Plant Molecular Biology

, Volume 29, Issue 5, pp 921–932 | Cite as

Nuclear mutants of Chlamydomonas reinhardtii defective in the biogenesis of the cytochrome b6f complex

  • Nicola J. Gumpel
  • Louise Ralley
  • Jacqueline Girard-Bascou
  • Francis-André Wollman
  • Jonathan H. A. Nugent
  • Saul Purton


The random integration of transforming DNA into the nuclear genome of Chlamydomonas has been employed as an insertional mutagen to generate a collection of photosynthetic mutants that display abnormal steady-state fluorescence levels and an acetate-requiring phenotype. Electron paramagnetic resonance spectroscopy was then used to identify those mutants that specifically lack a functional cytochrome b6f complex. Our analysis of RNA and protein synthesis in five of these mutants reveals four separate phenotypes. One mutant fails to accumulate transcript for cytochrome f, whilst a second displays a severely reduced accumulation of the cytochrome b6 transcript. Two other mutants appear to be affected in the insertion of the haem co-factor into cytochrome b6. The fifth mutant displays no detectable defect in the synthesis of any of the known subunits of the complex. Genetic analysis of the mutants demonstrates that in three cases, the mutant phenotype co-segregates with the introduced DNA. For the mutant affected in the accumulation of the cytochrome f transcript, we have used the introduced DNA as a tag to isolate the wild-type version of the affected gene.

Key words

Chlamydomonas reinhardtii chloroplast gene expression insertional mutagenesis cytochrome b6f complex nuclear-encoded factors 


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  1. 1.
    Adam M, Lentz KE, Loppes R: Insertional mutagenesis to isolate acetate-requiring mutants in Chlamydomonas reinhardtii. FEMS Microbiol Lett 110: 265–268 (1993).Google Scholar
  2. 2.
    Atteia A, deVitry C, Pierre Y, Popot J-L: Identification of mitochondrial proteins in membrane preparations from Chlamydomonas reinhardtii. J Biol Chem 267: 226–234 (1992).Google Scholar
  3. 3.
    Barkan A, Voelker R, Mendel-Hartvig J, Johnson D, Walker M: Genetic analysis of chloroplast biogenesis in higher plants. Physiol Plant 93: 163–170 (1995).Google Scholar
  4. 4.
    Bennoun P, Delepelaire P: Isolation of photosynthetic mutants in Chlamydomonas. In: Edelman M, Hallick R, Chua N-H (eds) Methods in Chloroplast Molecular Biology, pp. 25–38. Elsevier, Amsterdam (1982).Google Scholar
  5. 5.
    Buschlen S, Choquet Y, Kuras R, Wollman F-A: Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii. FEBS Lett 284: 257–262 (1991).Google Scholar
  6. 6.
    Cook WB, Miles D: Transposon mutagenesis of nuclear photosynthetic genes in Zea mays. In: Govindjee (ed) Molecular Biology of Photosynthesis, pp. 77–103. Kluwer Academic Publishers, Dordrecht (1988).Google Scholar
  7. 7.
    Danon A, Mayfield SPY: Light regulated translational activators: Identification of chloroplast gene specific mRNA binding proteins. EMBO J 10: 3993–4001 (1991).Google Scholar
  8. 8.
    Davies JP, Yildiz F, Grossman AR: Mutants of Chlamydomonas with aberrant responses to sulfur deprivation. Plant Cell 6: 53–63 (1994).Google Scholar
  9. 9.
    Debuchy R, Purton S, Rochaix J-D: The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J 8: 2803–2809 (1989).Google Scholar
  10. 10.
    Delepelaire P: Characterisation of additional thylakoid membrane polypeptides synthesized inside the chloroplast in Chlamydomonas reinhardtii. Photobiochem Photobiophys 6: 279–291 (1983).Google Scholar
  11. 11.
    Diner B, Wollman F-A: Isolation of highly active photosystem II particles from a mutant of C. reinhardtii. Eur J Biochem 110: 521–526 (1980).Google Scholar
  12. 12.
    Drapier D, Girard-Bascou J, Wollman F-A: Evidence for nuclear control of the expression of the atpA and atpB chloroplast genes in Chlamydomonas. Plant Cell 4: 283–295 (1992).Google Scholar
  13. 13.
    Evans MCW, Hallahan BJ, Hanley JA, Heathcote P, Gumpel NJ, Purton S: Isolation and characterisation of photosynthetic reaction centres from eukaryotic organisms. In: Brown G, Cooper C (eds) Bioenergetics: A Practical Approach, pp. 183–217. IRL Press, Oxford (1994).Google Scholar
  14. 14.
    Fong SE, Surzycki SJ: Organization and structure of plastome psbF, psbL, petG and ORF712 genes in Chlamydomonas reinhardtii. Curr Genet 21: 527–530 (1992).Google Scholar
  15. 15.
    Girard-Bascou J, Pierre Y, Drapier D: A nuclear mutant affects the synthesis of the chloroplast psbA gene production in C. reinhardtii. Curr Genet 22: 47–52 (1992).Google Scholar
  16. 16.
    Gruissem W: Chloroplast gene expression: how plants turn their plastids on. Cell 56: 161–170 (1988).Google Scholar
  17. 17.
    Gumpel NJ, Purton S: Playing tag with Chlamydomonas. Trends Cell Biol 4: 299–301 (1994).Google Scholar
  18. 18.
    Gumpel NJ, Rochaix J-D, Purton S: Studies on homologous recombination in the green alga Chlamydomonas reinhardtii. Curr Genet 26: 438–442 (1994).Google Scholar
  19. 19.
    Harris EH: The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego, FL (1989).Google Scholar
  20. 20.
    Kindle KL: High frequency transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87: 1228–1232 (1990).Google Scholar
  21. 21.
    Klaff P, Gruissem W: Changes in chloroplast mRNA stability during barley chloroplast development. Plant Cell 3: 517–529 (1991).Google Scholar
  22. 22.
    Kuchka MR, Goldschmidt-Clermont M, vanDillewijn J, Rochaix J-D: Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II. Cell 58: 869–876 (1989).Google Scholar
  23. 23.
    Kuras R, Wollman F-A: The assembly of cytochrome b6/f comoplexes: An approach using genetic transformation of the green alga Chlamydomonas reinhardtii. EMBO J 13: 1019–1027 (1994).Google Scholar
  24. 24.
    Laemmli UK: Cleavage of structural protein during assembly of the head of bacteriophage T4. Nature 227: 680–682 (1970).Google Scholar
  25. 25.
    Lemaire C, Girard-Bascou J, Wollman F-A, Bennoun P: Studies on the cytochrome b6/f complex. I. Characterisation of the complex subunits in Chlamydomonas reinhardtii. Biochim Biophys Acta 851: 229–238 (1986).Google Scholar
  26. 26.
    Mieszczak M, Klahre U, Levy JH, Goodall GJ, Filipowicz W: Multiple plant RNA binding proteins identified by PCR: expression of cDNAs encoding RNA binding proteins targeted to chloroplasts in Nicotiana plumbaginifolia. Mol Gen Genet 234: 390–400 (1992).Google Scholar
  27. 27.
    Monod C, Goldschmidt-Clermont M, Rochaix J-D: Accumulation of chloroplast psbB RNA requires a nuclear factor in Chlamydomonas reinhardtii. Mol Gen Genet 231: 449–459 (1992).Google Scholar
  28. 28.
    Mullet J E: Dynamic regulation of chloroplast transcription. Plant Physiol 103: 309–313 (1993).Google Scholar
  29. 29.
    Nelson JAE, Savereide PB, Lefebvre PA: The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. Mol Cell Biol 14: 4011–4019 (1994).Google Scholar
  30. 30.
    Nugent JHA, Bendall DS: Functional size measurements on the chloroplast cytochrome bf complex. Biochim Biophys Acta 893: 177–183 (1987).Google Scholar
  31. 31.
    Okayama H, Kawaichi M, Brownstein M, Lee F, Yokota T, Arai K: High efficiency cloning of full length cDNA; construction and screening of cDNA expression libraries for mammalian cells. Meth Enzymol 154: 3–27 (1987).Google Scholar
  32. 32.
    Pasquale SM, Goodenough UW: Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii. J Cell Biol 105: 2279–2292 (1987).Google Scholar
  33. 33.
    Pierre Y, Popot J-L: Identification of two 4 kDa miniproteins in the cytochrome b6f complex from Chlamydomonas reinhardtii. C R Acad Sci Paris 316: 1404–1409 (1993).Google Scholar
  34. 34.
    Purton S, Rochaix J-D: Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol Biol 24: 533–537 (1994).Google Scholar
  35. 35.
    Purton S, Rochaix J-D: Characterization of the ARG7 gene of Chlamydomonas reinhardtii and its application to nuclear transformation. Eur J Phycol 30: 141–148 (1995).Google Scholar
  36. 36.
    Rochaix J-D: Post-transcriptional steps in the expression of chloroplast genes. Annu Rev Cell Biol 8: 1–28 (1992).Google Scholar
  37. 37.
    Rochaix J-D, Mayfield S, Goldschmidt-Clermont M, Erickson J: Molecular biology of Chlamydomonas. In: Shaw CH (ed) Plant Molecular Biology: A Practical Approach, pp. 253–275. IRL Press, Oxford (1988).Google Scholar
  38. 38.
    Rochaix J-D, Kuchka M, Mayfield S, Schirmer-Rahire M, Girard-Bascou J, Bennoun P: Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii. EMBO J 8: 1013–1021 (1989).Google Scholar
  39. 39.
    Salvador ML, Klein U, Bogorad L: 5′ sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts. Proc Natl Acad Sci USA 90: 1556–1560 (1993).Google Scholar
  40. 40.
    Sambrook J, Frisch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  41. 41.
    Schmidt CL, Malkin R: Low molecular weight subunits associated with the cytochrome b6f complexes from spinach and Chlamydomonas reinhardtii. Photosyn Res 38: 73–81 (1993).Google Scholar
  42. 42.
    Schuster G, Gruissem W: Chloroplast mRNA 3′ processing requires a nuclear-encoded RNA-binding protein. EMBO J 10: 1493–1502 (1991).Google Scholar
  43. 43.
    Sieburth LE, Berry-Lowe S, Schmidt GW: Chloroplast RNA stability in Chlamydomonas. Rapid degradation of psbB and psbC transcripts in two nuclear mutants. Plant Cell 3: 175–190 (1991).Google Scholar
  44. 44.
    Stern DB, Radwanski ER, Kindle KL: A 3′ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell 3: 285–297 (1991).Google Scholar
  45. 45.
    Stevens DR, Purton S: Development of a dominant selectable marker for nuclear transformation of Chlamydomonas reinhardtii. J Exp Bot 46 (suppl): 37 (1995).Google Scholar
  46. 46.
    Tam L-W, Lefebvre PA: Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. Genetics 135: 375–384 (1993).Google Scholar
  47. 47.
    Thomas PE, Ryan D, Levin W: An improved staining procedure for the detection of peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem 75: 168–176 (1976).Google Scholar
  48. 48.
    Zerges W, Rochaix J-D: The 5′ leader of a chloroplast mRNA mediates the translational requirements for two nuclear encoded functions in Chlamydomonas reinhardtii. Mol Cell Biol 14: 5268–5277 (1994).Google Scholar
  49. 49.
    Zhang H, Herman PL, Weeks DP: Gene isolation through complementation using an indexed library of Chlamydomonas reinhardtii DNA. Plant Mol Biol 24: 663–672 (1994).Google Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Nicola J. Gumpel
    • 1
  • Louise Ralley
    • 1
  • Jacqueline Girard-Bascou
    • 1
  • Francis-André Wollman
    • 2
  • Jonathan H. A. Nugent
    • 1
  • Saul Purton
    • 1
  1. 1.UCL Photosynthesis Research Group, Department of BiologyUniversity College LondonLondonUK
  2. 2.Service de PhotosynthèseInstitut de Biologie Physico-ChimiqueParisFrance

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