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Effects of selective inactivation of individual genes for low-molecular-mass subunits on the assembly of photosystem II, as revealed by chloroplast transformation: the psbEFLJ operon in Nicotiana tabacum

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Abstract

Photosystem (PSII) is a supramolecular polypeptide complex found in oxygenic photosynthetic membranes, which is capable of extracting electrons from water for the reduction of plastoquinone. An intriguing feature of this assembly is the fact that it includes more than a dozen low-mass polypeptides of generally unknown function. Using a transplastomic approach, we have individually disrupted the genes of the psbEFLJ operon in Nicotiana tabacum, which encode four such polypeptides, without impairing expression of downstream loci of the operon. All four mutants exhibited distinct phenotypes; none of them was capable of photoautotrophic growth. All mutants bleached rapidly in the light. Disruption of psbE and psbF, which code for the α and β apoproteins of cytochrome b 559 , abolished PSII activity, as expected; ΔpsbL and ΔpsbJ plants displayed residual PSII activity in young leaves. Controlled partial solubilisation of thylakoid membranes uncovered surprisingly severe impairment of PSII structure, with subunit and assembly patterns varying depending on the mutant considered. In the ΔpsbL mutant PSII was assembled primarily in a monomeric form, the homodimeric form was preponderant in ΔpsbJ, and, unlike the case in ΔpsbZ, the thylakoids of both mutants released some PSII supercomplexes. On the other hand, Photosystem I (PSI), the cytochrome b6f complex, ATP synthase, LHCII, and CP24/CP26/CP29 antennae were present in near wild-type levels. The data are discussed in terms of their implications for structural, biogenetic and functional aspects of PSII.

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References

  • Anbudurai PR, Pakrasi HB (1993) Mutational analysis of the PsbL protein of photosystem II in the cyanobacterium Synechocystis sp PCC 6803. Z Naturforsch 48:267–274

    CAS  Google Scholar 

  • Andersson J, Walters RG, Horton P Jansson S (2001) Antisense inhibition of the photosynthetic antenna proteins CP29 and CP26: implications for the mechanism of protective energy dissipation. Plant Cell 13:1193–1204

    Article  CAS  PubMed  Google Scholar 

  • Babcock GT, Widger WR, Cramer WA, Oertling WA, Metz JG (1985) Axial ligands of chloroplast cytochrome b-559: identification and requirement for a heme-cross-linked polypeptide structure. Biochemistry 24:3638–3645

    Google Scholar 

  • Baena-Gonzalez E, Gray JC, Tyystjarvi E, Aro EM, Maenpaa P (2001) Abnormal regulation of photosynthetic electron transport in a chloroplast ycf9inactivation mutant. J Biol Chem 276:20795–20802

    Article  CAS  PubMed  Google Scholar 

  • Barber J, Kühlbrandt W (1999) Photosystem II. Curr Opin Struct Biol 9:469–475

    Article  CAS  PubMed  Google Scholar 

  • Barry BA, Boerner RJ, de Paula JC (1994) The use of cyanobacteria in the study of the structure and function of photosystem II. In: Bryant DA (ed) The Molecular biology of cyanobacteria. Kluwer Academic, Dordrecht, pp 217–257

  • Bock R, Kossel H, Maliga P (1994) Introduction of a heterologous editing site into the tobacco plastid genome: the lack of RNA editing leads to a mutant phenotype. EMBO J 13:4623–4628

    CAS  PubMed  Google Scholar 

  • Boekema EJ, Hankamer B, Bald D, Kruip J, Nield J, Boonstra AF, Barber J, Rögner M (1995) Supramolecular structure of the photosystem II complex from green plants and cyanobacteria. Proc Natl Acad Sci USA 92:175–179

    CAS  PubMed  Google Scholar 

  • Boekema EJ, Van Roon H, Van Breemen JF, Dekker JP (1999) Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Eur J Biochem 266:444–452

    CAS  PubMed  Google Scholar 

  • Chad (2003)

  • De Santis-MaicIossek G, Kofer W, Bock A, Schoch S, Maier RM, Wanner G, Rudiger W, Koop HU, Herrmann RG (1999) Targeted disruption of the plastid RNA polymerase genes rpoA,band C1: molecular biology, biochemistry and ultrastructure. Plant J 18:477–89

    PubMed  Google Scholar 

  • Debus RJ (2000) The polypeptides of photosystem II and their influence on manganotyrosyl-based oxygen evolution. Met Ions Biol Syst 37:657–711

    CAS  PubMed  Google Scholar 

  • Dovzhenko A Bergen U, Koop HU (1998) Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts: shoot formation in less than two weeks. Protoplasma 20:114–118

    Google Scholar 

  • Gau AE, Thole HH, Sokolenko A, Altschmied L, Hermann RG, Pistorius EK (1998) PsbY, a novel manganese-binding, low-molecular-mass protein associated with photosystem II. Mol Gen Genet 260:56–68

    Article  CAS  PubMed  Google Scholar 

  • Haehnel W, Jansen T, Gause K, Klösgen RB, Stahl B, Michl D, Huvermann B, Karas M, Herrmann RG (1994) Electron transfer from plastocyanin to photosystem I. EMBO J 13:1028–1038

    CAS  PubMed  Google Scholar 

  • Hager M, Hermann M, Biehler K, Krieger-Liszkay A, Bock R (2002) Lack of the small plastid-encoded PsbJ polypeptide results in a defective water-splitting apparatus of photosystem II, reduced photosystem I levels, and hypersensitivity to light. J Biol Chem 277:14031–14039

    Article  CAS  PubMed  Google Scholar 

  • Hankamer B, Nield J, Zheleva D, Boekema E, Jansson S, Barber J (1997) Isolation and biochemical characterisation of monomeric and dimeric photosystem II complexes from spinach and their relevance to the organisation of photosystem II in vivo. Eur J Biochem 243:422–429

    CAS  PubMed  Google Scholar 

  • Hankamer B, Morris EP, Barber J (1999) Revealing the structure of the oxygen-evolving core dimer of photosystem II by cryoelectron crystallography. Nat Struct Biol 6:560–564

    Article  CAS  PubMed  Google Scholar 

  • Hankamer B, Morris E, Nield J, Carne A, Barber J (2001) Subunit positioning and transmembrane helix organisation in the core dimer of photosystem II. FEBS Lett 504:142–151

    Article  CAS  PubMed  Google Scholar 

  • Harbinson J, Woodward FI (1987) The use of light-induced absorbance changes at 820 nm to monitor the oxidation state of P700 in leaves. Plant Cell Environ 10:131–140

    CAS  Google Scholar 

  • Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 55–116

    Google Scholar 

  • Herrmann RG (1996) Photosynthesis research: aspects and perspectives. In: Andersson B, Salter AH, Barber J (eds) Frontiers in molecular biology: molecular genetics in photosynthesis. Oxford Univ Press, pp 1–44

    Google Scholar 

  • Herrmann RG, Alt J, Schiller B, Widger WR, Cramer WA (1984) Nucleotide sequence of the gene for apocytochrome b-559on the spinach plastid chromosome: implications for the structure of the membrane protein. FEBS Lett 176:239–244

    Article  CAS  Google Scholar 

  • Herrmann RG, Westhoff P, Link G (1992) Biogenesis of plastids in higher plants. In: Herrmann RG (ed) Plant gene research: cell organelles. Springer Verlag, Wien-New York, pp 275–349

    Google Scholar 

  • Hupfer H, Swiatek M, Hornung S, Herrmann RG, Maier RM, Chiu WL, Searsb(2000) Complete nucleotide sequence of the Oenothera elata plastid chromosome, representing plastome I of the five distinguishable Euoenothera plastomes. Mol Gen Genet 263:581–585

    Google Scholar 

  • Ikeuchi M, Eggers B, Shen GZ, Webber A, Yu JJ, Hirano A, Inoue Y, Vermaas W (1991) Cloning of the psbK gene from Synechocystis sp PCC 6803 and characterization of photosystem II in mutants lacking PSII-K. J Biol Chem 266:11111–11115

    CAS  PubMed  Google Scholar 

  • Keren N, Berg A, van Kann PJ, Levanon H, Ohad I (1997) Mechanism of photosystem II photoinactivation and D1 protein degradation at low light: the role of back electron flow. Proc Natl Acad Sci USA 94:1579–1584

    Article  CAS  PubMed  Google Scholar 

  • Kitamura K, Ozawa S, Shiina T, Toyoshima Y (1994) L protein, encoded by psbL, restores normal functioning of the primary quinone acceptor, QA, in isolated D1/D2/CP47/Cytb-559/I photosystem II reaction center core complex. FEBS Lett 354:113–116

    Article  CAS  PubMed  Google Scholar 

  • Koop H-U, Kofer W (1995) Plastid transformation by polyethylene glycol treatment of protoplasts and regeneration of transplastomic tobacco plants In: Potrykus I, Spangenberg G (eds) Gene transfer to plants. Springer Verlag, Berlin, pp 75–82

    Google Scholar 

  • Koop H-U, Steinmüller K, Wagner H, Rossler C, Eibl C, Sacher L (1996) Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199:193–201

    CAS  PubMed  Google Scholar 

  • Krause GH, Weis E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu Rev Plant Physiol Plant Mol Biol 42:313–349

    CAS  Google Scholar 

  • Kretzer F, Ohad I, Bennoun P (1976) Ontogeny, insertion and activation of two thylakoid peptides required for photosystem II activity in the nuclear, temperature sensitive T4 mutant of Chlamydomonas reinhardtii. In: Bücher T (eds) Genetics and biogenesis of chloroplasts and mitochondria. Elsevier/North Holland Biomedical Press, Amsterdam, pp 25–32

  • Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492

    CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage. T4 Nature 227:680–685

    CAS  Google Scholar 

  • Lind LK, Shukla VK, Nyhus KJ, Pakrasi HB (1993) Genetic and immunological analyses of the cyanobacterium Synechocystis sp PCC 6803 show that the protein encoded by the psbJ gene regulates the number of photosystem II centers in thylakoid membranes. J Biol Chem 268:1575–1579

    CAS  PubMed  Google Scholar 

  • Mayers SR, Dubbs JM, Vass I, Hideg E, Nagy L, Barber J (1993) Further characterization of the psbH locus of Synechocystis sp PCC 6803: inactivation of psbH impairs QA to QB electron transport in photosystem 2. Biochemistry 32:1454–1465

    CAS  PubMed  Google Scholar 

  • Morais F, Barber J, Nixon PJ (1998) The chloroplast-encoded alpha subunit of cytochrome b-559 is required for assembly of the photosystem two complex in both the light and the dark in Chlamydomonas reinhardtii. J Biol Chem 273:29315–29320

    Article  CAS  PubMed  Google Scholar 

  • Morais F, Kuhn K, Stewart DH, Barber J, Brudvig GW, Nixon PJ (2001) Photosynthetic water oxidation in cytochrome b(559) mutants containing a disrupted heme-binding pocket. J Biol Chem 276:31986–31993

    Article  CAS  PubMed  Google Scholar 

  • Müller B, Eichacker LA (1999) Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley etioplasts. Plant Cell 11:2365–2377

    Article  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    CAS  Google Scholar 

  • Nanba O, Satoh K (1987) Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome. Proc Natl Acad Sci USA 84:109–112

    CAS  Google Scholar 

  • Nield J, Funk C, Barber J (2000a) Supermolecular structure of photosystem II and location of the PsbS protein. Philos Trans R Soc LondbBiol Sci 355:1337–1344

    Article  CAS  Google Scholar 

  • Nield J, Kruse O, Ruprecht J, da Fonseca P, Büchel C, Barber J (2000b) Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcus elongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization. J Biol Chem 275:27940–27946

    CAS  PubMed  Google Scholar 

  • Ozawa S, Kobayashi T, Sugiyama R, Hoshida H, Shiina T, Toyoshima Y (1997) Role of PSII-L protein ( psbL gene product) in the electron transfer in photosystem II complex 1. Over-production of wild-type and mutant versions of PSII-L protein and reconstitution into the PSII core complex. Plant Mol Biol 34:151–161

    Article  CAS  PubMed  Google Scholar 

  • Pakrasi HB, Nyhus KJ, Granok H (1990) Targeted deletion mutagenesis of the beta subunit of cytochrome b559 protein destabilizes the reaction center of photosystem II. Z Naturforsch 45:423–429

    CAS  Google Scholar 

  • Palomares R, Herrmann RG, Oelmüller R (1993) Post-transcriptional and post-translational regulatory steps are crucial in controlling the appearance and stability of thylakoid polypeptides during the transition of etiolated tobacco seedlings to white light. Eur J Biochem 217:345–352

    CAS  PubMed  Google Scholar 

  • Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a andbwith four different solvents: verification of the concentration of chlorophyll by atomic absorption spectroscopy. Biochem Biophys Acta 975, 385–394

    Google Scholar 

  • Regel RE, Ivleva NB, Zer H, Meurer J, Shestakov SV, Herrmann RG, Pakrasi HB, Ohad I (2001) Deregulation of electron flow within photosystem II in the absence of the PsbJ protein. J Biol Chem 276:41473–41478

    Article  CAS  PubMed  Google Scholar 

  • Rosenblum BB, Lee LG, Spurgeon SL, Khan SH, Menchen SM, Heiner CR, Chen SM (1997) New dye-labeled terminators for improved DNA sequencing patterns. Nucleic Acids Res 25:4500–4504

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989): Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Sandona D, Croce R, Pagano A, Crimi M, Bassi R (1998) Higher plants light harvesting proteins. Structure and function as revealed by mutation analysis of either protein or chromophore moieties. Biochim Biophys Acta 1365:207–214

    CAS  PubMed  Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1992) DNA sequencing with chain-terminating inhibitors 1997. Biotechnology 24:104–108

    CAS  PubMed  Google Scholar 

  • Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    PubMed  Google Scholar 

  • Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231

    PubMed  Google Scholar 

  • Stewart DH, Brudvig GW (1998) Cytochrome b559of photosystem II. Biochim Biophys Acta 1367, 63–87

    Google Scholar 

  • Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917

    CAS  PubMed  Google Scholar 

  • Swiatek M, Kuras R, Sokolenko A, Higgs D, Olive J, Cinque G, Müller B, Eichacker LA, Stern DB, Bassi R, Herrmann RG, Wollman FA (2001) The chloroplast gene ycf9encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture. Plant Cell 13:1347–1367

    Article  CAS  PubMed  Google Scholar 

  • Swiatek M, Greiner S, Kemp S, Drescher A, Koop H-U, Herrmann RG, Maier RM (2003) PCR analysis of pulse-field gelelectrophoresis purified plastid DNA, a sensitive tool to judge the hetero- or homoplastomic status of plastid transformation. Curr Genet, in press

  • Takahashi Y, Matsumoto H, Goldschmidt-Clermont M, Rochaix JD (1994) Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex. Plant Mol Biol 24:779–788

    CAS  PubMed  Google Scholar 

  • Thidholm E, Lindstrom V, Tissier C, Robinson C, Schröder WP, Funk C (2002) Novel approach reveals localisation and assembly pathway of the PsbS and PsbW proteins into the photosystem II dimer. FEBS Lett 513:217–222

    Article  CAS  PubMed  Google Scholar 

  • Westhoff P, Alt J, Herrmann RG (1983) Localization of the genes for the two chlorophyll a-conjugated polypeptides (mol wt 51 and 44 kd) of the photosystem II reaction center on the spinach plastid chromosome. EMBO J 2:2229–2237

    CAS  Google Scholar 

  • Westhoff P, Alt J, Nelson N, Herrmann RG (1985) Genes and transcripts of the ATP synthase CF0 subunits I and II from spinach thylakoid membranes. Mol Gen Genet 199:290–299

    CAS  Google Scholar 

  • Wollman FA, Minai L, Nechushtai R (1999) The biogenesis and assembly of photosynthetic proteins in thylakoid membranes. Biochim Biophys Acta 1411:21–85

    Article  CAS  PubMed  Google Scholar 

  • Zer H, Vink M, Keren N, Dilly-Hartwig HG, Paulsen H, Herrmann RG, Anderssonband Ohad I (1999) Regulation of thylakoid protein phosphorylation at the substrate level: reversible light-induced conformational changes expose the phosphorylation site of the light-harvesting complex II. Proc Natl Acad Sci USA 96:8277–8282

    Article  CAS  PubMed  Google Scholar 

  • Zhang L, Paakkarinen V, Suorsa M, Aro EM (2001) A SecY homolog is involved in chloroplast-encoded D1 protein biogenesis. J Biol Chem 276:37809–37814

    CAS  PubMed  Google Scholar 

  • Zouni A, Witt HT, Kern J, Fromme P, Krauss N, Saenger W, Orth P (2001) Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature 409:739–743

    Google Scholar 

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Acknowledgements

We are most grateful to Ms. Martina Reymers and Ms. Claudia Nickel for expert technical assistence, to Dr. Roberto Barbato for providing antisera against D1, D2, CP43 and CP47, to Dr. Peter Westhoff and Dr. Wolfgang Schröder for antisera against PsbH and PsbW, respectively. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 184, SFB-TR1), the Human Frontier Science Programme (HFSP), the Fonds der Chemischen Industrie, the Department of Energy, USA (DOE), the National Institutes of Health (NIH) and the Israeli Science Foundation (ISF)

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Authors and Affiliations

  1. Department Biologie I, Bereich Botanik der Universität München, Menzinger Str. 67, 80638 , München, Germany

    M. Swiatek, R. E. Regel, J. Meurer, G. Wanner & R. G. Herrmann

  2. Department of Biology, Washington University, P.O. Box 1137, St. Louis, MO 63130, USA

    H. B. Pakrasi

  3. Department of Biological Chemistry, The Hebrew University of Jerusalem, 91904 , Jerusalem, Israel

    I. Ohad

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  1. M. Swiatek
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  2. R. E. Regel
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  3. J. Meurer
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  4. G. Wanner
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  5. H. B. Pakrasi
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  6. I. Ohad
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  7. R. G. Herrmann
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Correspondence to R. G. Herrmann.

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Communicated by R. Hagemann

The first two authors contributed equally to this work

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Swiatek, M., Regel, R.E., Meurer, J. et al. Effects of selective inactivation of individual genes for low-molecular-mass subunits on the assembly of photosystem II, as revealed by chloroplast transformation: the psbEFLJ operon in Nicotiana tabacum . Mol Gen Genomics 268, 699–710 (2003). https://doi.org/10.1007/s00438-002-0791-1

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