develop macroscopic necrotic lesions. Several suppressor mutants, dubbed singlet oxygen-linked death activator (soldat), were identified that abrogate ¹O₂-mediated cell death of flu seedlings. One of the soldat mutations, soldat10, affects a gene encoding a plastid-localized protein related to the human mitochondrial transcription termination factor mTERF. As a consequence of this mutation, plastid-specific rRNA levels decrease and protein synthesis in plastids of soldat10 is attenuated. This disruption of chloroplast homeostasis in soldat10 seedlings affects communication between chloroplasts and the nucleus and leads to changes in the steady-state concentration of nuclear gene transcripts. The soldat10 seedlings suffer from mild photo-oxidative stress, as indicated by the constitutive up-regulation of stress-related genes. Even though soldat10/flu seedlings overaccumulate the photosensitizer protochlorophyllide in the dark and activate the expression of ¹O₂-responsive genes after a dark-to-light shift they do not show a ¹O₂-dependent cell death response. Disturbance of chloroplast homeostasis in emerging soldat10/flu seedlings seems to antagonize a subsequent ¹O₂-mediated cell death response without suppressing ¹O₂-dependent retrograde signaling. The results of this work reveal the unexpected complexity of what is commonly referred to as 'plastid signaling'.

2006, Vidi, Pierre-Alexandre, Kanwischer, Marion, Baginsky, Sacha, Austin, Jotham R., Csucs, Gabor, Dörmann, Peter, Kessler, Félix, Bréhélin, Claire

Chloroplasts contain lipoprotein particles termed plastoglobules. Plastoglobules are generally believed to have little function beyond lipid storage. Here we report on the identification of plastoglobule proteins using mass spectrometry methods in Arabidopsis thaliana. We demonstrate specific plastoglobule association of members of the plastid lipid-associated proteins/fibrillin family as well as known metabolic enzymes, including the tocopherol cyclase (VTE1), a key enzyme of tocopherol (vitamin E) synthesis. Moreover, comparative analysis of chloroplast membrane fractions shows that plastoglobules are a site of vitamin E accumulation in chloroplasts. Thus, in addition to their lipid storage function, we propose that plastoglobules are metabolically active, taking part in tocopherol synthesis and likely other pathways.

2002, Bauer, Jörg, Hiltbrunner, Andreas, Weibel, Petra, Vidi, Pierre-Alexandre, Alvarez-Huerta, Mayte, Smith, Matthew, Schnell, Danny, Kessler, Félix

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.

2010, Zbierzak, Anna Maria, Kanwischer, Marion, Wille, Christina, Vidi, Pierre-Alexandre, Giavalisco, Patrick, Lohmann, Antje, Briesen, Isabel, Porfirova, Svetlana, Bréhélin, Claire, Kessler, Félix, Dörmann, Peter

Plastoglobules, lipid–protein bodies in the stroma of plant chloroplasts, are enriched in non-polar lipids, in particular prenyl quinols. In the present study we show that, in addition to the thylakoids, plastoglobules also contain a considerable proportion of the plastidial PQ-9 (plastoquinol-9), the redox component of photosystem II, and of the cyclized product of PQ-9, PC-8 (plastochromanol-8), a tocochromanol with a structure similar to γ-tocopherol and γ-tocotrienol, but with a C-40 prenyl side chain. PC-8 formation was abolished in the Arabidopsis thaliana tocopherol cyclase mutant vte1, but accumulated in VTE1-overexpressing plants, in agreement with a role of tocopherol cyclase (VTE1) in PC-8 synthesis. VTE1 overexpression resulted in the proliferation of the number of plastoglobules which occurred in the form of clusters in the transgenic lines. Simultaneous overexpression of VTE1 and of the methyltransferase VTE4 resulted in the accumulation of a compound tentatively identified as 5-methyl-PC-8, the methylated form of PC-8. The results of the present study suggest that the existence of a plastoglobular pool of PQ-9, along with the partial conversion of PQ-9 into PC-8, might represent a mechanism for the regulation of the antioxidant content in thylakoids and of the PQ-9 pool that is available for photosynthesis.

2007, Kessler, Félix, Vidi, Pierre-Alexandre

2006, Vidi, Pierre-Alexandre, Kessler, Félix

Les chloroplastes contiennent des corps protéo-lipidiques connus sous le nom de plastoglobules. Les plastoglobules ont longtemps été considérés comme des sites de stockage de lipides. Cependant, des études ont montré que leur nombre et leur volume augmente dans des situations de stress oxydatif. Ces structures ont donc un comportement dynamique. Des protéines structurelles appartenant à la famille des PAP/fibrillines, sont associées aux plastoglobules et leur expression est positivement régulée par plusieurs types de stress environnementaux. Nous avons identifié AtPGL35, une protéine marqueur pour les plastoglobules dans la plante Arabidopsis thaliana, et montré que la synthèse de cette protéine est induite par divers stress oxydatifs. En parallèle, des observations microscopiques ont montré une augmentation de la taille des plastoglobules dans des chloroplastes exposés à un stress oxydatif. Dans le dessein de découvrir de nouveaux composants et de nouvelles fonctions des plastoglobules, un extrait protéique issu de plastoglobules purifiés a été analysé par spectrométrie de masse. Les peptides identifiés appartenaient à des PAP/fibrillines ainsi qu’à des enzymes dont la tocopherol cyclase (AtVTE1), catalysant la pénultième étape de la biosynthèse de l’-tocopherol (la vitamine E). De plus, un fort enrichissement en tocophérols a été mesuré dans les plastoglobules. Des résultats suggérant que AtVTE1 est la seule enzyme de la voie de synthèse des tocophérols à être localisée dans les plastoglobules sont présentés. La présence de AtVTE1 en association avec les plastoglobules indique que ces corps lipidiques ne sont pas uniquement des sites de stockage, mais qu’ils participent à la synthèse de la vitamine E. Actuellement, les protéines recombinantes destinées à la médecine et à l’industrie sont principalement produites par des microorganismes ou des cultures de cellules animales. Dans l’optique de limiter les coûts de production et les risques liés aux pathogènes dans les systèmes animaux, les plantes représentent une alternative intéressante ("l’agriculture moléculaire"). Si la production de matériel végétal est peu coûteuse, les étapes de purifications sont un obstacle pour cette technologie. Afin de simplifier les procédures de purification, nous proposons de tirer parti de la faible densité des plastoglobules pour une première étape d’enrichissement. Comme preuve de principe, nous avons généré des plantes exprimant la protéine fluorescente jaune (YFP) fusionnée à une PAP/fibrilline. La protéine chimérique a été localisée dans les plastoglobules des plantes transgéniques et a pu être fortement enrichie par une simple centrifugation sur gradient. L’accumulation de la protéine recombinante n’a pas eu de conséquence néfaste apparente pour les plantes. Ces résultats démontrent que les plastoglobules représentent un site d’adressage prometteur pour l’agriculture moléculaire., Chloroplasts contain lipoprotein particles termed plastoglobules. Plastoglobules are generally believed to have little function beyond lipid storage. However, increased plastoglobule size and number has been correlated with oxidative stress conditions indicating dynamic behaviour. Structural proteins from the PAP/fibrillin family are associated with plastoglobules and are upregulated under various environmental stresses. We have identified AtPGL35 as an Arabidopsis thaliana plastoglobule marker protein, and shown that its upregulation under oxidative stress conditions parallels increase of plastoglobule size in chloroplasts. In order to discover new components and functions of plastoglobules, proteins from purified Arabidopsis plastoglobules have been identified by tandem mass spectrometry. The identified peptides belonged to PAP/fibrillins and to known metabolic enzymes including the tocopherol cyclase (AtVTE1), catalysing the penultimate step of  tocopherol (Vitamin E) biosynthesis. In addition, plastoglobules were shown to be a major site of -tocopherol accumulation. The data presented suggest that the cyclase reaction is the only step of the -tocopherol biosynthesis pathway occurring in plastoglobules. Association of AtVTE1 with plastoglobules indicates that these lipid-bodies are not mere storage sites for tocopherols but that they are involved in their synthesis. Plants are emerging as cost-effective alternatives for the production of recombinant proteins. Extraction and purification of transgene products are however obstacles for so-called molecular farming. We propose to take advantage of the low-density of plastoglobules for efficient recovery of recombinant proteins. As a proof-of-concept, the yellow fluorescent protein (YFP), fused to an Arabidopsis PAP/fibrillin, was targeted to plastoglobules and purified by a simple gradient flotation centrifugation procedure. Accumulation of the recombinant protein had no apparent effect on plant viability. These results identify plastoglobules as a promising sub-organellar compartment for molecular farming.

2009, Meskauskiene, Rasa, Würsch, Marco, Laloi, Christophe, Vidi, Pierre-Alexandre, Coll, Núria S, Kessler, Félix, Baruah, Aiswarya, Kim, Chanhong, Apel, Klaus

2007, Vidi, Pierre-Alexandre, Bréhélin, Claire, Kessler, Félix

Background
The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures) to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary.
Results
Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34) as the carrier. Similar to adipocyte differentiation related protein (ADRP) in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism.
Conclusion
Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming.

2006, Austin, Jotham R., Frost, Elizabeth, Vidi, Pierre-Alexandre, Kessler, Félix, Staehelin, L. Andrew

Plastoglobules are lipoprotein particles inside chloroplasts. Their numbers have been shown to increase during the upregulation of plastid lipid metabolism in response to oxidative stress and during senescence. In this study, we used state-of-the-art high-pressure freezing/freeze-substitution methods combined with electron tomography as well as freeze-etch electron microscopy to characterize the structure and spatial relationship of plastoglobules to thylakoid membranes in developing, mature, and senescing chloroplasts. We demonstrate that plastoglobules are attached to thylakoids through a half-lipid bilayer that surrounds the globule contents and is continuous with the stroma-side leaflet of the thylakoid membrane. During oxidative stress and senescence, plastoglobules form linkage groups that are attached to each other and remain continuous with the thylakoid membrane by extensions of the half-lipid bilayer. Using three-dimensional tomography combined with immunolabeling techniques, we show that the plastoglobules contain the enzyme tocopherol cyclase (VTE1) and that this enzyme extends across the surface monolayer into the interior of the plastoglobules. These findings demonstrate that plastoglobules function as both lipid biosynthesis and storage subcompartments of thylakoid membranes. The permanent structural coupling between plastoglobules and thylakoid membranes suggests that the lipid molecules contained in the plastoglobule cores (carotenoids, plastoquinone, and tocopherol [vitamin E]) are in a dynamic equilibrium with those located in the thylakoid membranes.