title: Towards in vivo mutation analysis: knock-out of specific chlorophylls bound to the light-harvesting complexes of Arabidopsis thaliana - the case of CP24 (Lhcb6).
abstract: In the last ten years, a large series of studies have targeted antenna complexes of plants (Lhc) with the aim of understanding the mechanisms of light harvesting and photoprotection. Combining spectroscopy, modeling and mutation analyses, the role of individual pigments in these processes has been highlighted in vitro. In plants, however, these proteins are associated with multiple complexes of the photosystems and function within this framework. In this work, we have envisaged a way to bridge the gap between in vitro and in vivo studies by knocking out in vivo pigments that have been proposed to play an important role in excitation energy transfer between the complexes or in photoprotection. We have complemented a CP24 knock-out mutant of Arabidopsis thaliana with the CP24 (Lhcb6) gene carrying a His-tag and with a mutated version lacking the ligand for chlorophyll 612, a specific pigment that in vitro experiments have indicated as the lowest energy site of the complex. Both complexes efficiently integrated into the thylakoid membrane and assembled into the PSII supercomplexes, indicating that the His-tag does not impair the organization in vivo. The presence of the His-tag allowed the purification of CP24-WT and of CP24-612 mutant in their native states. It is shown that CP24-WT coordinates 10 chlorophylls and 2 carotenoid molecules and has properties identical to those of the reconstituted complex, demonstrating that the complex self-assembled in vitro assumes the same folding as in the plant. The absence of the ligand for chlorophyll 612 leads to the loss of one Chl a and of lutein, again as in vitro, indicating the feasibility of the method. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.
authors.affiliation: Department of Biophysical chemistry, Groningen Biological Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Francesca
authors.initials: F
authors.lastname: Passarini
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
authors.forename: Pengqi
authors.initials: P
authors.lastname: Xu
authors.affiliation: Aix Marseille Université, CNRS, CEA, UMR 7265 Biologie Végétale et Microbiologie Environnementales, 13009 Marseille, France.
authors.forename: Stefano
authors.initials: S
authors.lastname: Caffarri
authors.affiliation: Department Molecular Biology of Plants, Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Linnaeusborg, 9747 AG Groningen, The Netherlands.
authors.forename: Jacques
authors.initials: J
authors.lastname: Hille
authors.affiliation: Department of Biophysical chemistry, Groningen Biological Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands; Department of Physics and Astronomy, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. Electronic address: r.croce@vu.nl.
authors.forename: Roberta
authors.initials: R
authors.lastname: Croce
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001665:Binding Sites; D002734:Chlorophyll / Q000378:metabolism*; D061146:Chlorophyll Binding Proteins / Q000378:metabolism*; D045342:Light-Harvesting Protein Complexes / Q000378:metabolism*; D009154:Mutation; D020524:Thylakoids / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D061146:Chlorophyll Binding Proteins; C529795:Lhcb6 protein, Arabidopsis; D045342:Light-Harvesting Protein Complexes; D002734:Chlorophyll
keywords: Arabidopsis thaliana; In vivo mutation analysis; Lhcb6; Light-harvesting complex; Photosystem II; Reconstituted complex