Abstract
Three different β-1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were obtained by RT-PCR from RNA isolated from Cichorium hybrid `474' leaf fragments cultured for 11 days under somatic embryogenesis-inducing conditions. When expressed in Escherichia coli the proteins encoded by the three cDNAs were recognized by antibodies raised against 38 kDa extracellular β-1,3-glucanases studied previously (Helleboid et al., Planta 205 (1998) 56–63). The CG2 and CG3 cDNAs may represent expressed alleles of one gene because their sequences showed a very high identity (98.5%) and are only 70% identical with CG1. Southern blot analysis revealed the presence of 3–4 genes coding for β-1,3-glucanases in the Cichorium genome. Expression analysis of the genes corresponding to the three clones analysed by semi-quantitative RT-PCR indicated that CG1 mRNAs were only detectable in Cichorium hybrid `474' leaf fragments from day 3 of somatic embryogenesis induction, whereas CG2-CG3 mRNAs were already present in non-induced leaf tissue of both the embryogenic hybrid `474' and a non-embryogenic genotype. The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embryos, and when the amount of callose deposited in cell walls surrounding embryogenic cells and young embryos decreased. These results indicate that expression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38 kDa β-1,3-glucanase protein that may be involved in the degradation of callose localized around embryogenic cells and young embryos. A full-length CG1 cDNA clone was obtained using 3′ and 5′ RACE-PCR, and its sequence revealed that it encodes a β-1,3-glucanase that is equally homologous to both class III and class IV plant β-1,3-glucanases.
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References
Bairoch, A., Bucher, P. and Hofmann, K. 1997. The PROSITE database: its status in 1997. Nucl. Acids Res. 25: 217–221.
Bellamy, A. 1992. Application du polymorphisme moléculaire de l'ADN à l'identification variétale de Cichorium intybus. Ph. D. dissertation, Université de Paris-Sud, Orsay, France.
Blervacq, A.S., Dubois, T., Dubois, J. and Vasseur, J. 1992. First divisions of somatic embryogenesis cells in Cichorium hybrid '474'. Protoplasma 186: 163–168.
Bucciaglia, P.A. and Smith, A.G. 1994. Cloning and characterization of Tag1, a tobacco anther β-1,3-glucanase expressed during tetrad dissolution. Plant Mol. Biol. 24: 903–914.
Casacuberta, J.M., Raventos, D., Puigdomè nech, P. and San Segundo, B. 1992. Expression of the gene encoding the PR-like protein PRms in germinating maize embryos. Mol. Gen. Genet. 234: 97–104.
Chirgwin, J.M., Przybyla, A.E., Macdonald, R.J. and Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry 19: 5294–5299.
Cote, F., Cutt, J.R., Asselin, A. and Klessig, D.F. 1991. Pathogenesis-related acidic β-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals. Mol. Plant-Microbe Interact. 4: 173–181.
Gheysen, G., Inzé, D., Soetaert, P., Van Montagu, M. and Castresana, C. 1990. Sequence of Nicotiana plumbaginifolia β-1,3-glucanase gene encoding a vacuolar isoform. Nucl. Acids Res. 18: 6685.
Gruner, R. and Pfitzner, U.M. 1994. The upstream region of the gene for the pathogenesis-related protein 1a from tobacco responds to environmental as well as to developmental signals in transgenic plants. Eur. J. Biochem. 220: 247–255.
Hanfrey, C., Fife, M. and Buchanan-Wollaston, V. 1996. Leaf senescence in Brassica napus: expression of genes encoding pathogenesis-related proteins. Plant Mol. Biol. 30: 597–609.
Helleboid, S. 1998. Caractérisation de protéines extracellulaires reliées aux phases précoces de l'embryogenè se somatique d'un Cichorium hybride. Analyses immunocytologique et moléculaire de l'implication de β-1,3-glucanases accumulées au cours du processus embryogè ne. Ph.D. dissertation, Université de Technologie de Compiè gne, Compiè gne, France.
Helleboid, S., Couillerot, J.P., Hilbert, J.-L. and Vasseur, J. 1995. Effects of α-difluoromethylarginine on embryogenesis, polyamine content and protein patterns in a Cichorium hybrid. Planta 196: 571–576.
Helleboid, S., Bauw, G., Belingheri, L., Vasseur, J. and Hilbert, J.-L. 1998. Extracellular β-1,3-glucanases are induced during early somatic embryogenesis in Cichorium. Planta 205: 56–63.
Keefe, D., Hinz, U. and Meins, F. 1990. The effect of ethylene on the cell-type-specific and intercellular localization of the β-1,3-glucanase and chitinase in tobacco leaves. Planta 182: 43–51.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Linthorst, H., Melchers, M., Mayer, A., van Roekel, J., Cornelissen, B. and Bol, J. 1990. Analysis of gene families encoding acidic and basic β-1,3-glucanases of tobacco. Proc. Natl. Acad. Sci. USA 87: 8756–8760.
MacGregor, A. and Ballance, G. 1991. Possible secondary structure in plant and yeast β-glucanases. Biochem. J. 274: 41–43.
Lotan, T., Ori, N. and Fluhr, R. 1989. Pathogenesis-related proteins are developmentally regulated in tobacco flowers. Plant Cell 1: 881–887.
Melchers, L.S., Sela-Buurlage, M.B., Vloemans, S.A., Woloshuk, C.P., van Roekel, J.S., Pen, J., van den Elzen, P.J. and Cornelissen, B.J. 1993. Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants. Plant Mol. Biol. 21: 583–593.
Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavages sites. Protein Eng. 10: 1–6.
Ori, N., Sessa, G., Lotan, T., Himmelhoch, S. and Fluhr, R. 1990. A major stylar matrix polypeptide (sp41) is a member of the pathogenesis-related proteins superclass. EMBO J. 9: 3429–3426.
Payne, G., Ward, E., Gaffney, T., Ahl Goy, P., Moyer, M., Harper, A., Meins, F.M. and Ryals, J. 1990. Evidence for a third structural class of β-1,3-glucanase in tobacco. Plant Mol. Biol. 15: 797–808.
Regalado, A.P. and Ricardo, C.P. 1996. Study of the intercellular fluid of healthy Lupinus albus organs. Presence of a chitinase and a thaumatin-like protein. Plant Physiol. 110: 227–232.
Robatche-Claive, A.S., Couillerot, J.P., Dubois, J., Dubois, T. and Vasseur, J. 1992. Embryogenè se somatique directe dans les feuilles du Cichorium hybride '474': synchronisation de l'induction. C.R. Acad. Sci. Paris 314: 371–377.
Steiglitz, H. 1973. Role of β-1,3-glucanases activity in developing anther of Lilium. Dev. Biol. 34: 169–173.
Tahiri-Alaoui, A., Dumas, E. and Gianinazzi, S. 1990. Detection of PR-b proteins in tobacco roots infected with Chalara elegans. Plant Mol. Biol. 14: 869–871.
Tornero, P., Conejero, V. and Vera, P. 1994. A gene encoding a novel isoform of the PR-1 protein family from tomato is induced upon viroid infection. Mol. Gen. Genet. 243: 47–53.
van Eldik, G.J., Wingens, M., Ruiter, R.K., Van Herpen, M.M., Schrauwen, J.A. and Wullems, G.J. 1996. Molecular analysis of a pistil-specific gene expressed in the stigma and cortex of Solanum tuberosum. Plant Mol. Biol. 30: 171–176.
Ward, E.R., Payne, G.B., Moyer, M.B., Williams, S.C., Dincher, S.S., Sharkey, K.C., Beck, J.J., Taylor, H.T., Ahl-Goy, P., Meins, F.M. and Ryals, J.A. 1991. Differential regulation of β-1,3-glucanase in tobacco. Plant Mol. Biol. 15: 797–808.
Worrall, D., Hird, D.L., Hodge, R., Paul, W., Draper, J. and Scott, R. 1992. Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco. Plant Cell 4: 759–771.
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Helleboid, S., Chapman, A., Hendriks, T. et al. Cloning of β-1,3-glucanases expressed during Cichorium somatic embryogenesis. Plant Mol Biol 42, 377–386 (2000). https://doi.org/10.1023/A:1006344024877
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DOI: https://doi.org/10.1023/A:1006344024877