Untersuchungen zur Biosynthese und Struktur von LHPP, des Light-Harvesting-POR-Protochlorophyllid-Komplexes etiolierter Pflanzen
Final Report Abstract
All POR proteins contain evolutionarily conserved cystein residues that were implicated in Pchlide-binding. cDNAs were constructed by site directed mutagenesis that encode PORA and PORB mutant proteins with defined Cys->Ala exchanges (the four Cys-residues of each POR were either exchanged separately or as a combination of several or even all, by Ala residues). Mutant Cys->Ala PORA and Cys->Ala PORB were created as the precursor (pPORA/pPORB) as well as mature forms (PORA/PORB) of the polypeptide. In PORB, two of the four Cys residues, Cys276 and Cys303, were shown to establish distinct pigment binding sites. While Cys276 constituted the Pchlide binding site in the active site of the enzyme, Cys303 established a second, low affinity pigment binding site that was involved in the assembly and stabilization of imported PORB enzyme inside etioplasts. Also in the (p)PORA, 2 distinctive Pchlide binding sites were identified. Two of the four evolutionarily conserved Cys residues, namely Cys-268/200 and Cys-295/227 (the first number refers to the precursor and the second to the mature protein), were identified as participating in the establishment of the photoactive enzyme state and LHPP assembly, respectively. Cys-268/200 (Cys-3) is adjacent to the NADPH binding site. Cys-295/227 (Cys-4) constitutes a second, low-affinity Pchlide b binding site. We observed that Pchlide b bound to Cys-4 participates in PORA:PORB interactions. Replacement of Cys-4 by an Ala residue reduced by half the amount of bound pigment per PORA enzyme monomer and gave rise to assembly-incompetent PORA molecules. Both Cys-3 and Cys-4 are involved in energy transfer. This is apparent from the lack of photoactive Pchlide in assays containing the (Cys-3->Ala)-PORA and (Cys-4->Ala)-PORA mutant proteins. It could be shown that the rates of 1O2 production were extremely low for functional LHPP complexes. Presumably because of energy transfer from Pchlide b to Pchlide a and subsequent Pchlide a to Chlide a reduction, no significant levels of 1O2 were produced. By contrast, perturbation of LHPP's normal function, such as assembly of (Cys-3->Ala)-PORA with PORB into larger, non-light-dissociable complexes, led to a marked increase in the rate of 1O2 evolution. Even more deleterious effects were observed for assays containing unassembled, but lipid-associated (Cys-4->Ala)-PORA and PORB. This experiment provided PORA's presumed photoprotective role during greening. Arabidopsis thaliana knock out mutants was isolated and characterized for both reductase genes. The porb-mutant was not able to express PORB due to a T-DNA insertion in the PORB gene. The pora-mutant (porA-Ds) had an insertion of a Ds element in the PORA gene and did not contain PORA protein. Plants of the porA-Ds mutant had a dwarf phenotyp and dramatically reduced seed yields. When grown under dark conditions they showed high levels of free Pchlide and photobleaching after irradiation (data not shown). Etiolated mutant porb--plants were characterized by the prolamellar bodies of reduced in size. In order to analyze barley d PORB and Cys->Ala PORB and d PORA and Cys->Ala PORA mutant proteins in planta we transformed Arabidopsis wild type and porb-plants (porA-Ds could not be used due to the low seed production) with the corresponding cDNAs cloned into the binary vector pGII 35S. Selection was performed with BASTA and transformed lines were investigated by Southern, Northern and Western blot analysis. Mutant porb-plants that had integrated wild-type and mutant barley PORBs into their genome expressed the corresponding mRNAs. However, only wild type barley PORB protein accumulated to substantial levels and led to the restauration of prolamellar bodies to normal levels. Neither d pPORB/PORB nor Cys->Ala pPORB/PORB were detected by Western blot analysis. The same observation was made in Arabidopsis wild-type plants that were successfully transformed with d pPORA and Cys->Ala pPORA. Since in vitro translation and subsequent immunoprecipitation with RNA isolated from these plants resulted in polypeptides of the correct size we hypothesize that the lack of d pPORA and Cys->Ala pPORA (and most likely also that of the analogous PORB mutant proteins) is due to degradation.
Publications
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LS Plant Physiology, University Bayreuth, Bayreuth, Germany: “Untersuchung und Charakterisierung des Lichtsammelkomplexes (LHPP) etiolierter Pflanzen”, 2006
Frank Buhr
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“In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: Two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly”, in Mol. Gen. Genomics, 275(2006), 540-552
C. Reinbothe, F. Buhr, S. Bartsch, C. Desvignes, F. Quigley, H. Pesey and S. Reinbothe
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“A substrate-independent, 14:3:3 protein-mediated plastid import pathway of NADPH:protochlorophyllide oxidoreductase A”, in Proc. Natl. Acad. Sci. USA, 104(2007), 8538-8543
A. Schemenewitz, S. Pollmann, Reinbothe, C. and Reinbothe, S.
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Photoprotective role of NADPH:protochlorophyllide oxidoreductase A”, in Proc. Natl. Acad. Sci. USA, 105(2008), 12629-12634
Frank Buhr, Majida El Bakkouri, Oscar Valdez, Stephan Pollmann, Nikolai Lebedev, Steffen Reinbothe, and Christiane Reinbothe
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LS Plant Physiology, University Bayreuth, Bayreuth, Germany: “Analyse der physiologischen Funktion von Mitgliedern der Rieske-Typ Eisen-Schwefel-Proteinfamilie in der inneren Plastidenhülle“, 2009
Sandra Bartsch
