Abstract
A cDNA encoding a metallothionein-like protein has been isolated from a cDNA library from the abscission zones of ethylene-treated Sambucus nigra leaflets. The precise function of this group of proteins in plants has yet to be confirmed but in animals there is convincing evidence that they bind heavy metals. Several of these proteins have recently been characterised from plants and it has been demonstrated that heavy metals have no stimulatory effect on their expression. In this paper we describe the isolation and characterisation of a metallothionein-like mRNA identified as a consequence of differentially screening a cDNA library for messages up-regulated during abscission. The accumulation of the mRNA occurred in the abscission zone tissue within 18 h of exposure to ethylene while, in contrast, no expression was detectable in adjacent non-abscission-zone tissue. The transcript size of the message was approximately 0.6 kb. Northern analysis revealed that the cDNA insert (JET12) did not hybridise to mRNA from either green or senescing leaflets but a signal was detectable with mRNA extracted from senescent tissue. The size of this hybridising transcript was approximately 0.5 kb. The predicted metallothionein-like protein encoded by JET12 was cysteinerich (18.4%) and had a molecular weight of approximately 7.5 kDa. Southern analysis of S. nigra genomic DNA showed that the mRNA was encoded by a small gene family. The protein exhibited greatest homology to other metallothioneins belonging to the Type 2 family including those from Mimulus (62%) and Arabidopsis (57%). This homology was greatest around the cysteine-rich amino and carboxy termini. The possible role of the protein encoded by JET12 during ethylene-promoted leaflet abscission is discussed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andrews GK (1990) Regulation of metallothionein expression. Prog Food Nutr Sci 14: 193–258
Bradley DJ, Kjellbom P, Lamb CJ (1992) Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defence response. Cell 70: 21–30
Buchanan-Wollaston V (1994) Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Plant Physiol 105: 839–846
Chongpraditnum P, Mori S, Chino M (1992) Excess copper induces a cytosolic Cu, Zn-superoxide dismutase in soybean root. Plant Cell Physiol 33: 239–244
Chubatsu L, Meneghini R (1993) Metallothionein protects DNA from oxidative damage. Biochem J 291: 193–198
Clarke BC, Moran LB, Appels R (1989) DNA analysis in wheat breeding. Genome 32: 334–339
Coupe SA, Taylor JE, Isaac PG, Roberts JA (1993) Identification and characterization of a proline-rich mRNA that accumulates during pod development in oilseed rape (Brassica napus L.). Plant Mol Biol 23: 1223–1232
del Campillo E, Lewis LN (1992a) Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones. Plant Physiol 98: 955–961
del Campillo E, Lewis LN (1992b) Occurrence of 9.5 cellulase and other hydrolases in flower reproductive organs undergoing major cell wall disruption. Plant Physiol 99: 1015–1020
de Miranda JR, Thomas MA, Thurman DA, Thomsett AB (1990) Metallothionein genes from the flowering plant Mimulus guttatus. FEBS Lett 260: 277–280
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12: 387–395
Foley RC, Singh KB (1994) Isolation of a Vicia faba metallothionein-like gene: expression in foliar trichomes. Plant Mol Biol 26: 435–444
Hamilton AJ, Lycett GW, Grierson D (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346: 284–287
Joshi CP (1987) Putative polyadenylation signals in nuclear genes of higher plants: acompilation and analysis. Nucleic Acids Res 15: 9627–9640
Kornfeld R, Kornfeld S (1985) Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 54: 631–664
Lamb CJ (1994) Plant disease resistance genes in signal perception and transduction. Cell 76: 419–422
Ledger SE, Gardner RC (1994) Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var. deliciosa). Plant Mol Biol 25: 877–886
Mackinney G (1941) Absorption of light by chlorophyll solutions. J Biol Chem 140: 315–323
McManus MT, Osborne DJ (1990) Identification of polypeptides specific to rachis abscission zone cells of Sambucus nigra. Physiol Plant 79: 471–478
McManus MT, Osborne DJ (1991) Identification and characterization of an ionically-bound cell wall glycoprotein expressed preferentially in the leaf rachis abscission zone of Sambucus nigra. J Plant Physiol 138: 63–67
Pearson WR, Lipman DJ (1988) Improved tools for sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448
Picton S, Barton SL, Bouzayen M, Hamilton AJ, Grierson D (1993a) Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene. Plant J 3: 469–481
Picton S, Gray JE, Barton S, AbuBakar U, Lowe A, Grierson D (1993b) cDNA cloning and characterization of novel ripeningrelated mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant. Plant Mol Biol 23: 193–207
Rigby PWJ, Dieckman M, Rhodes C, Berg P (1977) Labelling deoxyribonucleic acid in vitro by nick translation with DNA polymerase I. J Mol Biol 113: 237–251
Riordan JF, Vallee BL (1991) Metallobiochemistry part B, metallothionein and related molecules. Methods Enzymol 205: 8–10
Roberts JA, Tucker GA, Maunders MJ (1985) Ethylene and foliar senescence. In: Roberts JA, Tucker GA (eds) Ethylene and plant development. Butterworths, Sevenoaks, pp 267–275
Roberts JA, Taylor JE, Coupe SA, Harris N, Webb STJ (1993) Changes in gene expression during leaf abscission. In: Peche JC, Latche A, Balague C (eds) Cellular and molecular aspects of the plant hormone ethylene. Kluwer Academic Press, Dordecht, pp 272–277
Robinson NJ, Tommey AM, Kuske C, Jackson PJ (1993) Plant metallothioneins. Biochem J 295: 1–10
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual. Cold Spring Harbor Press, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467
Sexton R, Roberts JA (1982) Cell biology of abscission. Annu Rev Plant Physiol 33: 132–162
Stacey J, Isaac PG (1993) Restriction enzyme digestion gel electrophoresis and vacuum blotting of DNA to nylon membranes. In: Isaac PG (ed) Methods in molecular biology protocols for nucleic acid analysis by nonradioactive probes. Humana Press, New York
Taylor JE, Tucker GA, Lasslett Y, Smith CJS, Arnold CM, Watson CF, Schuch W, Grierson D, Roberts JA (1990) Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants. Planta 183: 133–138
Taylor JE, Webb STJ, Coupe SA, Tucker GA, Roberts JA (1993) Changes in polygalacturonase activity and solubility of polyuronides during ethylene-stimulated leaf abscission in Sambucus nigra. J Exp Bot 44: 93–98
Taylor JE, Coupe SA, Picton S, Roberts JA (1994) Characterization and accumulation pattern of an mRNA encoding an abscission-related β-1,4-glucanase from leaflets of Sambucus nigra. Plant Mol Biol 24: 961–964
Tucker ML, Sexton R, del Campillo E, Lewis LN (1988) Bean abscission cellulase. Characterization of a cDNA and regulation of gene expression by ethylene and auxin. Plant Physiol 88: 1257–1262
Wadsworth GJ, Rediinbaugh MG, Scandalios JG (1988) A procedure for the small scale isolation of plant RNA suitable for RNA blot analysis. Anal Biochem 172: 279–283
Webb STJ, Taylor JE, Coupe SA, Ferrarese L, Roberts JA (1993) Purification of β-1,4-glucanase from ethylene-treated leaflet abscission zones of Sambucus nigra. Plant Cell Environ 16: 329–333
Zhou J, Goldsbrough PB (1994) Functional homologs of fungal metallothionein genes from Arabidopsis. Plant Cell 6: 875–884
Author information
Authors and Affiliations
Additional information
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X83439
This work was supported by a grant from the Biotechnology and Biological Sciences Research Council to J.A.R. The authors would like to thank Dr. Nigel Robinson (University of Newcastle) for useful discussions during the preparation of this manuscript. Help in the preparation of computer-aided figures was provided by Dr. Isaac John and Alan Evans (University of Nottingham).
Rights and permissions
About this article
Cite this article
Coupe, S.A., Taylor, J.E. & Roberts, J.A. Characterisation of an mRNA encoding a metallothionein-like protein that accumulates during ethylene-promoted abscission of Sambucus nigra L. leaflets. Planta 197, 442–447 (1995). https://doi.org/10.1007/BF00196665
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00196665