Abstract
In order to identify markers of germination in Phaseolus vulgaris L., a cDNA–amplified fragment length polymorphism (AFLP) approach was conducted on mRNAs from embryo axes and from cotyledons. Among changes observed throughout the germination process, a cDNA fragment not detected 9 h after imbibition (HAI) but present specifically in axes 24 HAI was further studied. The complete cDNA was recovered by rapid amplification of cDNA ends, then cloned and sequenced. It includes an open reading frame predicting a 206-amino-acid polypeptide of 21.8 kDa. Analysis of the nucleotide sequence and deduced amino acid sequence revealed a high homology with germin-like proteins (GLPs), and particularly with an auxin-binding protein from peach, ABP19, that belongs to the GLP family. Thus, we propose that this cDNA encodes the first GLP described in P. vulgaris, designated PvGLP1. Northern blot analysis carried out on mRNAs from seed axes showed a dramatic increase in PvGLP1 expression a few hours before radicle emergence (17 HAI). Among mature vegetative tissues, PvGLP1 expression was very weak in pods and not detected in leaves, stems or roots. Immunoblot analysis using antibodies raised against AtGER3 from Arabidopsis thaliana showed that the protein could be detected only in axes from the dry seed stage onwards, at a steady-state level. Then, PvGLP1 expression seems to be associated with the early stages of embryo axis growth. The high homology indicated with ABP19 led us to study the effect of different concentrations of indole-3-acetic acid (IAA) on PvGLP1 expression during germination. Whereas no effect was noticed at low concentrations (1, 5, 10 μM), a marked decrease in PvGLP1 mRNA level was observed in axes of seeds imbibed with 100 μM IAA. Thus, PvGLP1 gene expression is not stimulated by auxin and, moreover, it might be inhibited by high concentrations of IAA.
Similar content being viewed by others
Abbreviations
- ABP:
-
auxin-binding protein
- AFLP:
-
amplified fragment length polymorphism
- GLP:
-
germin-like protein
- HAI:
-
hours after imbibition
- IAA:
-
indole-3-acetic acid
- TDF:
-
transcript-derived fragment
References
Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Bachem CW, van der Hoeven RS, de Bruijn SM, Vreugdenhil D, Zabeau M, Visser RG (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J 9:745–753
Baskin CC, Baskin JM (1998) Seeds—ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego
Bekman EP, Saibo NJ, Di Cataldo A, Regalado AP, Ricardo CP, Rodrigues-Pousada C (2000) Differential expression of four genes encoding 1-aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding. Planta 211: 663–672
Berna A, Bernier F (1997) Regulated expression of a wheat germin gene in tobacco: oxalate oxidase activity and apoplastic localization of the heterologous protein. Plant Mol Biol 33:417–429
Berna A, Bernier F (1999) Regulation by biotic and abiotic stress of a wheat germin gene encoding oxalate oxidase, a H2O2-producing enzyme. Plant Mol Biol 39:539–549
Bernier F, Berna A (2001) Germins and germin-like proteins: plant do-all proteins. But what do they do exactly? Plant Physiol Biochem 39:545–554
Bhalerao RP, Eklof J, Ljung K, Marchant A, Bennett M, Sandberg G (2002) Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings. Plant J 29:325–332
Bringezu K, Lichtenberger O, Leopold I, Neumann D (1999) Heavy metal tolerance of Silene vulgaris. J Plant Physiol 154:536–546
Caliskan M, Cuming AC (1998) Spatial specificity of H2O2-generating oxalate oxidase gene expression during wheat embryo germination. Plant J 15:165–171
Carter C, Thornburg RW (2000) Tobacco Nectarin Ⅰ. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defense of floral reproductive tissues. J Biol Chem 275:36726–36733
Carter C, Graham RA, Thornburg RW (1998) Arabidopsis thaliana contains a large family of germin-like proteins: characterization of cDNA and genomic sequences encoding 12 unique family members. Plant Mol Biol 38:929–943
Carter C, Graham RA, Thornburg RW (1999) Nectarin Ⅰ is a novel, soluble germin-like protein expressed in the nectar of Nicotiana sp. Plant Mol Biol 41:207–216
Chateigner AL, Le Deunff Y, Jalouzot R (1999) Germination-associated changes in the transcript content of pea seedlings lipoxigenases. Lipoxigenase-g: a new marker of axis growth resumption. Planta 208:606–613
Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA micropreparation: version Ⅱ. Plant Mol Biol Rep 1:19–21
Druka A, Kudrna D, Kannangara CG, von Wettstein D, Kleinhofs A (2002) Physical and genetic mapping of barley (Hordeum vulgare) germin-like cDNAs. Proc Natl Acad Sci USA 99:850–855
Dumas B, Sailland A, Cheviet JP, Freyssinet G, Pallett K (1993) Identification of barley oxalate oxidase as a germin-like protein. C R Acad Sci Life Sci 316:793–798
Dumas B, Freyssinet G, Pallett KE (1995) Tissue-specific expression of germin-like oxalate oxidase during development and fungal infection of barley seedlings. Plant Physiol 107:1091–1096
Epstein E, Cohen JD, Bandurski RS (1980) Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol 65:415–421
Fath A, Bethke PC, Jones RL (2001) Enzymes that scavenge reactive oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone. Plant Physiol 126:156–166
Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002
Gane PJ, Dunwell JM, Warwicker J (1998) Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase. J Mol Evol 46:488–493
Garciarrubio A, Legaria JP, Covarrubias AA (1997) Abscisic acid inhibits germination of mature Arabidopsis seeds by limiting the availability of energy and nutrients. Planta 203:182–187
Gómez-Cadenas A, Zentella R, Walker-Simmons MK, Ho THD (2001) Gibberellin/abscisic acid antagonism in barley aleurone cells: site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules. Plant Cell 13:667–679
Heintzen C, Fischer R, Melzer S, Kappeler S, Apel K, Staiger D (1994) Circadian oscillations of a transcript encoding a germin-like protein that is associated with cell walls in young leaves of the long-day plant Sinapis alba L. Plant Physiol 106:905–915
Huang Z, Fasco MJ, Kaminsky LS (1996) Optimization of DNaseⅠ removal of contaminating DNA from RNA for use in quantitative RNA–PCR. Biotechniques 20:1012–1020
Hurkman WJ, Tanaka CK (1996) Germin gene expression is induced in wheat leaves by powdery mildew infection. Plant Physiol 111:735–739
Inohara N, Shimomura S, Fukui T, Futai M (1989) Auxin-binding protein located in the endoplasmic reticulum of maize shoots: molecular cloning and complete primary structure. Proc Natl Acad Sci USA 86:3564–3568
Jones AM (1994) Auxin-binding proteins. Annu Rev Plant Physiol Plant Mol Biol 45:393–420
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lane BG, Dunwell JM, Ray JA, Schmitt MR, Cuming AC (1993) Germin, a protein marker of early plant development, is an oxalate oxidase. J Biol Chem 268:12239–12242
Limami M A, Rouillon C, Glevarec G, Gallais A, Hirel B (2002) Genetic and physiological analysis of germination efficiency in maize in relation to nitrogen metabolism reveals the importance of cytosolic glutamine synthetase. Plant Physiol 130:1860–1870
Ljung K, Ostin A, Lioussanne L, Sandberg G (2001) Developmental regulation of indole-3-acetic acid turnover in scots pine seedlings. Plant Physiol 125:464–75
Lomax TL, Muday GK, Rubery P (1995) Auxin transport. In: Davies PJ (ed) Plant hormones: physiology, biochemistry, and molecular biology, 2nd edn. Kluwer, Dordrecht, pp 509–530
Membré N, Berna A, Neutelings G, David A, David H, Staiger D, Vasquez JS, Raynal M, Delseny M, Bernier F (1997) cDNA sequence, genomic organization and differential expression of three Arabidopsis genes for germin/oxalate oxidase-like proteins. Plant Mol Biol 35:459–469
Membré N, Bernier F, Staiger D, Berna A (2000) Arabidopsis thaliana germin-like proteins: common and specific features point to a variety of functions. Planta 211:345–354
Métais I, Aubry C, Hamon B, Jalouzot R, Peltier D (2000) Description and analysis of genetic diversity between commercial bean lines (Phaseolus vulgaris L.), Theor Appl Genet 101:1207–1214
Michalczuk L, Ribnicky DM, Cooke TJ, Cohen JD (1992) Regulation of indol-3-acetic acid pathways in carrot cell cultures. Plant Physiol 100:1346–1353
Michalowski CB, Bohnert HJ (1992) Nucleotide sequence of a root-specific transcript encoding a germin-like protein from the halophyte Mesembryanthemum crystallinum. Plant Physiol 100:537–538
Neutelings G, Domon JM, Membré N, Bernier F, Meyer Y, David A, David H (1998) Characterization of a germin-like protein gene expressed in somatic and zygotic embryos of pine (Pinus caribaea Morelet). Plant Mol Biol 38:1179–1190
Nielsen H, Engelbrecht J, Brunak S, Von Heijne G (1997) Identification of prokaryotic and eucaryotic signal peptides and prediction of their cleavage sites. Prot Eng 10:1–6
Ohmiya A, Motoyuki K, Sakai S, Hayashi T (1993) Purification and properties of an auxin-binding protein from the shoot apex of peach tree. Plant Cell Physiol 34:177–183
Ohmiya A, Tanaka Y, Kadowaki KI, Hayashi T (1998) Cloning of genes encoding auxin-binding proteins (ABP19/20) from peach; significant peptide sequence similarity with germin-like proteins. Plant Cell Physiol 39:492–499
Ono M, Sage-Ono K, Inoue M, Kamada H, Harada H (1996) Transient increase in the level of mRNA for a germin-like protein in leaves of the short-day plant Pharbitis nil during the photoperiodic induction of flowering. Plant Cell Physiol 37:855–61
Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448
Poupard P, Strullu DG, Simoneau P (1998) Two members of the Bet v 1 gene family encoding birch pathogenesis-related proteins display different patterns of roots expression and wound-inducibility. Aust J Plant Physiol 25:459–464
Pritchard SL, Charlton WL, Baker A, Graham IA (2002) Germination and storage reserve mobilization are regulated independently in Arabidopsis. Plant J 31:639–647
Rodríguez-López M, Baroja-Fernández E, Zandueta-Criado A, Moreno-Bruna B, Muňoz FJ, Akazawa T, Pozueta-Romero J (2001) Two isoforms of a nucleotide-sugar pyrophosphatase/phosphodiesterase from barley leaves (Hordeum vulgare L.) are distinct oligomers of HvGLP1, a germin-like protein. FEBS Lett 490:44–48
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Schweizer P, Christoffel A, Dudler R (1999) Transient expression of members of the germin-like gene family in epidermal cells of wheat confers disease resistance. Plant J 20:541–552
Swart S, Logman TJJ, Smit G, Lugtenberg BJJ, Kijne JW (1994) Purification and partial characterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin, an attachment protein of Rhizobiaceae. Plant Mol Biol 24:171–183
Tamas IA (1987) Hormonal regulation of apical dominance. In: Davies PJ (ed) Plant hormones and their role in plant growth and development. Nijhoff, Boston, pp 393–410
Thompson EW, Lane BG (1980) Relation of protein synthesis in imbibing wheat embryos to the cell-free translational capacities of bulk mRNA from dry and imbibing embryos. J Biol Chem 255:5965–5970
Vallelian-Bindschedler L, Mösinger E, Métraux JP, Schweizer P (1998) Structure, expression and localization of a germin-like protein in barley (Hordeum vulgare L.) that is insolubilized in stressed leaves. Plant Mol Biol 37:297–308
Vassilevskaia TD, Bekman E, Jackson P, Pinto Ricardo C, Rodrigues-Pousada C (1996) Developmental expression and regulation by light of two closely related beta-tubulin genes in Lupinus albus. Plant Mol Biol 32:1185–1189
Verwoerd TC, Dekker BM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17:2362
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–4414
Wojtaszek P, Stobiecki M, Bolwell GP (1997) Changes in the composition of exocellular proteins of suspension-cultured Lupinus albus cells in response to fungal elicitors or CuCl2. J Exp Bot 48:2015–2021
Woo EJ, Dunwell JM, Goodenough PW, Pickersgill RW (1998) Barley oxalate oxidase is a hexameric protein related to seed storage proteins: evidence from X-ray crystallography. FEBS Lett 437: 87–90
Yamahara T, Shiono T, Suzuki T, Tanaka K, Takio S, Sato K, Yamazaki S, Satoh T (1999) Isolation of a germin-like protein with manganese superoxide dismutase activity from cells of a moss, Barbula unguiculata. J Biol Chem 274:33274–33278
Ziadi S, Poupard P, Brisset MN, Paulin JP, Simoneau P (2001) Characterization in apple leaves of two subclasses of PR-10 transcripts inducible by acibenzolar-S-methyl, a functional analogue of salicylic acid. Physiol Mol Plant Pathol 59:33–43
Acknowledgments
We thank Prof. F. Bernier (IBMP, Strasbourg, France) for providing anti-AtGER3 polyclonal antibodies and for critical comments on the manuscript. C. Aubry and M.-C. Morère-Le Paven contributed equally to this work.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Aubry, C., Morère-Le Paven, MC., Chateigner-Boutin, AL. et al. A gene encoding a germin-like protein, identified by a cDNA–AFLP approach, is specifically expressed during germination of Phaseolus vulgaris . Planta 217, 466–475 (2003). https://doi.org/10.1007/s00425-003-1004-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-003-1004-9