Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1993 Sep;12(9):3627–3635. doi: 10.1002/j.1460-2075.1993.tb06036.x

Initiation codon mutations in the Chlamydomonas chloroplast petD gene result in temperature-sensitive photosynthetic growth.

X Chen 1, K Kindle 1, D Stern 1
PMCID: PMC413638  PMID: 8253086

Abstract

The chloroplast petD gene encodes subunit IV of the cytochrome b6/f complex and is required for photosynthetic electron transport. We have created Chlamydomonas strains in which the initiation codon of the petD gene has been changed to AUU or AUC. These mutants can grow photosynthetically at room temperature, but not at 35 degrees C. The accumulation of subunit IV during photosynthetic or heterotrophic growth at room temperature is reduced to 10-20% of the wild-type level; petD mRNA abundance is reduced to approximately 50% of the wild-type amount. Pulse labeling experiments indicate that at room temperature, subunit IV translation proceeds at 10-20% of the wild-type rate. Cells grown heterotrophically at 35 degrees C accumulate < 5% as much subunit IV as wild-type cells grown under the same conditions, and < 1% as much subunit IV as wild-type cells grown at room temperature. We conclude that translation initiation in these mutants is inefficient, leading to decreased translation and accumulation of subunit IV. At 35 degrees C, translational inefficiency leads directly or indirectly to insufficient accumulation of subunit IV to support photosynthetic growth.

Full text

PDF
3630

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Atwater J. A., Wisdom R., Verma I. M. Regulated mRNA stability. Annu Rev Genet. 1990;24:519–541. doi: 10.1146/annurev.ge.24.120190.002511. [DOI] [PubMed] [Google Scholar]
  2. Barkan A., Miles D., Taylor W. C. Chloroplast gene expression in nuclear, photosynthetic mutants of maize. EMBO J. 1986 Jul;5(7):1421–1427. doi: 10.1002/j.1460-2075.1986.tb04378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barkan A. Nuclear Mutants of Maize with Defects in Chloroplast Polysome Assembly Have Altered Chloroplast RNA Metabolism. Plant Cell. 1993 Apr;5(4):389–402. doi: 10.1105/tpc.5.4.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barkan A. Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs. EMBO J. 1988 Sep;7(9):2637–2644. doi: 10.1002/j.1460-2075.1988.tb03116.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bonham-Smith P. C., Bourque D. P. Translation of chloroplast-encoded mRNA: potential initiation and termination signals. Nucleic Acids Res. 1989 Mar 11;17(5):2057–2080. doi: 10.1093/nar/17.5.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boni I. V., Isaeva D. M., Musychenko M. L., Tzareva N. V. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1. Nucleic Acids Res. 1991 Jan 11;19(1):155–162. doi: 10.1093/nar/19.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boynton J. E., Gillham N. W., Harris E. H., Hosler J. P., Johnson A. M., Jones A. R., Randolph-Anderson B. L., Robertson D., Klein T. M., Shark K. B. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science. 1988 Jun 10;240(4858):1534–1538. doi: 10.1126/science.2897716. [DOI] [PubMed] [Google Scholar]
  8. Bruce B. D., Malkin R. Biosynthesis of the chloroplast cytochrome b6f complex: studies in a photosynthetic mutant of Lemna. Plant Cell. 1991 Feb;3(2):203–212. doi: 10.1105/tpc.3.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Butler J. S., Springer M., Grunberg-Manago M. AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4022–4025. doi: 10.1073/pnas.84.12.4022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Büschlen 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. 1991 Jun 24;284(2):257–262. doi: 10.1016/0014-5793(91)80698-3. [DOI] [PubMed] [Google Scholar]
  11. Danon A., Mayfield S. P. Light regulated translational activators: identification of chloroplast gene specific mRNA binding proteins. EMBO J. 1991 Dec;10(13):3993–4001. doi: 10.1002/j.1460-2075.1991.tb04974.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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. 1989 Oct;8(10):2803–2809. doi: 10.1002/j.1460-2075.1989.tb08426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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. 1992 Mar;4(3):283–295. doi: 10.1105/tpc.4.3.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Folley L. S., Fox T. D. Site-directed mutagenesis of a Saccharomyces cerevisiae mitochondrial translation initiation codon. Genetics. 1991 Nov;129(3):659–668. doi: 10.1093/genetics/129.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fong S. E., Surzycki S. J. Organization and structure of plastome psbF, psbL, petG and ORF712 genes in Chlamydomonas reinhardtii. Curr Genet. 1992 May;21(6):527–530. doi: 10.1007/BF00351664. [DOI] [PubMed] [Google Scholar]
  16. Franzetti B., Carol P., Mache R. Characterization and RNA-binding properties of a chloroplast S1-like ribosomal protein. J Biol Chem. 1992 Sep 25;267(27):19075–19081. [PubMed] [Google Scholar]
  17. Girard-Bascou J., Choquet Y., Schneider M., Delosme M., Dron M. Characterization of a chloroplast mutation in the psaA2 gene of Chlamydomonas reinhardtii. Curr Genet. 1987;12(7):489–495. doi: 10.1007/BF00419557. [DOI] [PubMed] [Google Scholar]
  18. Gold J. C., Spremulli L. L. Euglena gracilis chloroplast initiation factor 2. Identification and initial characterization. J Biol Chem. 1985 Dec 5;260(28):14897–14900. [PubMed] [Google Scholar]
  19. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  20. Goldschmidt-Clermont M. Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucleic Acids Res. 1991 Aug 11;19(15):4083–4089. doi: 10.1093/nar/19.15.4083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Haley J., Bogorad L. A 4-kDa maize chloroplast polypeptide associated with the cytochrome b6-f complex: subunit 5, encoded by the chloroplast petE gene. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1534–1538. doi: 10.1073/pnas.86.5.1534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hoch B., Maier R. M., Appel K., Igloi G. L., Kössel H. Editing of a chloroplast mRNA by creation of an initiation codon. Nature. 1991 Sep 12;353(6340):178–180. doi: 10.1038/353178a0. [DOI] [PubMed] [Google Scholar]
  23. Howe G., Merchant S. The biosynthesis of membrane and soluble plastidic c-type cytochromes of Chlamydomonas reinhardtii is dependent on multiple common gene products. EMBO J. 1992 Aug;11(8):2789–2801. doi: 10.1002/j.1460-2075.1992.tb05346.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hudson G. S., Mason J. G., Holton T. A., Koller B., Cox G. B., Whitfeld P. R., Bottomley W. A gene cluster in the spinach and pea chloroplast genomes encoding one CF1 and three CF0 subunits of the H+-ATP synthase complex and the ribosomal protein S2. J Mol Biol. 1987 Jul 20;196(2):283–298. doi: 10.1016/0022-2836(87)90690-5. [DOI] [PubMed] [Google Scholar]
  25. Hurt E., Hauska G. Identification of the polypeptides in the cytochrome b6/f complex from spinach chloroplasts with redox-center-carrying subunits. J Bioenerg Biomembr. 1982 Dec;14(5-6):405–424. doi: 10.1007/BF00743067. [DOI] [PubMed] [Google Scholar]
  26. Jensen K. H., Herrin D. L., Plumley F. G., Schmidt G. W. Biogenesis of photosystem II complexes: transcriptional, translational, and posttranslational regulation. J Cell Biol. 1986 Oct;103(4):1315–1325. doi: 10.1083/jcb.103.4.1315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kindle K. L. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1228–1232. doi: 10.1073/pnas.87.3.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kindle K. L., Richards K. L., Stern D. B. Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1721–1725. doi: 10.1073/pnas.88.5.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kindle K. L., Schnell R. A., Fernández E., Lefebvre P. A. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol. 1989 Dec;109(6 Pt 1):2589–2601. doi: 10.1083/jcb.109.6.2589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Klaff P., Gruissem W. Changes in Chloroplast mRNA Stability during Leaf Development. Plant Cell. 1991 May;3(5):517–529. doi: 10.1105/tpc.3.5.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kraus B. L., Spremulli L. L. Chloroplast initiation factor 3 from Euglena gracilis. Identification and initial characterization. J Biol Chem. 1986 Apr 15;261(11):4781–4784. [PubMed] [Google Scholar]
  32. Kudla J., Igloi G. L., Metzlaff M., Hagemann R., Kössel H. RNA editing in tobacco chloroplasts leads to the formation of a translatable psbL mRNA by a C to U substitution within the initiation codon. EMBO J. 1992 Mar;11(3):1099–1103. doi: 10.1002/j.1460-2075.1992.tb05149.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Maina C. V., Riggs P. D., Grandea A. G., 3rd, Slatko B. E., Moran L. S., Tagliamonte J. A., McReynolds L. A., Guan C. D. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene. 1988 Dec 30;74(2):365–373. doi: 10.1016/0378-1119(88)90170-9. [DOI] [PubMed] [Google Scholar]
  35. Mayfield S. P., 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. 1987 Feb;6(2):313–318. doi: 10.1002/j.1460-2075.1987.tb04756.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Newman S. M., Boynton J. E., Gillham N. W., Randolph-Anderson B. L., Johnson A. M., Harris E. H. Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterization of integration events. Genetics. 1990 Dec;126(4):875–888. doi: 10.1093/genetics/126.4.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Newman S. M., Gillham N. W., Harris E. H., Johnson A. M., Boynton J. E. Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii. Mol Gen Genet. 1991 Nov;230(1-2):65–74. doi: 10.1007/BF00290652. [DOI] [PubMed] [Google Scholar]
  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. 1989 Apr;8(4):1013–1021. doi: 10.1002/j.1460-2075.1989.tb03468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rochaix J. D. Post-transcriptional steps in the expression of chloroplast genes. Annu Rev Cell Biol. 1992;8:1–28. doi: 10.1146/annurev.cb.08.110192.000245. [DOI] [PubMed] [Google Scholar]
  40. Rock C. D., Barkan A., Taylor W. C. The maize plastid psbB-psbF-petB-petD gene cluster: spliced and unspliced petB and petD RNAs encode alternative products. Curr Genet. 1987;12(1):69–77. doi: 10.1007/BF00420729. [DOI] [PubMed] [Google Scholar]
  41. Romero A., García P. Initiation of translation at AUC, AUA and AUU codons in Escherichia coli. FEMS Microbiol Lett. 1991 Dec 1;68(3):325–330. doi: 10.1016/0378-1097(91)90377-m. [DOI] [PubMed] [Google Scholar]
  42. Sakamoto W., Kindle K. L., Stern D. B. In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):497–501. doi: 10.1073/pnas.90.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shepherd H. S., Boynton J. E., Gillham N. W. Mutations in nine chloroplast loci of Chlamydomonas affecting different photosynthetic functions. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1353–1357. doi: 10.1073/pnas.76.3.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  45. Staub J. M., Maliga P. Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA. EMBO J. 1993 Feb;12(2):601–606. doi: 10.1002/j.1460-2075.1993.tb05692.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stern D. B., Radwanski E. R., Kindle K. L. A 3' stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell. 1991 Mar;3(3):285–297. doi: 10.1105/tpc.3.3.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sugiura M. The chloroplast genome. Plant Mol Biol. 1992 May;19(1):149–168. doi: 10.1007/BF00015612. [DOI] [PubMed] [Google Scholar]
  48. Westhoff P., Herrmann R. G. Complex RNA maturation in chloroplasts. The psbB operon from spinach. Eur J Biochem. 1988 Feb 1;171(3):551–564. doi: 10.1111/j.1432-1033.1988.tb13824.x. [DOI] [PubMed] [Google Scholar]
  49. Yu W., Spreitzer R. J. Sequences of trnR-ACG and petD that contain a tRNA-like element within the chloroplast genome of Chlamydomonas reinhardtii. Nucleic Acids Res. 1991 Feb 25;19(4):957–957. doi: 10.1093/nar/19.4.957. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES