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Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex

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Abstract

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reihardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O2 evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111–1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.

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References

  1. Arnon DI: Copper enzymes in isolated chloroplasts. Plant Physiol 24: 1–15 (1949).

    Google Scholar 

  2. Bennoun P, Delepelaire P: Isolation of photosynthesis mutants in Chlamydomonas. In: Edelman M, Hallick RB, Chua N-H (eds) Methods in Chloroplast Molecular Biology, pp. 25–38. Elsevier Biomedical Press, Amsterdam (1982).

    Google Scholar 

  3. Bennoun P, Spierer-Herz M, Erickson J, Girard-Bascou J, Pierre Y, Delosme M, Rochaix J-D: Characterization of photosystem II mutants of Chlamydomonas reinhardtii lacking the psbA gene. Plant Mol Biol 6: 151–160 (1986).

    Google Scholar 

  4. Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB, Sanford JC: Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240: 1534–1538 (1988).

    Google Scholar 

  5. Burnap RL, Sherman LA: Deletion mutagenesis in Synechocystis sp. PCC6803 indicates that the Mn-stabilizing protein of photosystem II is not essential for O2 evolution. Biochemistry 30: 30440–30446 (1991).

    Google Scholar 

  6. Chua N-H, Bennoun P: Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: wild-type and mutant strains deficient in photosystem II reaction center. Proc Natl Acad Sci USA 72: 2175–2179 (1975).

    Google Scholar 

  7. de Vitry C, Diner B, Popot J-L: Photosystem II particles from Chlamydomonas reinhardtii: purification, molecular weight, small subunit composition, and protein phosphorylation. J Biol Chem 266: 16614–16621 (1991).

    Google Scholar 

  8. Erickson JM, Rochaix J-D: The molecular biology of photosystem II. In: Barber J (ed) Topics in Photosynthesis, vol 11, pp. 101–177. Elsevier, Amsterdam (1992).

    Google Scholar 

  9. Fling SP, Gregerson DS: Peptide and protein molecular weight determination by electrophoresis using a high-molarity Tris buffer system without urea. Anal Biochem 155: 83–88 (1986).

    Google Scholar 

  10. Goldschmidt-Clermont M: Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of chlamydomonas. Nucl Acids Res 19: 4083–4089 (1991).

    Google Scholar 

  11. Gorman DS, Levine RP: Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardti. Proc Natl Acad Sci USA 54: 1665–1669 (1965).

    Google Scholar 

  12. Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji Y, Sun CR, Meng BY, Li YQ, Kanno A, Nishizawa Y, Hirai A, Shinozaki K, Sugiura M: The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet 217: 185–194 (1989).

    Google Scholar 

  13. Ikeuchi M: Subunit proteins of photosystem II. Bot Mag Tokyo 105: 327–374 (1992).

    Google Scholar 

  14. Ikeuchi M, Eggers B, Shen G, Webber A, Yu J, Hirano A, Inoue Y, Vermaas W: 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 (1991).

    Google Scholar 

  15. Ikeuchi M, Inoue Y: A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome. FEBS Lett 241: 99–104 (1988).

    Google Scholar 

  16. Ikeuchi M, Takio K, Inoue Y: N-terminal sequencing of photosystem II low-molecular-mass proteins: 5 and 4.1 kDa components of the O2-evolving core complex from higher plants. FEBS Lett 242: 263–269 (1989).

    Google Scholar 

  17. Koike H, Mamada K, Ikeuchi M, Inoue Y: Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing. FEBS Lett 244: 391–396 (1989).

    Google Scholar 

  18. Kuchka MR, Goldschmidt-Clermont M, van Dillewijn J, Rochaix J-D: Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PSII. Cell 58: 869–876 (1989).

    Google Scholar 

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

    Google Scholar 

  20. Mannan RM, Whitmarsh J, Nyman P, Pakrasi H: Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. Proc Natl Acad Sci USA 88: 10168–10172 (1991).

    Google Scholar 

  21. Mayfield SP, Bennoun P, Rochaix J-D: Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of photosystem II particles in Chlamydomonas reinhardtii. EMBO J 6: 313–318 (1987).

    Google Scholar 

  22. Murata N, Miyao M, Hayashida N, Hidaka T, Sugiura M: Identification of a new gene in the chloroplast genome encoding a low-molecular-mass polypeptide of photosystem II complex. FEBS Lett 235: 283–288 (1988).

    Google Scholar 

  23. Newman SM, Gillham NW, Harris EH, Johnson AM, Boynton JE: Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii. Mol Gen Genet 230: 65–74 (1991).

    Google Scholar 

  24. Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H, Ozeki H: Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572–574 (1986).

    Google Scholar 

  25. Pakrasi MB, Vermaas WFJ: Protein engineering of photosystem II. In: Barber J (ed) Topics in Photosynthesis, vol 11, pp. 231–257. Elsevier, Amsterdam (1992).

    Google Scholar 

  26. Rochaix J-D, Mayfield S, Goldschmidt-Clermont M, Erickson J: Molecular biology of Chlamydomonas. In: Shaw CH (eds) Plant Molecular Biology: A Practical Approach, pp. 253–275. IRL Press, Oxford (1988).

    Google Scholar 

  27. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  28. Sandra LB, Palmer JD: Evolutionary transfer of the chloroplast tufA gene to the nucleus. Nature 344: 262–265 (1990).

    Google Scholar 

  29. Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Shinozaki KY, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Susuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M: The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5: 2043–2049 (1986).

    Google Scholar 

  30. Silk GW, Cruz FD, Wu M: Nucleotide sequence of the chloroplast gene for the 4 kD K polypeptide of photosystem II (psbK) and the psbK-tufA intergenic region of Chalmydomonas reinhardtii. Nucl Acids Res 18: 4930 (1990).

    Google Scholar 

  31. Silk GW, Wu M: 3′ transcript mapping of the Chlamydomonas reinhardtii chloroplast psbK mRNA using a novel method to prepare 3′ end-labeled single-stranded DNA probes. Res Report 14: 830–833 (1993).

    Google Scholar 

  32. Takahashi Y, Goldschmidt-Clermont M, Soen S-Y, Franzén LG, Rochaix JD: Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I. EMBO J 10: 2033–2040 (1991).

    Google Scholar 

  33. Tang X-S, Satoh K: Characterization of a 47-kilodalton chlorophyll-binding polypeptide (CP-47) isolated from a photosystem II core complex. Plant Cell Physiol 25: 935–945 (1984).

    Google Scholar 

  34. Weeks DP, Beerman N, Griffith OM: A small-scale fivehour procedure for isolating multiple samples of CsCl-purified DNA: application to isolations from mammalian, insect, higher plant, algal, yeast, and bacterial sources. Anal Biochem 152: 376–385 (1986).

    Google Scholar 

  35. Yamagishi A, Katoh S: A photoactive photosystem-II reaction-center complex lacking a chlorophyll-binding 40 kilodalton subunit from the thermophilic cyanobacterium Synechococcus sp. Biochim Biophys Acta 765: 118–124 (1984).

    Google Scholar 

  36. Yamaguchi N, Takahashi Y, Satoh K: Isclation and characterization of a photosystem II core complex depleted in the 43 kDa chlorophyll-binding subunit. Plant Cell Physiol 29: 123–129 (1988).

    Google Scholar 

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Takahashi, Y., Matsumoto, H., Goldschmidt-Clermont, M. et al. Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex. Plant Mol Biol 24, 779–788 (1994). https://doi.org/10.1007/BF00029859

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  • DOI: https://doi.org/10.1007/BF00029859

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