title: Functional complementation of the Arabidopsis thaliana psbo1 mutant phenotype with an N-terminally His6-tagged PsbO-1 protein in photosystem II.
abstract: The Arabidopsis thaliana mutant psbo1 has recently been described and characterized. Loss of expression of the PsbO-1 protein leads to a variety of functional perturbations including elevated levels of the PsbO-2 protein and defects on both the oxidizing- and reducing-sides of Photosystem II. In this communication, two plant lines were produced using the psbo1 mutant as transgenic host, which contained an N-terminally histidine(6)-tagged PsbO-1 protein. This protein was expressed and correctly targeted into the thylakoid lumen. Immunological analysis indicated that different levels of expression of the modified PsbO-1 protein were obtained in different transgenic plant lines and that the level of expression in each line was stable over several generations. Examination of the Photosystem II closure kinetics demonstrated that the defective double reduction of Q(B) and the delayed exchange of Q(B)H(2) with the plastoquinone pool which were observed during the characterization of the psbo1 mutant were effectively restored to wild-type levels by the His(6)-tagged PsbO-1 protein. Flash fluorescence induction and decay were also examined. Our results indicated that high expression of the modified PsbO-1 was required to increase the ratio of PS II(alpha)/PS II(beta) reaction centers to wild-type levels. Fluorescence decay kinetics in the absence of DCMU indicated that the expression of the His(6)-tagged PsbO-1 protein restored efficient electron transfer to Q(B), while in the presence of DCMU, charge recombination between Q(A)(-) and the S(2) state of the oxygen-evolving complex occurred at near wild-type rates. Our results indicate that high expression of the His(6)-tagged PsbO-1 protein efficiently complements nearly all of the photochemical defects observed in the psbo1 mutant. Additionally, this study establishes a platform on which the in vivo consequences of site-directed mutagenesis of the PsbO-1 protein can be examined.
authors.affiliation: Department of Biological Sciences, Division of Biochemistry and Molecular Biology, Louisiana State University, Baton Rouge, LA 70803, USA.
authors.forename: Haijun
authors.initials: H
authors.lastname: Liu
authors.forename: Laurie K
authors.initials: LK
authors.lastname: Frankel
authors.forename: Terry M
authors.initials: TM
authors.lastname: Bricker
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002734:Chlorophyll / Q000378:metabolism; D000077194:Chlorophyll A; D004237:Diuron / Q000494:pharmacology; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D009842:Oligopeptides / Q000235:genetics; D045332:Photosystem II Protein Complex / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020524:Thylakoids / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D029681:Arabidopsis Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D045332:Photosystem II Protein Complex; D011993:Recombinant Fusion Proteins; C054266:photosystem II manganese-stabilizing protein; D002734:Chlorophyll; D006639:Histidine; D004237:Diuron; D000077194:Chlorophyll A