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NADPH:protochlorophyllide oxidoreductase B (PORB) action in Arabidopsis thaliana revisited through transgenic expression of engineered barley PORB mutant proteins

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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.

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Acknowledgements

We are grateful to P.M. Mullineaux (School of Biological Sciences, University of Essex, UK) for a gift of pGreen and other transformation vectors. We thank K. Kálai and E. Hideg (Institute for Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary) for a gift of the DanePy reagent. We are indebted to Claudia Rossig for help with the construction of the PORB mutant cDNAs and Iga Samol for advice with regard to the seedling viability tests. This work was supported by a Chaire d’Excellence granted to CR by the French Ministry of Research and Education.

Author contributions

FB carried out experiments. AL carried out experiments, AS carried out electron microscopy, SR and DvW analyzed data, CR designed research, carried out experiments analyzed data, StR designed research, carried out research, analyzed data, wrote the paper.

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  1. Armin Springer

    Present address: Medical Biology and Electron Microscopy Centre, University Medical Center Rostock, Strempelstraße 14, 18057, Rostock, Germany

Authors and Affiliations

  1. Biologie Environnementale et Systémique (BEeSy), Université Grenoble-Alpes, LBFA, BP53F, 38041, Grenoble cedex 9, France

    Frank Buhr, Abderrahim Lahroussi, Armin Springer, Christiane Reinbothe & Steffen Reinbothe

  2. Department of Plant and Environmental Sciences, Clemson University Pee Dee Research and Education Center, Florence, SC, 29506, USA

    Sachin Rustgi

  3. Department of Crop and Soil Sciences, Washington State University, Pullman, WA, 99164-6420, USA

    Sachin Rustgi & Diter von Wettstein

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  1. Frank Buhr
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Correspondence to Steffen Reinbothe.

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Buhr, F., Lahroussi, A., Springer, A. et al. NADPH:protochlorophyllide oxidoreductase B (PORB) action in Arabidopsis thaliana revisited through transgenic expression of engineered barley PORB mutant proteins. Plant Mol Biol 94, 45–59 (2017). https://doi.org/10.1007/s11103-017-0592-x

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  • DOI: https://doi.org/10.1007/s11103-017-0592-x

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