Journal of Experimental Botany, Vol. 53, No. 366, pp. 147-148,
January 1, 2002
© 2002 Oxford University Press
Gene Notes |
PM19, a barley (Hordeum vulgare L.) gene encoding a putative plasma membrane protein, is expressed during embryo development and dormancy
2Cellular Microbiology Group, Department of Oral Surgery Research, Eastman Dental Institute, University College London, 256 Gray's Inn Road, London WC1X 8LD, UK
3Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK
Received 6 April 2001; Accepted 17 August 2001
Abstract
A barley (Hordeum vulgare L.) cDNA, PM19, encoding a putative plasma membrane protein was isolated through differential screening of a dormant wild oat embryo library. PM19 is expressed in barley embryos from mid-embryogenesis up to maturity. PM19 mRNA levels decline upon germination, whereas dormant embryos retained high levels of message for up to 72 h of imbibition. PM19 mRNA levels also remained high or were reinduced in non-dormant embryos by treatments that prevented germination (250 mm NaCl, 10% sorbitol, or 50 µm ABA). The PM19 protein sequence is highly conserved in monocotyledonous and dicotyledonous plants.
Key words: Barley, embryo, dormancy, ABA, stress.
Cereal grains commonly possess coat-imposed dormancy, which in barley results in uneven germination during malting with consequent production losses. Genetic factors play a major role in dormancy (Han et al., 1996
; Li and Foley, 1997), and the identification of such genes will aid in the recognition of dormant varieties and allow the development of methods to control dormancy.
In order to isolate cereal embryo genes associated with dormancy, a dormant wild oat embryo cDNA library was differentially screened with DIG-labelled heterologous cDNA probes derived from embryos of dormant and non-dormant barley (cv. Triumph). This approach will preferentially pick out conserved sequences, expected of genes encoding proteins with critical functions. One oat clone that was detected by the dormant embryo probe was a partial cDNA showing homology to WTABAPM (accession No. U80037), an ABA-inducible gene encoding a plasma membrane localized protein (PM-19), isolated from a winter wheat cell suspension culture (Koike et al., 1997). A full-length sequence of the barley cDNA homologue, PM19, (AF218627) was obtained by 3'/5' race. PM19 is 985 nucleotides in length excluding the poly-A tail, and encodes a putative protein of 181 amino acids. The deduced protein sequence is 92.8% identical to that of the wheat PM-19 and also has high homology with protein sequences encoded by clones derived from dry seeds of Arabidopsis thaliana L. (EST clone PAP111, Z27032, Z29867), rice embryos (Oryza sativa L.) (U57639) and immature soybean cotyledons (Glycine max L.) (GmPM3, L20806) (Hsing et al., 1995) (Fig. 1). There are no further similarities with any other DNA or protein sequences in the databases.
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Expression of the PM19 gene in barley was studied by RT-PCR and Northern analysis. During grain development, the transcript was present from 28 DPA through to maturity (Fig. 2A
). Northern blots showed that dormant embryos contain mRNA for PM19 at a similar level to dry mature embryos for up to 72 h after imbibition, but the transcript was almost absent in germinating embryos after 24 h. No signal was seen in root or leaf RNA from seedlings (Fig. 2B). This expression pattern and the provenance of the homologous cDNAs indicate that embryo-specific expression of this gene is conserved between both monocotyledonous and dicotyledonous plants.
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The regulation of PM19 gene expression by plant growth regulators and environmental stress was investigated (Fig. 2C
). Non-dormant grains were put on basal medium or media containing additives for 48 h. Imbibed grains were also transferred from basal medium to additive-containing media after 24 h and left for a further 24 h. Grains were also incubated at 4 °C for 24 or 48 h. Germination was inhibited by both 48 and 24 h treatment with either 250 mM NaCl, 10% (w/v) sorbitol or 50 µM ABA. Root and coleoptile growth was reduced by 50 µM jasmonic acid and cold treatment. Expression of the PM19 gene was retained or reinduced (with respect to the germinating control) by treatment with salt, sorbitol and ABA. ABA is implicated in seed dormancy, osmotic stress and freezing tolerance in plants (Bravo et al., 1988). Koike et al. showed that the wheat WTABAPM gene is induced in suspension culture cells by ABA, which also promoted freezing tolerance of the cells (Koike et al., 1997). Jasmonic acid and cold treatment also gave slightly stronger PM19 signals than the control, but much less than those for salt, sorbitol or ABA.
The function of the PM19 protein is not apparent from sequence homologies, however, the expression pattern of the gene indicates that it is associated with embryo development, dormancy and also with environmental stress during germination. Although the expression pattern is similar to many LEA genes, PM19 is not an LEA protein as described previously (Dure et al., 1989), since it is hydrophobic, of relatively low abundance and present from mid-embryogenesis onward.
Acknowledgments
JCR acknowledges the financial support of the Lindisfarne Trust. We thank Dr Mike Holdsworth, Long Ashton Research Station, for providing the wild oat embryo library.
Notes
1 The nucleotide sequence data reported are in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession number AF218627. 
4 To whom correspondence should be addressed. Fax: +44 131 451 3009. E-mail: p.c.morris{at}hw.ac.uk
References
Bravo LA, Zuniga GE, Alberdi M, Corcuera LJ. 1998. The role of ABA in freezing tolerance and cold acclimation in barley. Physiologia Plantarum 103, 17–23.
Dure LS, Crouch M, Harada J, Ho T-HD, Mundy J, Quatrano R, Thomas T, Sung ZR. 1989. Common amino acid sequence domains among the LEA proteins of higher plants. Plant Molecular Biology 12, 475–486.
Han F, Ullrich SE, Clancy JA, Jitkov V, Kilian A, Romagosa I. 1996. Verification of barley seed dormancy loci via linked molecular markers. Theoretical and Applied Genetics 92, 87–91.
Hsing YIC, Chen ZY, Cho TY. 1995. A soybean cDNA (Accession No. L20806) encoding a hydrophobic embryogenesis abundant protein. Plant Physiology 109, 1125.[Medline]
Koike M, Takezawa D, Arakawa K, Yoshida S. 1997. Accumulation of a 19 kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid. Plant Cell Physiology 38, 707–716.
Li B, Foley ME. 1997. Genetic and molecular control of seed dormancy. Trends in Plant Science 2, 384–389.
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