Skip to main content
NCBI home page
The Plant Cell logoLink to The Plant Cell
. 1993 Dec;5(12):1817–1829. doi: 10.1105/tpc.5.12.1817

Chloroplast-encoded chlB is required for light-independent protochlorophyllide reductase activity in Chlamydomonas reinhardtii.

J Li 1, M Goldschmidt-Clermont 1, M P Timko 1
PMCID: PMC160407  PMID: 8305874

Abstract

A chloroplast-encoded gene, designated chlB, has been isolated from Chlamydomonas reinhardtii, its nucleotide sequence determined, and its role in the light-independent reduction of protochlorophyllide to chlorophyllide demonstrated by gene disruption experiments. The C. reinhardtii chlB gene is similar to open reading frame 563 (orf563) of C. moewusii, and its encoded protein is a homolog of the Rhodobacter capsulatus bchB gene product that encodes one of the polypeptide components of bacterial light-independent protochlorophyllide reduction. To determine whether the chlB gene product has a similar role in light-independent protochlorophyllide reduction in this alga, a series of plasmids were constructed in which the aadA gene conferring spectinomycin resistance was inserted at three different sites within the chlB gene. The mutated chlB genes were introduced into the Chlamydomonas chloroplast genome using particle gun-mediated transformation, and homoplasmic transformants containing the disrupted chlB genes were selected on the basis of conversion to antibiotic resistance. Individual transformed strains containing chlB disruptions were grown in the dark or light, and 17 of the 18 strains examined were found to have a "yellow-in-the-dark" phenotype and to accumulate the chlorophyll biosynthetic precursor protochlorophyllide. RNA gel blot analysis of chlB gene expression in wild-type cells indicated that the gene was transcribed at low levels in both dark- and light-grown cells. The results of these studies support the involvement of the chlB gene product in light-independent protochlorophyllide reduction, and they demonstrate that, similar to its eubacterial predecessors, this green alga requires at least three components (i.e., chlN, chlL, and chlB) for light-independent protochlorophyllide reduction.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

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

  1. Apel K., Santel H. J., Redlinger T. E., Falk H. The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Isolation and characterization of the NADPH:protochlorophyllide oxidoreductase. Eur J Biochem. 1980 Oct;111(1):251–258. doi: 10.1111/j.1432-1033.1980.tb06100.x. [DOI] [PubMed] [Google Scholar]
  2. Bauer C. E., Bollivar D. W., Suzuki J. Y. Genetic analyses of photopigment biosynthesis in eubacteria: a guiding light for algae and plants. J Bacteriol. 1993 Jul;175(13):3919–3925. doi: 10.1128/jb.175.13.3919-3925.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bauer C. E., Buggy J. J., Yang Z. M., Marrs B. L. The superoperonal organization of genes for pigment biosynthesis and reaction center proteins is a conserved feature in Rhodobacter capsulatus: analysis of overlapping bchB and puhA transcripts. Mol Gen Genet. 1991 Sep;228(3):433–444. doi: 10.1007/BF00260637. [DOI] [PubMed] [Google Scholar]
  4. Benli M., Schulz R., Apel K. Effect of light on the NADPH-protochlorophyllide oxidoreductase of Arabidopsis thaliana. Plant Mol Biol. 1991 Apr;16(4):615–625. doi: 10.1007/BF00023426. [DOI] [PubMed] [Google Scholar]
  5. Burke D. H., Alberti M., Hearst J. E. The Rhodobacter capsulatus chlorin reductase-encoding locus, bchA, consists of three genes, bchX, bchY, and bchZ. J Bacteriol. 1993 Apr;175(8):2407–2413. doi: 10.1128/jb.175.8.2407-2413.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burke D. H., Alberti M., Hearst J. E. bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants. J Bacteriol. 1993 Apr;175(8):2414–2422. doi: 10.1128/jb.175.8.2414-2422.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burke D. H., Hearst J. E., Sidow A. Early evolution of photosynthesis: clues from nitrogenase and chlorophyll iron proteins. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7134–7138. doi: 10.1073/pnas.90.15.7134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Choquet Y., Rahire M., Girard-Bascou J., Erickson J., Rochaix J. D. A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii. EMBO J. 1992 May;11(5):1697–1704. doi: 10.1002/j.1460-2075.1992.tb05220.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coomber S. A., Chaudhri M., Connor A., Britton G., Hunter C. N. Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster of Rhodobacter sphaeroides. Mol Microbiol. 1990 Jun;4(6):977–989. doi: 10.1111/j.1365-2958.1990.tb00670.x. [DOI] [PubMed] [Google Scholar]
  11. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ellsworth R. K., Aronoff S. Investigations on the biogenesis of chlorophyll a. 3. Biosynthesis of Mg-vinylpheoporphine a5 methylester from Mg-protoporphine IX monomethylester as observed in Chlorella mutants. Arch Biochem Biophys. 1968 Apr;125(1):269–277. doi: 10.1016/0003-9861(68)90661-9. [DOI] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Ford C., Wang W. Three new yellow loci in Chlamydomonas reinhardtii. Mol Gen Genet. 1980;179(2):259–263. doi: 10.1007/BF00425452. [DOI] [PubMed] [Google Scholar]
  15. Fujita Y., Matsumoto H., Takahashi Y., Matsubara H. Identification of a nifDK-like gene (ORF467) involved in the biosynthesis of chlorophyll in the cyanobacterium Plectonema boryanum. Plant Cell Physiol. 1993 Mar;34(2):305–314. [PubMed] [Google Scholar]
  16. Fujita Y., Takahashi Y., Kohchi T., Ozeki H., Ohyama K., Matsubara H. Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha. Plant Mol Biol. 1989 Nov;13(5):551–561. doi: 10.1007/BF00027315. [DOI] [PubMed] [Google Scholar]
  17. Goldschmidt-Clermont M., Choquet Y., Girard-Bascou J., Michel F., Schirmer-Rahire M., Rochaix J. D. A small chloroplast RNA may be required for trans-splicing in Chlamydomonas reinhardtii. Cell. 1991 Apr 5;65(1):135–143. doi: 10.1016/0092-8674(91)90415-u. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Griffiths W. T. Protochlorophyll and protochlorophyllide as precursors for chlorophyll synthesis in vitro. FEBS Lett. 1974 Dec 15;49(2):196–200. doi: 10.1016/0014-5793(74)80510-7. [DOI] [PubMed] [Google Scholar]
  20. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  21. Huang C., Liu X. Q. Nucleotide sequence of the frxC, petB and trnL genes in the chloroplast genome of Chlamydomonas reinhardtii. Plant Mol Biol. 1992 Mar;18(5):985–988. doi: 10.1007/BF00019214. [DOI] [PubMed] [Google Scholar]
  22. Kohchi T., Shirai H., Fukuzawa H., Sano T., Komano T., Umesono K., Inokuchi H., Ozeki H., Ohyama K. Structure and organization of Marchantia polymorpha chloroplast genome. IV. Inverted repeat and small single copy regions. J Mol Biol. 1988 Sep 20;203(2):353–372. doi: 10.1016/0022-2836(88)90004-6. [DOI] [PubMed] [Google Scholar]
  23. Lidholm J., Gustafsson P. Homologues of the green algal gidA gene and the liverwort frxC gene are present on the chloroplast genomes of conifers. Plant Mol Biol. 1991 Oct;17(4):787–798. doi: 10.1007/BF00037061. [DOI] [PubMed] [Google Scholar]
  24. Quail P. H. Phytochrome: a light-activated molecular switch that regulates plant gene expression. Annu Rev Genet. 1991;25:389–409. doi: 10.1146/annurev.ge.25.120191.002133. [DOI] [PubMed] [Google Scholar]
  25. Reith M., Munholland J. A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea. Plant Cell. 1993 Apr;5(4):465–475. doi: 10.1105/tpc.5.4.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Richard M., Bellemare G. Nucleotide sequence of Chlamydomonas moewusii chloroplastic tRNA(thr). Nucleic Acids Res. 1990 May 25;18(10):3061–3061. doi: 10.1093/nar/18.10.3061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Sager R. Inheritance in the Green Alga Chlamydomonas Reinhardi. Genetics. 1955 Jul;40(4):476–489. doi: 10.1093/genetics/40.4.476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schulz R., Steinmüller K., Klaas M., Forreiter C., Rasmussen S., Hiller C., Apel K. Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression in Escherichia coli. Mol Gen Genet. 1989 Jun;217(2-3):355–361. doi: 10.1007/BF02464904. [DOI] [PubMed] [Google Scholar]
  30. Spano A. J., He Z., Timko M. P. NADPH: protochlorophyllide oxidoreductases in white pine (Pinus strobus) and loblolly pine (P. taeda). Evidence for light and developmental regulation of expression and conservation in gene organization and protein structure between angiosperms and gymnosperms. Mol Gen Genet. 1992 Dec;236(1):86–95. [PubMed] [Google Scholar]
  31. Stanier R. Y., Cohen-Bazire G. Phototrophic prokaryotes: the cyanobacteria. Annu Rev Microbiol. 1977;31:225–274. doi: 10.1146/annurev.mi.31.100177.001301. [DOI] [PubMed] [Google Scholar]
  32. Suzuki J. Y., Bauer C. E. Light-independent chlorophyll biosynthesis: involvement of the chloroplast gene chlL (frxC). Plant Cell. 1992 Aug;4(8):929–940. doi: 10.1105/tpc.4.8.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tsudzuki J., Nakashima K., Tsudzuki T., Hiratsuka J., Shibata M., Wakasugi T., Sugiura M. Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: nucleotide sequences of trnQ, trnK, psbA, trnI and trnH and the absence of rps16. Mol Gen Genet. 1992 Mar;232(2):206–214. doi: 10.1007/BF00279998. [DOI] [PubMed] [Google Scholar]
  34. Umesono K., Inokuchi H., Shiki Y., Takeuchi M., Chang Z., Fukuzawa H., Kohchi T., Shirai H., Ohyama K., Ozeki H. Structure and organization of Marchantia polymorpha chloroplast genome. II. Gene organization of the large single copy region from rps'12 to atpB. J Mol Biol. 1988 Sep 20;203(2):299–331. doi: 10.1016/0022-2836(88)90002-2. [DOI] [PubMed] [Google Scholar]
  35. Weeks D. P., Beerman N., Griffith O. M. A small-scale five-hour procedure for isolating multiple samples of CsCl-purified DNA: application to isolations from mammalian, insect, higher plant, algal, yeast, and bacterial sources. Anal Biochem. 1986 Feb 1;152(2):376–385. doi: 10.1016/0003-2697(86)90423-9. [DOI] [PubMed] [Google Scholar]
  36. Yang Z. M., Bauer C. E. Rhodobacter capsulatus genes involved in early steps of the bacteriochlorophyll biosynthetic pathway. J Bacteriol. 1990 Sep;172(9):5001–5010. doi: 10.1128/jb.172.9.5001-5010.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zsebo K. M., Hearst J. E. Genetic-physical mapping of a photosynthetic gene cluster from R. capsulata. Cell. 1984 Jul;37(3):937–947. doi: 10.1016/0092-8674(84)90428-8. [DOI] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES