title: NADPH:protochlorophyllide oxidoreductase B (PORB) action in Arabidopsis thaliana revisited through transgenic expression of engineered barley PORB mutant proteins.
abstract: NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme for the light-induced greening of etiolated angiosperm plants. It belongs to the 'RED' family of reductases, epimerases and dehydrogenases. All POR proteins characterized so far contain evolutionarily conserved cysteine residues implicated in protochlorophyllide (Pchlide)-binding and catalysis. cDNAs were constructed by site-directed mutagenesis that encode PORB mutant proteins with defined Cys→Ala exchanges. These cDNAs were expressed in transgenic plants of a PORB-deficient knock-out mutant (porB) of Arabidopsis thaliana. Results show that porB plants expressing PORB mutant proteins with Ala substitutions of Cys276 or Cys303 are hypersensitive to high-light conditions during greening. Hereby, failure to assemble higher molecular weight complexes of PORB with its twin isoenzyme, PORA, as encountered with (Cys303→Ala)-PORB plants, caused more severe effects than replacing Cys276 by an Ala residue in the active site of the enzyme, as encountered in (Cys276→Ala)-PORB plants. Our results are consistent with the presence of two distinct pigment binding sites in PORB, with Cys276 establishing the active site of the enzyme and Cys303 providing a second, low affinity pigment binding site that is essential for the assembly of higher molecular mass light-harvesting PORB::PORA complexes and photoprotection of etiolated seedlings. Failure to assemble such complexes provoked photodynamic damage through the generation of singlet oxygen. Together, our data highlight the importance of PORB for Pchlide homoeostasis and greening in Arabidopsis.
authors.affiliation: Biologie Environnementale et Systémique (BEeSy), Université Grenoble-Alpes, LBFA, BP53F, 38041, Grenoble cedex 9, France.
authors.forename: Frank
authors.initials: F
authors.lastname: Buhr
authors.affiliation: Biologie Environnementale et Systémique (BEeSy), Université Grenoble-Alpes, LBFA, BP53F, 38041, Grenoble cedex 9, France.
authors.forename: Abderrahim
authors.initials: A
authors.lastname: Lahroussi
authors.affiliation: Biologie Environnementale et Systémique (BEeSy), Université Grenoble-Alpes, LBFA, BP53F, 38041, Grenoble cedex 9, France.|Medical Biology and Electron Microscopy Centre, University Medical Center Rostock, Strempelstraße 14, 18057, Rostock, Germany.
authors.forename: Armin
authors.initials: A
authors.lastname: Springer
authors.affiliation: Department of Plant and Environmental Sciences, Clemson University Pee Dee Research and Education Center, Florence, SC, 29506, USA.|Department of Crop and Soil Sciences, Washington State University, Pullman, WA, 99164-6420, USA.
authors.forename: Sachin
authors.initials: S
authors.lastname: Rustgi
authors.affiliation: Department of Crop and Soil Sciences, Washington State University, Pullman, WA, 99164-6420, USA.
authors.forename: Diter
authors.initials: D
authors.lastname: von Wettstein
authors.affiliation: Biologie Environnementale et Systémique (BEeSy), Université Grenoble-Alpes, LBFA, BP53F, 38041, Grenoble cedex 9, France.
authors.forename: Christiane
authors.initials: C
authors.lastname: Reinbothe
authors.affiliation: Biologie Environnementale et Systémique (BEeSy), Université Grenoble-Alpes, LBFA, BP53F, 38041, Grenoble cedex 9, France. sreinbot@ujf-grenoble.fr.
authors.forename: Steffen
authors.initials: S
authors.lastname: Reinbothe
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D001467:Hordeum / Q000201:enzymology*; D009154:Mutation; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified
chemical_list: D010940:Plant Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase
keywords: Greening; Singlet oxygen; Skotomorphogenesis; Tetrapyrrole biosynthesis