Plant Molecular Biology

, Volume 61, Issue 4–5, pp 615–627 | Cite as

Germin-like genes are expressed during somatic embryogenesis and early development of conifers

  • M. Mathieu
  • M. A. Lelu-Walter
  • A. S. Blervacq
  • H. David
  • S. Hawkins
  • G. NeutelingsEmail author


Germins and germin-like proteins (GLPs) are members of a superfamily of proteins widely distributed in plants. Their localization within the extracellular matrix and in some cases their hydrogen peroxide-producing activity suggests that these proteins are involved in cell wall metabolism during stress responses and developmental processes. Several very highly conserved conifer GLPs have been identified in somatic embryo tissues. In order to gain more knowledge on their potential involvement in the development of this particular tissue, we have characterized a new GLP gene, LmGER1 in hybrid larch. Anti-GLP immunserum and in-gel activity analyses suggested the presence of superoxide dismutase activity in apoplastic proteins from larch somatic embryos. These results could indicate a possible role for LmGER1 in this physiological process. The expression of LmGER1 has been followed during the maturation of somatic embryos and in different organs of young plantlets by homologous transformation with a promoter-gus construct. This promoter was activated in the root cap of young embryos and, later on, in the cotyledons and in the vascular procambium and xylem. Furthermore, the importance of this gene in embryo development was evaluated by transforming embryonal masses with a gene construct encoding a hairpin RNA leading to gene silencing. The potential role of LmGER1 in cross-linking of cell wall components is discussed.


Cell wall Conifers Cross-linking Germin-like proteins Somatic embryogenesis Superoxide dismutase 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Beauchamp, C, Fridovich, I 1971Superoxide dismutase: improved assays and an assay applicable to acrylamide gelsAnal Biochem44276287PubMedCrossRefGoogle Scholar
  2. Becwar, S, Nagmani, R, Wann, R 1990Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda)Can J For Res20810817Google Scholar
  3. Bernier, F, Berna, A 2001Germins and germin-like proteins: plant do-all proteins. But what do they do exactly?Plant Physiol Biochem39545554CrossRefGoogle Scholar
  4. Bishop-Hurley, SL, Gardner, RC, Walter, C 2003Isolation and molecular characterization of genes expressed during somatic embryo development in pinus radiataPlant Cell Tissue and Organ Culture74267281CrossRefGoogle Scholar
  5. Caliskan, M, Cuming, AC 1998Spatial specificity of H2O2-generating oxalate oxidase gene expression during wheat embryo germinationPlant J15165171PubMedCrossRefGoogle Scholar
  6. Caliskan, M, Turet, M, Cuming, AC 2004Formation of wheat (Triticum aestivum L.) embryogenic callus involves peroxide-generating germin-like oxalate oxidasePlanta219132140PubMedCrossRefGoogle Scholar
  7. Campbell, MA, Lara, MH, Jennifer, C 1998Plant Physiol. The characterization of a gene encoding a putative germin-like protein from Solamun tuberosum Plant Physiol118711CrossRefGoogle Scholar
  8. Carter, C, Graham, RA, Thornburg, RW 1999Nectarin I is a novel, soluble germin-like protein expressed in the nectar of Nicotiana spPlant Mol Biol41207216PubMedCrossRefGoogle Scholar
  9. Carter, C, Thornburg, RW 2000Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defensE of floral reproductive tissuesJ Biol Chem2753672636733PubMedCrossRefGoogle Scholar
  10. Christensen, A, Thordal-Christensen, H, Zimmermann, G, Gjetting, T, Lyngkjær, M, Dudler, R, Schweizer, P 2004The germinlike protein GLP4 exhibits superoxide dismutase activity and is an important component of quantitative resistance in wheat and barleyMol Plant–Microbe Interact17109117PubMedGoogle Scholar
  11. Domon, JM, Dumas, B, Laine, E, Meyer, Y, David, A, David, H 1995Three glycosylated polypeptides secreted by several embryogenic cell cultures of pine show highly specific serological affinity to antibodies directed against the wheat germin apoprotein monomerPlant Physiol108141148PubMedCrossRefGoogle Scholar
  12. Dumas, B, Freyssinet, G, Pallett, KE 1995Tissue-specific expression of germin-like oxalate oxidase during development and fungal infection of barley seedlingsPlant Physiol10710911096PubMedGoogle Scholar
  13. Dumas, B, Sailland, A, Cheviet, JP, Freyssinet, G, Pallett, K 1993Identification of barley oxalate oxidase as a germin-like proteinC R Acad Sci III316793798PubMedGoogle Scholar
  14. Dunwell, JM 1998Cupins: a new superfamily of functionally diverse proteins that include germins and plant storage proteinsBiotechnol Genet Eng Rev15132PubMedGoogle Scholar
  15. Dunwell, JM, Khuri, S, Gane, PJ 2000Microbial relatives of the seed storage proteins of higher plants: conservation of structure and diversification of function during evolution of the cupin superfamilyMicrobiol Mol Biol Rev64153179PubMedCrossRefGoogle Scholar
  16. Dunwell, JM, Purvis, A, Khuri, S 2004Cupins: the most functionally diverse protein superfamily?Phytochemistry65717PubMedCrossRefGoogle Scholar
  17. Dyrløv Bendtsen, J, Nielsen, H, Heijne, G, Brunak, S 2004Improved prediction of signal peptides: signalP 3.0J Mol Biol340783795CrossRefGoogle Scholar
  18. Egertsdotter, U, Arnold, S 1995Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea abies)Physiol Plant93334345CrossRefGoogle Scholar
  19. Fan, Z, Gu, H, Chen, X, Song, H, Wang, Q, Liu, M, Qu, LJ, Chen, Z 2005Cloning and expression analysis of Zmglp1, a new germin-like protein gene in maizeBiochem Biophys Res Commun33112571263PubMedCrossRefGoogle Scholar
  20. Filonova, LH, Bozhkov, PV, Brukhin, VB, Daniel, G, Zhivotovsky, B, Arnold, S 2000Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruceJ Cell Sci11343994411PubMedGoogle Scholar
  21. Fry, SC 1998Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicalsBiochem J332507515PubMedGoogle Scholar
  22. Fry, SC, Miller, J, Dumville, J 2002A proposed role for copper ions in cell wall looseningPlant Soil2475767CrossRefGoogle Scholar
  23. Gleeson, D, Lelu-Walter, MA, Parkinson, M 2005Overproduction of proline in transgenic hybrid larch (Larix×leptoeuropaea (Dengler)) cultures renders them tolerant to cold, salt and frostMol Breed152129CrossRefGoogle Scholar
  24. Helliwell, C, Waterhouse, P 2003Constructs and methods for high-throughput gene silencing in plantsMethods30289295PubMedCrossRefGoogle Scholar
  25. Jaikaran, AS, Kennedy, TD, Dratewka-Kos, E, Lane, BG 1990Covalently bonded and adventitious glycans in germinJ Biol Chem2651250312512PubMedGoogle Scholar
  26. Jefferson, RA, Kavanagh, TA, Bevan, MW 1987GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plantsEmbo J639013907PubMedGoogle Scholar
  27. Jones, L, Milne, JL, Ashford, D, McCann, MC, McQueen-Mason, SJ 2005A conserved functional role of pectic polymers in stomatal guard cells from a range of plant speciesPlanta221255264PubMedCrossRefGoogle Scholar
  28. Khuri, S, Bakker, FT, Dunwell, JM 2001Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteinsMol Biol Evol18593605PubMedGoogle Scholar
  29. Kim, HJ, Pesacreta, TC, Triplett, BA 2004Cotton-fiber germin-like protein. II: immunolocalization, purification, and functional analysisPlanta218525535PubMedCrossRefGoogle Scholar
  30. Kim, HJ, Triplett, BA 2004Cotton fiber germin-like protein. I. Molecular cloning and gene expressionPlanta218516524PubMedCrossRefGoogle Scholar
  31. Lane, BG 2002Oxalate, germins, and higher-plant pathogensIUBMB Life536775PubMedCrossRefGoogle Scholar
  32. Lelu, MA, Label, P 1994Changes in the levels of abscisic acid and its glucose ester conjugate during maturation of hybrid larch (Larix×leptoeuropaea) somatic embryos, in relation to germination and plantlet recoveryPhysiol Plant925360CrossRefGoogle Scholar
  33. Lelu, MA, Pilate, G 2000Transgenics in LarixJain, MMinocha, SC eds. Molecular biology of woody plantsKluwer Academic PublishersNetherlands119134Google Scholar
  34. Lelu, MA, Klimaszewska, K, Charest, P 1994Somatic embryogenesis from immature and mature zygotic embryos and from cotyledons and needles of somatic plantlets of LarixCan J For Res24100106Google Scholar
  35. Lo Schiavo, F, Giuliano, G, Vries, S, Genga, A, Bollini, R, Pitto, L, Cozzani, F, Nuti-Ronchi, V, Terzi, M 1990A carrot cell variant temperature sensitive for somatic embryogenesis reveals a defect in the glycosylation of extracellular proteinsMol Gen Genet223385393PubMedCrossRefGoogle Scholar
  36. Mathieu, M, Neutelings, G, Hawkins, S, Grenier, E, David, H 2003Cloning of a pine germin-like protein (GLP) gene promoter and analysis of its activity in transgenic tobacco bright yellow 2 cellsPhysiol Plant117425434PubMedCrossRefGoogle Scholar
  37. Membré, N, Berna, A, Neutelings, G, David, A, David, H, Staiger, D, Saez Vasquez, J, Raynal, M, Delseny, M, Bernier, F 1997cDNA sequence, genomic organization and differential expression of three Arabidopsis genes for germin/oxalate oxidase-like proteinsPlant Mol Biol35459469PubMedCrossRefGoogle Scholar
  38. Moller, SG, McPherson, MJ 1998Developmental expression and biochemical analysis of the Arabidopsis atao1 gene encoding an H2O2-generating diamine oxidasePlant J13781791PubMedCrossRefGoogle Scholar
  39. Neutelings, G, Domon, JM, Membre, N, Bernier, F, Meyer, Y, David, A, David, H 1998Characterization of a germin-like protein gene expressed in somatic and zygotic embryos of pine (Pinus caribaea Morelet)Plant Mol Biol3811791190PubMedCrossRefGoogle Scholar
  40. Ohmiya, A, Tanaka, Y, Kadowaki, K, Hayashi, T 1998Cloning of genes encoding auxin-binding proteins (ABP19/20) from peach: significant peptide sequence similarity with germin-like proteinsPlant Cell Physiol39492499PubMedGoogle Scholar
  41. Ros Barcelo, A 1998The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzymePlanta207207216CrossRefGoogle Scholar
  42. Sambrook, J, Fritsch, E, Maniatis, T 1989Molecular cloning: a laboratory manual2Cold Spring Harbor Laboratory PressCold Spring Harbor, NYGoogle Scholar
  43. Sanchez, M, Pena, MJ, Revilla, G, Zarra, I 1996Changes in dehydrodiferulic acids and peroxidase activity against ferulic acid associated with cell walls during growth of Pinus pinaster hypocotylPlant Physiol111941946PubMedGoogle Scholar
  44. Segarra, CI, Casalongue, CA, Pinedo, ML, Ronchi, VP, Conde, RD 2003A germin-like protein of wheat leaf apoplast inhibits serine proteasesJ Exp Bot5413351341PubMedCrossRefGoogle Scholar
  45. Tabuchi, T, Kumon, T, Azuma, T, Nanmori, T, Yasuda, T 2003The expression of a germin-like protein with superoxide dismutase activity in the halophyte Atriplex lentiformis is differentially regulated by wounding and abscisic acidPhysiol Plant118523531CrossRefGoogle Scholar
  46. Thompson, EW, Lane, BG 1980Relation of protein synthesis in imbibing wheat embryos to the cell-free translational capacities of bulk mRNA from dry and imbibing embryosJ Biol Chem25559655970PubMedGoogle Scholar
  47. Vallelian-Bindschedler, L, Mosinger, E, Metraux, JP, Schweizer, P 1998Structure, expression and localization of a germin-like protein in barley (Hordeum vulgare L.) that is insolubilized in stressed leavesPlant Mol Biol37297308PubMedCrossRefGoogle Scholar
  48. Aderkas, P, Label, P, Lelu, MA 2002aCharcoal affects early development and hormonal concentrations of somatic embryos of hybrid larchTree Physiol22431434Google Scholar
  49. Aderkas, P, Rhor, R, Sunberg, B, Gutmann, M, Dumont-Beboux, N, Lelu, MA 2002bABA and its influence on development of the embryonal root cap, storage product and secondary metabolite accumulation in hybrid larch somatic embryosPlant Cell Tissue Org Cult69111120CrossRefGoogle Scholar
  50. Wagner, A, Phillips, L, Narayan, RD, Moody, JM, Geddes, B 2005Gene silencing studies in the gymnosperm species Pinus radiata Plant Cell Rep2495102PubMedCrossRefGoogle Scholar
  51. Wesley, SV, Helliwell, CA, Smith, NA, Wang, MB, Rouse, DT, Liu, Q, Gooding, PS, Singh, SP, Abbott, D, Stoutjesdijk, PA, Robinson, SP, Gleave, AP, Green, AG, Waterhouse, PM 2001Construct design for efficient, effective and high-throughput gene silencing in plantsPlant J27581590PubMedCrossRefGoogle Scholar
  52. Whitmore, F 1976Binding of ferulic acid to cell walls by peroxidases of Pinus elliottii Phytochemistry15375378CrossRefGoogle Scholar
  53. Woo, EJ, Dunwell, JM, Goodenough, PW, Marvier, AC, Pickersgill, RW 2000Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activitiesNat Struct Biol710361040PubMedCrossRefGoogle Scholar
  54. Yamahara, T, Shiono, T, Suzuki, T, Tanaka, K, Takio, S, Sato, K, Yamazaki, S, Satoh, T 1999Isolation of a germin-like protein with manganese superoxide dismutase activity from cells of a moss, Barbula unguiculata J Biol Chem2743327433278PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • M. Mathieu
    • 1
  • M. A. Lelu-Walter
    • 2
  • A. S. Blervacq
    • 3
  • H. David
    • 1
  • S. Hawkins
    • 1
  • G. Neutelings
    • 1
  1. 1.Laboratoire de Physiologie des Parois Végétales UPRES EA3568-USC INRAUniversité des Sciences et Technologies de LilleVilleneuve d’Ascq CedexFrance
  2. 2.Unité Amélioration, Génétique et Physiologie ForestièresOlivet CedexFrance
  3. 3.Laboratoire de Physiologie de la Différenciation Végétale UPRES EA3567Université des Sciences et Technologies de LilleVilleneuve d’Ascq CedexFrance

Personalised recommendations