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Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression inEscherichia coli

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Summary

The primary structure of the NADPH-protochlorophyllide oxidoreductase of barley has been deduced from the nucleotide sequence of a cloned full-length cDNA. This cDNA hybridizes to a 1.7 kb RNA whose steady-state level in dark-grown seedlings is drastically reduced upon illumination. The predicted amino acid sequence (388 residues in length) includes a transit peptide of 74 amino acids whose end point has been delimited by sequencing the N-terminus of the mature protein. Expression of the cDNA inEscherichia coli leads to the synthesis of an enzymatically active precursor of the NADPH-protochlorophyllide oxidoreductase. Activity of this protein in bacterial lysates is completely dependent on the presence of NADPH and protochlorophyllide and requires light.

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References

  • Apel K (1981) The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Phytochrome-induced decrease of translatable mRNA coding for the NADPH-protochlorophyllide oxidoreductase. Eur J Biochem 120:89–93

    Article  PubMed  CAS  Google Scholar 

  • Apel K, Kloppstech K (1978) The plastid membranes of barley (Hordeum vulgare). Light-induced appearance of mRNA coding for the apoprotein of the light-harvesting chlorophyll a/b protein. Eur J Biochem 85:581–588

    Article  PubMed  CAS  Google Scholar 

  • Apel K, Santel H-J, Redlinger TE, Falk H (1980) The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Isolation and characterization of the NADPH-protochlorophyllide oxidoreductase. Eur J Biochem 111:251–258

    Article  PubMed  CAS  Google Scholar 

  • Batschauer A, Mösinger E, Kreuz K, Dörr I, Apel K (1986) The implication of a plastid-derived factor in the transcriptional control of nuclear genes encoding the light-harvesting chlorophyll a/b protein. Eur J Biochem 154:625–634

    Article  PubMed  CAS  Google Scholar 

  • Birnboim H, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    PubMed  CAS  Google Scholar 

  • Boardman NK, Anderson JM, Goodchild DJ (1978) Chlorophyll-protein complexes and structure of mature and developing chloroplasts. Curr Top Bioenerget 8:35–109

    CAS  Google Scholar 

  • Bogorad L (1976) Chlorophyll biosynthesis. In: Goodwin TW (ed) Chemistry and biochemistry of plant pigments, 2nd edn, vol 1. Academic Press, New York, London, pp 64–148

    Google Scholar 

  • Bohlmann H, Apel K (1987) Isolation and characterization of cDNAs coding for leaf-specific thionins closely related to the endosperm-specific hordothionin of barley (Hordeum vulgare L.). Mol Gen Genet 207:446–454

    Article  CAS  Google Scholar 

  • Bonner WM, Laskey RA (1974) A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83–88

    Article  PubMed  CAS  Google Scholar 

  • Castelfranco PA, Beale ST (1983) Chlorophyll biosynthesis: recent advances and areas of current interest. Annu Rev Plant Physiol 34:241–278

    Article  CAS  Google Scholar 

  • Chang J-Y (1983) Manual micro-sequence analysis of polypeptides using dimethylaminoazobenzene isothiocyanate. Methods Enzymol 91:455–466

    PubMed  CAS  Google Scholar 

  • Chang J-Y, Brauer D, Wittmann-Liebold B (1978) Micro-sequence analysis of peptides and proteins using 4-NN-dimethylamino-benzene 4′-isothiocyanate/phenylisothiocyanate double coupling method. FEBS Lett 93:205–214

    Article  CAS  Google Scholar 

  • Dehesh K, Ryberg M (1985) The NADPH-protochlorophyllide oxidoreductase is the major protein constituent of prolamellar bodies in wheat (Triticum aestivum L.) Planta 164:396–399

    Article  CAS  Google Scholar 

  • Dehesh K, Klaas M, Häuser I, Apel K (1986) Light-induced changes in the distribution of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase within different cellular compartments of barley (Hordeum vulgare L.). I. Localization by immunoblotting in isolated plastids and total leaf extracts. Planta 169:162–171

    Article  CAS  Google Scholar 

  • Fairbanks G, Steck TL, Wallach DFH (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617

    Article  PubMed  CAS  Google Scholar 

  • Gollmer I, Apel K (1983) The phytochrome-controlled accumulation of mRNA sequences encoding the light-harvesting chlorophyll a/b protein of barley (Hordeum vulgare L.). Eur J Biochem 133:309–313

    Article  PubMed  CAS  Google Scholar 

  • Griffiths WT (1974) Protochlorophyll and protochlorophyllide as precursors for chlorophyll synthesis in vitro. FEBS Lett 49:196–200

    Article  PubMed  CAS  Google Scholar 

  • Griffiths WT (1978) Reconstitution of chlorophyllide formation by isolated etioplast membranes. Biochem J 174:681–692

    PubMed  CAS  Google Scholar 

  • Häuser I, Dehesh K, Apel K (1984) The protoeolytic degradation in vitro of the NADPH-protochlorophyllide oxidoreductase of barley (Hordeum vulgare L.) Arch Biochem Biophys 228:577–586

    Article  PubMed  Google Scholar 

  • Heidecker G, Messing J (1986) Structural analysis of plant genes. Annu Rev Plant Physiol 37:439–466

    Article  CAS  Google Scholar 

  • Huynh T, Young RA, Davis RW (1985) In: Glover DM (ed) DNA cloning. A practical approach. Practical approach series, IRL Press, Oxford, pp 49–78

    Google Scholar 

  • Ikeuchi M, Murakami S (1982) Behavior of the 36000 dalton protein in the internal membranes of squash etioplasts during greening. Plant Cell Physiol 23:575–583

    CAS  Google Scholar 

  • Ikeuchi M, Murakami S (1983) Separation and characterization of prolamellar bodies and prothylakoids from squash etioplast. Plant Cell Physiol 24:71–80

    CAS  Google Scholar 

  • Kay SA, Griffiths WT (1983) Light-induced breakdown of NADPH-protochlorophyllide oxidoreductase in vitro. Plant Physiol 72:229–236

    PubMed  CAS  Google Scholar 

  • Kreuz K, Dehesh K, Apel K (1986) The light-dependent accumulation of the P700 chlorophylla protein of the photosystem I reaction center in barley. Evidence for translational control. Eur J Biochem 159:459–467

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Lindstein A, Ryberg M, Sundqvist C (1988) The polypeptide composition of highly purified prolamellar bodies and prothylakoids from wheat (Triticum aestivum) as revealed by silver staining. Physiol Plantarum 72:167–176

    Article  Google Scholar 

  • Lütcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA (1987) Selection of AUG initiation codons differs in plants and animals. EMBO J 6:43–48

    PubMed  Google Scholar 

  • Mapleston ER, Griffiths WT (1980) Light modulation of the activity of protochlorophyllide reductase. Biochem J 189:125–133

    PubMed  CAS  Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78

    Article  PubMed  CAS  Google Scholar 

  • Meyer G, Bliedung H, Kloppstech K (1983) NADPH-protochlorophyllide oxidoreductase: reciprocal regulation in mono- and dicotyledonean plants. Plant Cell Reports 2:26–29

    CAS  Google Scholar 

  • Mösinger E, Batschauer A, Schäfer E, Apel K (1985) Phytochrome control of in vitro transcription of specific genes in isolated nuclei from barley (Hordeum vulgare). Eur J Biochem 147:137–142

    Article  PubMed  Google Scholar 

  • Oliver RP, Griffiths WT (1981) Covalent labelling of the NADPH-protochlorophyllide oxidoreductase from etioplast membranes with (3H)N-phenylmaleimide. Biochem J 195:93–101

    PubMed  CAS  Google Scholar 

  • Pelham HRB, Jackson RJ (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem 67:247–256

    Article  PubMed  CAS  Google Scholar 

  • Poulsen C (1983) The barley chloroplast genome: Physical structure and transcriptional activity in vivo. Carlsberg Res Commun 48:57–80

    Article  CAS  Google Scholar 

  • Queen C, Korn LJ (1984) A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res 12:581–599

    PubMed  CAS  Google Scholar 

  • Röper U, Prinz H, Lütz C (1987) Amino acid composition of the enzyme NADPH-protochlorophyllide oxidoreductase. Plant Sci 52:15–19

    Article  Google Scholar 

  • Ryberg M, Dehesh K (1986) Localization of NADPH-protochlorophyllide oxidoreductase in dark-grown wheat (Triticum aestivum) by immuno-electron microscopy before and after transformation of the prolamellar bodies. Physiol Plant 66:616–624

    Article  CAS  Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Santel HJ, Apel K (1981) The protochlorophyllide holochrome of barley (Hordeum vulgare L.). The effect of light on the NADPH-protochlorophyllide oxidoreductase. Eur J Biochem 120:95–103

    Article  PubMed  CAS  Google Scholar 

  • Towbin M, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    Article  PubMed  CAS  Google Scholar 

  • von Heijne G (1985) Signal sequences. The limits of variation. J Mol Biol 184:99–105

    Article  Google Scholar 

  • Wickens MP, Buell GN, Schimke RT (1978) Synthesis of double-stranded cDNA complementary to lysozyme, ovomucoid and ovalbumin mRNAs. J Biol Chem 253:2483–2495

    PubMed  CAS  Google Scholar 

  • Young RA, Davis RW (1983) Efficient isolation of genes using antibody probes. Proc Natl Acad Sci USA 80:1194–1198

    Article  PubMed  CAS  Google Scholar 

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Communicated by R.G. Herrmannn

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Schulz, R., Steinmüller, K., Klaas, M. et al. Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression inEscherichia coli . Molec. Gen. Genet. 217, 355–361 (1989). https://doi.org/10.1007/BF02464904

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  • DOI: https://doi.org/10.1007/BF02464904

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