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Complexity and expression of the glutamine synthetase multigene family in the amphidiploid crop Brassica napus

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Abstract

In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families of the A and C genome types encoding a certain protein are now combined in one plant genome.

In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six different cDNA sequences were isolated from leaf and root specific libraries. One sequence pair (BnGSL1/BnGSL2) was characterized by the presence of amino- terminal transit peptides, a typical feature of all nuclear encoded chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high homology between each other were found in a root specific cDNA library and represent protein subunits for cytosolic glutamine synthetase isoforms.

Comparative PCR amplifications of genomic DNA isolated from B. napus, B. campestris and B. oleracea followed by sequence–specific restriction analyses of the PCR products permitted the assignment of the cDNA sequences to either the A genome type (BnGSL1/BnGSR1- 1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously in oilseed rape. This result was also confirmed by RFLP (restriction fragment length polymorphism) analysis of RT-PCR products.

In addition, the different GS genes showed tissue specific expression patterns which are correlated with the state of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a marked increase of expression could be observed after the onset of leaf senescence.

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References

  1. Bernhard W, Matile P: Differential expression of glutamine synthetase genes during the senescence of Arabidopsis thaliana rosette leaves. Plant Sci 98: 7–14(1994).

    Google Scholar 

  2. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of proteins using the principle of protein-dye-binding. Anal Biochem 72: 248–258(1976).

    Article  PubMed  Google Scholar 

  3. Buchanan-Wollaston V: Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Plant Physiol 105: 839–846(1994).

    Article  PubMed  Google Scholar 

  4. Buchanan-Wollaston V: The molecular biology of leaf senescence. J Exp Bot 48: 181–199(1997). 199 (1997).

    Google Scholar 

  5. Buchanan-Wollaston V, Ainsworth C: Leaf senescence in Brassica napus: cloning of senescence related genes by subtractive hybridisation. Plant Mol Biol 33: 821–834(1997).

    PubMed  Google Scholar 

  6. Croy EJ, lkemura T, Shirsat A, Croy RRD: Plant nucleic acids. In: Croy RRD (ed) Plant Molecular Biology Labfax, pp. 21–48. BIOS Scientific Publishers, Oxford (1993).

    Google Scholar 

  7. Davis LG, Dibner MD, Battey JF: BasicMethods in Molecular Biology. Elsevier Science Publishers, New York (1986).

    Google Scholar 

  8. Feller U, Fischer A: Nitrogen metabolism in senescing leaves. Crit Rev Plant Sci 13: 241–273(1994).

    Google Scholar 

  9. Forde BG, Cullimore JV: The molecular biology of glutamine synthetase in higher plants. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, pp. 246–296. Oxford University Press, Oxford (1989).

    Google Scholar 

  10. Frommer WB, Kwart M, Hirner B, Fischer WN, Hummel S, Ninnemann O: Transporters for nitrogenous compounds in plants. Plant Mol Biol 26: 165–670(1994).

    PubMed  Google Scholar 

  11. Green PD, Wong PP: Subunit analysis of glutamine synthetase isozymes from root nodules of tepary bean (Phaseolus acutifolius Gray). Plant Sci 82: 179–186(1992).

    Google Scholar 

  12. Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269(1983).

    Article  PubMed  Google Scholar 

  13. Higgins DG, Sharp PM: CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Gene 73: 237–244(1988).

    Article  PubMed  Google Scholar 

  14. Höpfner M, Ochs G, Wild A: Glutamine synthetase of green and etiolated leaves of Sinapis alba. Evidence of the identity of the respective enzyme proteins. Planta 181:155–161 (1990).

    Google Scholar 

  15. Huffaker RC: Proteolytic activity during senescence of plants. New Phytol 116: 199–231 (1990).

    PubMed  Google Scholar 

  16. Kamachi K, Yamaya T, Mae T, Ojima K: A role for glutamine synthetase in the remobilization of leaf nitrogen during natural senescence in rice leaves. Plant Physiol 96: 411–417(1991).

    Google Scholar 

  17. Kamachi K, Yamaya T, Hayakawa T, Mae T, Ojima K: Changes in cytosolic glutamine synthetase polypeptide and its mRNA in a leaf blade of rice plants during natural senescence. Plant Physiol 98: 1323–1329(1992).

    Google Scholar 

  18. Kawakami N, Watanabe A: Senescence-specific increase in cytosolic glutamine synthetase and its mRNA in radish cotyledons. Plant Physiol 88: 1430–1434(1988).

    Google Scholar 

  19. Kozaki A, Takeba G: Photorespiration protects C3 plants from photooxidation. Nature 384: 557–560(1996).

    Google Scholar 

  20. Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y: Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci USA 90: 3009–3013 (1993).

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  22. Lam HM, Coschigano KT, Oliveira IC, Melo-Oliveira R, Coruzzi GM: Molecular-genetic dissection of ammonium assimilation in Arabidopsis thaliana. Plant Physiol Biochem 35: 185–198(1997).

    Google Scholar 

  23. Lea PJ: Nitrogen metabolism. In: Lea PJ, Leegood RC (eds) Plant Biochemistry and Molecular Biology, pp. 155–180. John Wiley, New York (1993).

    Google Scholar 

  24. Lea PJ, Miflin BJ: Transport and metabolism of asparagine and other nitrogen compounds within the plant. In: Miflin BJ (ed) The Biochemistry of Plants Amino Acids and Derivatives, pp. 569–607. Academic Press, New York (1980).

    Google Scholar 

  25. Li M, Villemur R, Hussey PJ, Silflow CD, Gantt JS, Snustad DP: Differential expression of six glutamine synthetase genes in Zea mays. Plant Mol Biol 23: 401–407 (1993).

    PubMed  Google Scholar 

  26. Murray MG, Thompson WF: Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 8: 4321–4325 (1980).

    PubMed  Google Scholar 

  27. Ochs G, Schock G, Wild A: Chloroplastic glutamine synthetase from Brassica napus. Plant Physiol 103: 303–304 (1993).

    PubMed  Google Scholar 

  28. Ochs G, Schock G, Wild A: Purification and characterization of glutamine synthetase isoenzymes from leaves and roots of Brassica napus (L.). J Plant Physiol 147: 1–8(1995).

    Google Scholar 

  29. Oliveira IC, Lam HM, Coschigano K, Melo-Oliveira R, Coruzzi GM: Molecular-genetic dissection of ammonium assimilation in Arabidopsis thaliana. Plant Physiol Biochem 35: 185–198(1997).

    Google Scholar 

  30. Peoples MB, Gifford RM: Long-distance transport of carbon and nitrogen from sources to sinks in higher plants. In: Dennis DT, Turpin DH (eds) Plant Physiology, Biochemistry and Molecular Biology, pp. 434–447. John Wiley, New York (1993).

    Google Scholar 

  31. Peterman TK, Goodman HM: The glutamine synthetase gene family of Arabidopsis thaliana: light regulation and differential expression in leaves, roots and seeds. Mol Gen Genet 230: 145–154(1991).

    Google Scholar 

  32. Roulin S, Feller U: Light-induced proteolysis of stromal proteins in pea (Pisum sativum L.) chloroplasts: requirement for intact organelles. Plant Sci 128: 31–41(1997).

    Google Scholar 

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

    PubMed  Google Scholar 

  34. Schenck HR, Röbbelen G: Somatic hybrids by fusion of protoplasts from Brassica oleracea and B. campestris. Z Pflanzenzücht 89: 278–288(1982).

    Google Scholar 

  35. Schock G, Ochs G, Wild A: Glutamine synthetase from roots of Brassica napus. Plant Physiol 105: 757–758(1994).

    PubMed  Google Scholar 

  36. Shen B, Davis LC: Nodulation and nodulin gene expression in an interspecific hybrid between Glycine max and Glycine tomentella. Aust J Plant Physiol 19: 693–707(1992).

    Google Scholar 

  37. Smart CM: Gene expression during leaf senescence. New Phytol 126: 419–448(1994).

    Google Scholar 

  38. Tingey SV, Walker EL, Coruzzi GM: Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules. EMBO J 6: 1–9 (1987).

    PubMed  Google Scholar 

  39. Tingey SV, Tsai FJ, Edwards JW, Walker EL, Coruzzi GM: Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressed in vivo. J Biol Chem 263: 9651–9657(1988).

    PubMed  Google Scholar 

  40. Tsuprun VL, Boekema EJ, Pushkin AV, Tagunova IV: Electron microscopy and image analysis of the GroEL-like protein and its complexes with glutamine synthetase from pea leaves. Biochim Biophys Acta 1099: 67–73 (1992).

    PubMed  Google Scholar 

  41. Vierstra RD: Protein degradation in plants. Annu Rev Plant Physiol Plant Mol Biol 44: 385–410(1993).

    Article  Google Scholar 

  42. Wallsgrove RM, Turner JG, Hall NP, Kendall AC, Bright SWJ: Barley mutants lacking chloroplast glutamine synthetasebiochemical and genetic analysis. Plant Physiol 83: 155–158 (1987).

    Google Scholar 

  43. Watanabe A, Hamada K, Yokoi H, Watanabe A: Biphasic and differential expression of cytosolic glutamine synthetase genes of radish during seed germination and senescence of cotyledons. Plant Mol Biol 26: 1807–1817(1994).

    PubMed  Google Scholar 

  44. Weising K, Beyermann B, Ramser J, Kahl G: Plant DNA fingerprinting with radioactive and digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences. Electrophoresis 12: 155–169(1991).

    Google Scholar 

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Authors and Affiliations

  1. Institut für Allgemeine Botanik, Johannes Gutenberg-, Universität Mainz, 55099 , Mainz, Germany

    Günther Ochs, Gerald Schock, Martin Trischler, Kirstin Kosemund & Aloysius Wild

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  1. Günther Ochs
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  2. Gerald Schock
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  3. Martin Trischler
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  4. Kirstin Kosemund
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  5. Aloysius Wild
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Ochs, G., Schock, G., Trischler, M. et al. Complexity and expression of the glutamine synthetase multigene family in the amphidiploid crop Brassica napus. Plant Mol Biol 39, 395–405 (1999). https://doi.org/10.1023/A:1006193717093

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