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Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.

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Summary

Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.

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Abbreviations

BSA:

bovine serum albumin

EM:

electron microscope

GOGAT:

glutamate synthase

GS:

glutamine synthetase

GUS:

β-glucuronidase

IgG:

immunoglobulin

PBS:

phosphate buffer saline

SDS-PAGE:

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

References

  • Botella JR, Canovas FM, Avila C, Nuñez de Castro I, Valpuesta V (1986) Immunochemical comparison of glutamine synthetases from some Solanaceae plants. Plants Physiol 82: 585–587

    Google Scholar 

  • —, Verbelen JP, Valpuesta V (1988) Immunocytolocalization of glutamine synthetase in green leaves and cotyledons ofLycopersicon esculentum. Plant Physiol 88: 943–946

    Google Scholar 

  • Brangeon J, Hirel B, Forchioni A (1989) Immunogold localization of glutamine synthetase in soybean leaves, roots and nodules. Protoplasma 151: 88–97

    Google Scholar 

  • Cassab GI, Varner JE (1987) Immunocytolocalization of extensin in developing soybean seedcoat by immunogold-silver staining and by tissue printing on nitrocellulose paper. J Cell Biol 105: 2581–2588

    Google Scholar 

  • Cullimore JV, Bennett MJ (1988) The molecular biology and biochemistry of plant glutamine synthetase from root nodules ofPhaseolus vulgaris L. and other legumes, J Plant Physiol 132: 387–393

    Google Scholar 

  • —, Miflin BJ (1984) Immunological studies on glutamine synthetase using antisera raised to the two plant forms of the enzyme fromPhaseolus root nodules. J Exp Bot 35: 581–587

    Google Scholar 

  • Edwards JW, Walker El, Coruzzi GM (1990) Cell-specific expression in transgenic plants reveals non-overlapping roles for chloroplast and cytosolic glutamine synthetase. Proc Natl Acad Sci USA 87: 3459–3463

    Google Scholar 

  • Esaguy N, Aguas AP, Silva MT (1989) High-resolution localization of lactoferrin in human neutrophils: labelling of secondary granules and cell heterogeneity. J Leukocyte Biol 46: 51–62

    Google Scholar 

  • Esau K (1977) Anatomy of seed plants. Wiley, New York

    Google Scholar 

  • Forde BG, Day HM, Turton JF, Wen-jun S, Cullimore JV, Oliver JE (1989) Two glutamine synthetase genes fromPhaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenicLotus corniculatus plants. Plant Cell 1: 391–401

    Google Scholar 

  • Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Herman EM (1988) Immunocytochemical localization of macromolecules with the electron microscope. Annu Rev Plant Physiol Plant Mol Biol 39: 139–155

    Google Scholar 

  • Hirel B, Gadal P (1980) Glutamine synthetase in rice. A comparative study of the enzymes from roots and leaves. Plant Physiol 66: 619–623

    Google Scholar 

  • —, Bouet C, King B, Layzell D, Jacobs F, Verma D (1987) Glutamine synthetase genes are regulated by ammonia provided externally or by symbiotic nitrogen fixation. EMBO J 6: 1167–1171

    Google Scholar 

  • —, Perrot-Rechenmann C, Suzuki A, Vidal J, Gadal P (1982) Glutamine synthetase in spinach leaves. Immunological studies and immunocytochemical localization. Plant Physiol 69: 983–987

    Google Scholar 

  • —, Weatherley C, Certin C, Bergounioux C, Gadal P (1984) Multiple subunit composition of chloroplastic glutamine synthetase ofNicotiana tabacum L. Plant Physiol 74: 448–450

    Google Scholar 

  • Hoagland DR, Arnon DI (1938) The water culture for growing plants without soil. Circ Calif Agricult Exp Stat 347: 461–462

    Google Scholar 

  • Howes C (1988) Subcellular localization of macromolecules by microscopy. In: Shaw CH (ed) Plant molecular biology, a practical approach. IRL Press, Oxford, pp 103–130

    Google Scholar 

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

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227: 680–685

    Google Scholar 

  • Lara M, Porta H, Padilla J, Folch J Sanchez F (1984) Heterogeneity of glutamine synthetase polypeptides inPhaseolus vulgaris L. Plant Physiol 76: 1019–1023

    Google Scholar 

  • Lea PJ, Miflin BJ (1980) Transport and metabolism of asparagin and other nitrogen compounds within the plant. In: Milfin BJ (ed) The biochemistry of plants, vol 5. Academic Press, San Diego, pp 569–607

    Google Scholar 

  • Lightfoot DA, Green NK, Cullimore JV (1988) The chloroplastlocated glutamine synthetase ofPhaseolus vulgaris L.: nucleotide sequence, expression in different organs and uptake into isolated chloroplasts Plant Mol Biol 11: 191–202

    Google Scholar 

  • Mann AF, Fentem PA, Stewart GR (1979) Identification of two forms of glutamine synthetase in barley (Hordeum vulgare). Biochem Biophys Res Commun 88: 515–521

    Google Scholar 

  • McNally SF, Hirel B, Gadal P, Mann AF, Stewart GR (1983) Glutamine synthetases of higher plants. Plant Physiol 72: 22–25

    Google Scholar 

  • Miflin BJ (1974) The location on nitrite reductase and other enzymes related to amino acid biosynthesis in the plastids of roots and leaves. Plant Physiol 54: 550–555

    Google Scholar 

  • —, Lea PJ (1977) Amino acid metabolism. Annu Rev Plant Physiol 28: 299–329

    Google Scholar 

  • — — (1980) Ammonia assimilation. In: Miflin BJ (ed) The biochemistry of plants, vol 5. Academic Press, San Diego, pp 169–202

    Google Scholar 

  • Reid PD, Campillo E, Lewis LN (1990) Anatomical changes and immunolocalization of cellulase during abscission as observed on nitrocellulose tissue prints. Plant Physiol 93: 160–165

    Google Scholar 

  • Thalouarn P, Rey L, Hirel B, Renaudin S, Fer A (1987) Activity and immunocytochemical localization of glutamine synthetase inLathraea clandestina L. Protoplasma 141: 95–100

    Google Scholar 

  • Tingey SV, Coruzzi GM (1987) Glutamine synthetase ofNicotiana plumbaginifolia. Plant Physiol 84: 366–373

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  • Towbin H, Staehlin 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

    Google Scholar 

  • Vezina LP, Langlois JR (1989) Tissue and cellular distribution of glutamine synthetase in roots of pea (Pisum sativum) seedlings. Plant Physiol 90: 1129–1133

    Google Scholar 

  • Walker EL, Coruzzi GM (1989) Developmentally regulated expression of the gene family for cytosolic glutamine synthetase inPisum sativum. Plant Physiol 91: 702–708

    Google Scholar 

  • Wallgrove RM, Turner JC, Hall NP, Kendall AC, Bright SWJ (1989) Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis. Plant Physiol 83: 155–158

    Google Scholar 

  • Ye Z-H, Varner JE (1991) Tissue-specific expression of cell-wall proteins in developing soybean tissues. Plant Cell 3: 23–37

    Google Scholar 

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Authors and Affiliations

  1. Centro de Citologia Experimental (INIC), Universidade do Porto, Rua Campo Alegre 823, P-4100, Porto, Portugal

    Susana Pereira, Helena Carvalho, C. Sunkel & R. Salema

  2. Instituto de Botânica, Universidade do Porto, Porto

    Susana Pereira & R. Salema

Authors
  1. Susana Pereira
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  2. Helena Carvalho
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  3. C. Sunkel
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  4. R. Salema
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Pereira, S., Carvalho, H., Sunkel, C. et al. Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.. Protoplasma 167, 66–73 (1992). https://doi.org/10.1007/BF01353582

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  • DOI: https://doi.org/10.1007/BF01353582

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