Plant Molecular Biology 18: 1195-1198, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
1195
Update section
Sequence
Molecular cloning of a novel barley seed protein gene that is repressed by abscisic acid Ru Liu 1, Odd-Arne Olsen 2, Martin Kreis 3 and Nigel G. H alford 4,
1Biochemistry and Physiology Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, UK,"2plant Molecular Biology Laboratory NLVH, P.O.B. 51, N-1432,4S-NLH, Norway," 3 Universitd de Paris-Sud, Biologie du DOveloppementdes Plantes, Bdtiment 430, F-91400 Orsay Cedex, France; 4Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, UK (* authorfor correspondence) Received 20 December 1991; accepted 14 January 1992
Key words:abscisic acid, barley, endosperm, seed protein
Abscisic acid (ABA) plays an important role in the control of seed development. Its level gradually rises to reach a peak just before or at the onset of seed desiccation [9] and it has been implicated in the development of desiccation tolerance [8]. It is also required for the onset of dormancy, since mutants that are ABA-deficient or non-responsive produce seeds that germinate precociously [6, 7]. At high concentrations, ABA induces the expression of a number of genes, some of which encode proteins associated with osmotic stress [14], including the early-methionine-labelled (Em) proteins [ 12, 15]. These genes are expressed in other organs of the plant during water stress. It also induces the expression of a barley gene encoding aldose reductase [1] which may be involved in osmotic regulation. ABA has also been shown to repress several genes that are usually expressed during germination, including a-amylase [ 17, 2], and aleurain, a thiol protease expressed in the aleurone layer of germinating barley seeds [13]. It also represses synthesis of the small subunit ofribulose bisphosphate carboxylase [ 12].
In this paper we report the characterization of a novel cDNA and a corresponding genomic clone of a gene that is expressed in the endosperm of developing barley seeds. It is also highly expressed in cultured embryos but this expression is reduced to undetectable levels by treatment with ABA. Clone cB32E was isolated from a cDNA library prepared from barley cv. Bomi aleurone mRNA as described by Jakobsen et al. [5]. The c D N A insert was cloned into the Eco RI site of pUC18 [16] to produce the plasmid pcB32E. The insert was used to screen a genomic library of cultivar Sunbar (obtained from Clontech). A hybridising clone was isolated and found to contain a 5.4kb Barn HI fragment which was then inserted into pUC18 to produce plasmid pgB32E. The insert of pcB32E was entirely sequenced. It is 909 bp long (disregarding the poly(A) tail) and contains an open reading frame of 516 bp beginning at the 5' end. The insert ofpgB32E was sequenced from its 5' end to a position 3' to the polyadenylation site, a total of 2384 bp (Fig. 1). Despite the fact that they originate from different cultivars, the two sequences are identical where
1196 1 GTGCGATTACCACGATGTACTACACCATGAGATGCCTGAGATAACATACCTGAATCCGTTACATGTTAATGCTAT 76 G C T A T T A T G T G T C A T G C A T G T C A A T A A A T G A G A T C A C C T A T G A T A C T A G T T T A T G A T A C T A T G C A G T A T A A A G G T 151
AGTATCATACAATAGTATCATATGCATGATGCTACTATATGTTACTCCCCACTATGATCAGCCTAACTCAGTTGT
226
TTCCTGCAAGTTTAGATGATTTAAAGTATCCCATTTTGACGGAATTGAAACATTCTTAACATCATACCATGTCCA
301
AGACACATGAAGGCACATGAGCGTTAATTCACCCACCAGGATTTGACTTGCATTAATGGAATTATTTCGTGATTG
376
TAATGGTGCCTCCCTTAAAGTGTTACAAAGTTTGATGATAGAAAGGAGAACCATAGTGACATAATAATCTTCTGG
451 A A A T T G A G T T A T T C T A T T T A C A T T A T G C T T G C C T T G A G C T A C T T G T A C T T G A C G T T T T G G C C T T G T T G T T T T T T A 526 A A G A C A A C A A A C T G A T A T T C T T A G A A A A G T A G T A G A A C T T A T A T T G G T G T A T G C C C T T T T G C T T C T C C G T T T A A C 601
TGATGTCATTCATGTGATGGTGCTGTTTACTGTAAATATTAATTTGATTGCTTCATGCTATAGGGTGTGAGAGAG
676
GAGGTTCATTGTTGACCTTGTTGGGCAGTGTCGAATCTTTACCATGCTTCGTGTGATGGATCTTGTTAGCGATAC
751
TGGGTATGCATCTTTTCACTCTGCCTTTCGGTTCTGTGCTTTTTGCATTGTCATGTGACAGGACTTCATGTATTC
826
CCTTGATGAGAGCCTTCTGTTTCAGGCTCTAGGACTAAATGATGAGTTGCAGCGTGTTCTTCAGCGTCATGATGA
901
CATAGCAAAAGGAGTTCCTCCAAACATACCAGTGCCTGTTGCTGGTAATGTAAACCAAGGGGCAGCCCCTCCTCG
976
GCCTGCCGGGGTGTCATTTTCCCCTCTTCTTAATGTACACCATGAGGACGATGAACCAGAAGATGAGTTCTCTGT
1051
GCTTTCTCGCAGGTAAGCTATATTTGTTTTTTCCCATTACATTTGACTCTCATCTTTTCATGCTTATTGGTGTTC
1126
CATGCTTTGGATAGTTTCTGCAACCTCAATTCGTATTATCGTTTTCTCAAATTGCGCTCCTCATGACTAAAATTG
1201
TAGGTCTGCACGGGACGCCGCAGCAGCACAGGGCAACATGTCTTCTGTTCCGAGAAATGAAAGGCCATACCCGAG
25 1276
CCCACTTCTTCCCCCACCACCATCGACAAAAAGGCCTGTTTACACAGAGGCAAGCAGTGTCGACTATCTAAGTGG
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50 D S Y K S E K V P D D F V N P T A P A N I S A P S 1351 A G A C T C C T A C A A A T C G G A A A A A G T A C C A G A T G A T T T T G T C A A C C C A A C A G C C C C C G C A A A T A T A T C T G C A C C A T C >cB32E 75 Y S K T E T D H E P K Y D S R S E Y V P D D F I N 1426 T T A T T C A A A A A C A G A G A C G G A T C A C G A G C C A A A A T A T G A T A G C A G G T C A G A A T A T G T G C C T G A T G A C T T C A T A A A 100 1501
CCCAACTGCAGCACCCAGTTTTTCCATGCCCTCTCGCCCTACGAGTGAGCCGAATAATTCATCTGTGAATAAGCT
P
125 1576
E S L P D D D F I N P T A L P G F S S S S T S E TGAGAGCCTTCCTGATGATGACTTCATAAACCCCACTGCACTTCCTGGCTTTTCTTCGTCTTCAACTAGTGAAGA
150 1651
TCTTCCAAAAGCTCCATGGGAAGCGCAAGCTCCAGTTTCCCTACCACCGCCTCCTGCAAGGCATGGCCAAAGACA
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175 Q F F E Q Q H G F P G G N N E G G Y D E M L T Q T 1726 ACAGTTCTTTGAGCAACAGCATGGCTTTCCTGGTGGGAACAATGAAGGTGGCTATGATGAAATGCTGACGCAGAC 200 G S L S L N Q R N T E N G K S A S V S T A S R Q P 1801 A G G A A G C C T T T C T C T T A A T C A A A G G A A C A C T G A G A A T G G G A A G A G T G C A T C G G T G T C G A C T G C A T C C C G C C A G C C 225 1876
CAAACATGAGGATGCCCTTTTTAAGGATCTTGTTGATTTTGCCAAGAAGAATCCGTCTTCTCCATCTAAACCAGC
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250 1951
N S R R T R * CAACAGCCGCAGGACTCGCTGAGTGATGAGTGTTCCAGTCATGTAACACACATAATTGAGTTGCCGGCCTGCTGG
2026
TTTTGCTACAGGCTAGCGAATTAGATATACCGTCAAATAAGAGGGTTCGGAGTTTGGACTGTTTCACATCACAAC
2101
ATTGGCAGTTATTTGATTTGCGCATTGGACAATTAAAATCACAGGTTCAGATACATAGTCATGATACTGGGTGTG
2176
GTGTGTAGTGAGCTGCCTTTCACCTTTTTCTTGTGTGGTATTGGTTATCGATTCTGGCTTTATTTTGCTTAGCTG
2251
TATATATTTCTCCATTTGACAGCAGTATTCTATTCATAGTTGAGTTTACGATTGATCTTGTAAGACTTAGTTAAC
2326
AGTTTATTTTCAACGTGGACGCTCCCATTTGTTCTTGTACTGAGCAGTAACCTTGGCTA
I Polyadenylation
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1197 they overlap. The open reading frame that was identified in the c D N A sequence extends a further 259 bps in the 5' direction in the genomic sequence. The derived amino acid sequence of this ORF from the first possible intron 3' splice site is shown in Fig. 1, however, there are no experimental data to confirm that this is the actual intron splice site. Subsequent northern blot analysis showed that the transcript of the B32E gene is approximately 3 kb long, indicating that there must be other ORFs further upstream and that gB32E does not contain the whole gene. The longest ORF upstream of the one indicated runs from position 809 to 1063. The function of the protein encoded by B32E remains unknown. Searches of the PIR protein sequence database and the EMBL and G E N B A N K nucleic acid databases failed to find significant similarities to any other sequences. The amino acid sequence of 255 residues is rich in serine (14.9~o) and proline (13.7~o) and is relatively hydrophilic. The sequence Asp-Asp-PheVal/Ile-Asn-Pro-Thr-Ala occurs three times, at positions 59, 95 and 130. The chromosomal location of the B32E gene was determined by Southern blot analysis of genomic D N A isolated from six wheat-barley disomic addition lines and ditelosomic chromosome 4S and 4L addition lines [4, 3]. The results showed clearly that the B32E gene is located on the short arm of chromosome 4. Southern blot analysis of seven barley cultivars (Andrea, Betzes, CarlsbergII, Hiproly, Sundance, Teo and Vada) showed that they all contain the 5.4 kb Barn HI fragment corresponding to that of pgB 32E. Single hybridising fragments were also detected after digestion with Eco RI (5.2 kb), Kpn I (7.0 kb) and Sst I (approximately 23 kb). The only restriction fragment length polymorphism detected resulted from a Hind III digest. This gave fragments of 2.2 and 2.7 kb in all the cultivars except Teo, which contained fragments of 2.2 kb and 4 kb. Reconstruction experiments suggested that B32E is a single-copy gene.
Northern blot analyses indicated that the B32E gene is expressed predominantly in the developing endosperm, although transcripts were detectable in aleurone, shoot, root and leafRNA after amplification by the polymerase chain reaction. The length of the transcript is approximately 3 kb. It was also expressed in embryos cultured in the dark at 25 ° C on medium containing 1 ~o sucrose, 1 mg/ml thiamine, half-strength MS [10] solidified with 0.5~o agarose, but this expression was reduced to undetectable levels when ABA was added at 50 #M. The repression of expression of B32E by ABA is similar to that reported for a second cDNA clone, B22E, isolated from the same library [11]. B22E m R N A accumulates in the developing seed but is not detectable in the mature seed, it then accumulates again upon germination. As with B32E, its expression in cultured embryos is severely repressed by ABA treatment. However, unlike B32E, B22E transcripts are only detectable in the embryo and aleurone. References 1. Barrels D, Engelhardt K, Roncarati R, Schneider K, Rotter M, Salamini F: An ABA and AG modulated gene expressed in the barley embryo encodes an aldose reductase related protein. EMBO J 10:1037-1043 (1991). 2. Garcia-Maya M, Chapman JM, Black M: Regulation of e-amylase formation and gene expression in the developing wheat embryo. Planta 181:296-303 (1990). 3. Islam AKMR: Ditelosomic additions of barley chromosomes to wheat. In: Proceedings of the 6th International Wheat Genetics Symposium, Kyoto, Japan, pp. 233-238 (1983). 4. Islam AKMR, Shepherd KW: Wheat-barley addition lines: their use in genetic and evolutionary studies of barley. In: Proceedings of the 4th International Barley Genetics Symposium, Edinburgh, Scotland, pp. 729-739 (1981). 5. Jakobsen K, Klemsdal SS, Aalen RB, Bosnes M, Alexander D, Olsen O-A: Barley aleurone cell development: molecular cloning of aleurone-specific cDNAs from immature grains. Plant Mol Biol 12:285-293 (1989). 6. Karssen CM, Lacka E: A revision of the hormone balance theory of seed dormancy: studies on gibberellin and/
Fig. 1. The nucleotide sequence of gB32E and the derived amino acid sequence of the longest ORF. The region corresponding to cB32E is indicated.
1198
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or ABA-deficient mutants of Arabidopsis thaliana. In: Bopp M (ed) Plant Growth Substances, pp. 315-323. Springer-Verlag, Berlin (1986). Karssen CM, Brinkhorst-van der Swan DLC, Breekland AE, Koorneef M: Induction of dormancy during seed development by endogenous abscisic acid: studies on abscisic acid deficient genotypes ofArabidopsis thaliana (L.) Heynh. Planta 157:158-165 (1983). Kermode AR, Bewley JD: Regulatory processes involved in the switch from seed development to germination: possible roles for desiccation and ABA. In; Monti L, Porceddu E (eds) Drought Resistance in Plants; Physiological and Genetic Aspects, pp. 59-76. EEC (1987). King RW: Abscisic acid in developing wheat grains and its relationship to grain growth and maturation. Planta 132:43-51 (1976). Murashige T, Skoog F: A revised medium for rapid growth and bioassay of tobacco tissue cultures. Physiol Plant 15:473-497 (1962). Olsen O-A, Jakobsen KS, Schmelzer E: Development of barley aleurone cells: temporal and spatial patterns of accumulation of cell-specific mRNAs. Planta 181: 462466 (1990).
12. Quatrano BS, Ballo BL, Williamson JD, Hamblin MT, Mansfield M: ABA controlled expression of embryospecific genes during wheat grain development. In: Goldberg R (ed) Plant Molecular Biology, pp. 343-353. Alan R. Liss, New York (1983). 13. Rogers JC, Dean D, Heck G: Aleurain: a barley thiol protease closely related to mammalian cathepsin H. Proc Natl Acad Sci USA 82:6512-6516 (1985). 14. Skriver K, Mundy J: Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503-512 (1990). 15. Williamson D, Quatrano RS, Cuming AC: Em polypeptide and its messenger RNA levels are modulated by abscisic acid during embryogenesis in wheat. Eur J Biochem 152:501-507 (1985). 16. Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mpl8 and pUC19 vectors. Gene 33: 103-119 (1985). 17. Yomo H, Varner JE: Control of the formation of amylases and proteases in the cotyledons of germinating peas. Plant Physiol 51:708-713 (1973).
1195
Update section
Sequence
Molecular cloning of a novel barley seed protein gene that is repressed by abscisic acid Ru Liu 1, Odd-Arne Olsen 2, Martin Kreis 3 and Nigel G. H alford 4,
1Biochemistry and Physiology Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, UK,"2plant Molecular Biology Laboratory NLVH, P.O.B. 51, N-1432,4S-NLH, Norway," 3 Universitd de Paris-Sud, Biologie du DOveloppementdes Plantes, Bdtiment 430, F-91400 Orsay Cedex, France; 4Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, UK (* authorfor correspondence) Received 20 December 1991; accepted 14 January 1992
Key words:abscisic acid, barley, endosperm, seed protein
Abscisic acid (ABA) plays an important role in the control of seed development. Its level gradually rises to reach a peak just before or at the onset of seed desiccation [9] and it has been implicated in the development of desiccation tolerance [8]. It is also required for the onset of dormancy, since mutants that are ABA-deficient or non-responsive produce seeds that germinate precociously [6, 7]. At high concentrations, ABA induces the expression of a number of genes, some of which encode proteins associated with osmotic stress [14], including the early-methionine-labelled (Em) proteins [ 12, 15]. These genes are expressed in other organs of the plant during water stress. It also induces the expression of a barley gene encoding aldose reductase [1] which may be involved in osmotic regulation. ABA has also been shown to repress several genes that are usually expressed during germination, including a-amylase [ 17, 2], and aleurain, a thiol protease expressed in the aleurone layer of germinating barley seeds [13]. It also represses synthesis of the small subunit ofribulose bisphosphate carboxylase [ 12].
In this paper we report the characterization of a novel cDNA and a corresponding genomic clone of a gene that is expressed in the endosperm of developing barley seeds. It is also highly expressed in cultured embryos but this expression is reduced to undetectable levels by treatment with ABA. Clone cB32E was isolated from a cDNA library prepared from barley cv. Bomi aleurone mRNA as described by Jakobsen et al. [5]. The c D N A insert was cloned into the Eco RI site of pUC18 [16] to produce the plasmid pcB32E. The insert was used to screen a genomic library of cultivar Sunbar (obtained from Clontech). A hybridising clone was isolated and found to contain a 5.4kb Barn HI fragment which was then inserted into pUC18 to produce plasmid pgB32E. The insert of pcB32E was entirely sequenced. It is 909 bp long (disregarding the poly(A) tail) and contains an open reading frame of 516 bp beginning at the 5' end. The insert ofpgB32E was sequenced from its 5' end to a position 3' to the polyadenylation site, a total of 2384 bp (Fig. 1). Despite the fact that they originate from different cultivars, the two sequences are identical where
1196 1 GTGCGATTACCACGATGTACTACACCATGAGATGCCTGAGATAACATACCTGAATCCGTTACATGTTAATGCTAT 76 G C T A T T A T G T G T C A T G C A T G T C A A T A A A T G A G A T C A C C T A T G A T A C T A G T T T A T G A T A C T A T G C A G T A T A A A G G T 151
AGTATCATACAATAGTATCATATGCATGATGCTACTATATGTTACTCCCCACTATGATCAGCCTAACTCAGTTGT
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TTCCTGCAAGTTTAGATGATTTAAAGTATCCCATTTTGACGGAATTGAAACATTCTTAACATCATACCATGTCCA
301
AGACACATGAAGGCACATGAGCGTTAATTCACCCACCAGGATTTGACTTGCATTAATGGAATTATTTCGTGATTG
376
TAATGGTGCCTCCCTTAAAGTGTTACAAAGTTTGATGATAGAAAGGAGAACCATAGTGACATAATAATCTTCTGG
451 A A A T T G A G T T A T T C T A T T T A C A T T A T G C T T G C C T T G A G C T A C T T G T A C T T G A C G T T T T G G C C T T G T T G T T T T T T A 526 A A G A C A A C A A A C T G A T A T T C T T A G A A A A G T A G T A G A A C T T A T A T T G G T G T A T G C C C T T T T G C T T C T C C G T T T A A C 601
TGATGTCATTCATGTGATGGTGCTGTTTACTGTAAATATTAATTTGATTGCTTCATGCTATAGGGTGTGAGAGAG
676
GAGGTTCATTGTTGACCTTGTTGGGCAGTGTCGAATCTTTACCATGCTTCGTGTGATGGATCTTGTTAGCGATAC
751
TGGGTATGCATCTTTTCACTCTGCCTTTCGGTTCTGTGCTTTTTGCATTGTCATGTGACAGGACTTCATGTATTC
826
CCTTGATGAGAGCCTTCTGTTTCAGGCTCTAGGACTAAATGATGAGTTGCAGCGTGTTCTTCAGCGTCATGATGA
901
CATAGCAAAAGGAGTTCCTCCAAACATACCAGTGCCTGTTGCTGGTAATGTAAACCAAGGGGCAGCCCCTCCTCG
976
GCCTGCCGGGGTGTCATTTTCCCCTCTTCTTAATGTACACCATGAGGACGATGAACCAGAAGATGAGTTCTCTGT
1051
GCTTTCTCGCAGGTAAGCTATATTTGTTTTTTCCCATTACATTTGACTCTCATCTTTTCATGCTTATTGGTGTTC
1126
CATGCTTTGGATAGTTTCTGCAACCTCAATTCGTATTATCGTTTTCTCAAATTGCGCTCCTCATGACTAAAATTG
1201
TAGGTCTGCACGGGACGCCGCAGCAGCACAGGGCAACATGTCTTCTGTTCCGAGAAATGAAAGGCCATACCCGAG
25 1276
CCCACTTCTTCCCCCACCACCATCGACAAAAAGGCCTGTTTACACAGAGGCAAGCAGTGTCGACTATCTAAGTGG
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50 D S Y K S E K V P D D F V N P T A P A N I S A P S 1351 A G A C T C C T A C A A A T C G G A A A A A G T A C C A G A T G A T T T T G T C A A C C C A A C A G C C C C C G C A A A T A T A T C T G C A C C A T C >cB32E 75 Y S K T E T D H E P K Y D S R S E Y V P D D F I N 1426 T T A T T C A A A A A C A G A G A C G G A T C A C G A G C C A A A A T A T G A T A G C A G G T C A G A A T A T G T G C C T G A T G A C T T C A T A A A 100 1501
CCCAACTGCAGCACCCAGTTTTTCCATGCCCTCTCGCCCTACGAGTGAGCCGAATAATTCATCTGTGAATAAGCT
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125 1576
E S L P D D D F I N P T A L P G F S S S S T S E TGAGAGCCTTCCTGATGATGACTTCATAAACCCCACTGCACTTCCTGGCTTTTCTTCGTCTTCAACTAGTGAAGA
150 1651
TCTTCCAAAAGCTCCATGGGAAGCGCAAGCTCCAGTTTCCCTACCACCGCCTCCTGCAAGGCATGGCCAAAGACA
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175 Q F F E Q Q H G F P G G N N E G G Y D E M L T Q T 1726 ACAGTTCTTTGAGCAACAGCATGGCTTTCCTGGTGGGAACAATGAAGGTGGCTATGATGAAATGCTGACGCAGAC 200 G S L S L N Q R N T E N G K S A S V S T A S R Q P 1801 A G G A A G C C T T T C T C T T A A T C A A A G G A A C A C T G A G A A T G G G A A G A G T G C A T C G G T G T C G A C T G C A T C C C G C C A G C C 225 1876
CAAACATGAGGATGCCCTTTTTAAGGATCTTGTTGATTTTGCCAAGAAGAATCCGTCTTCTCCATCTAAACCAGC
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250 1951
N S R R T R * CAACAGCCGCAGGACTCGCTGAGTGATGAGTGTTCCAGTCATGTAACACACATAATTGAGTTGCCGGCCTGCTGG
2026
TTTTGCTACAGGCTAGCGAATTAGATATACCGTCAAATAAGAGGGTTCGGAGTTTGGACTGTTTCACATCACAAC
2101
ATTGGCAGTTATTTGATTTGCGCATTGGACAATTAAAATCACAGGTTCAGATACATAGTCATGATACTGGGTGTG
2176
GTGTGTAGTGAGCTGCCTTTCACCTTTTTCTTGTGTGGTATTGGTTATCGATTCTGGCTTTATTTTGCTTAGCTG
2251
TATATATTTCTCCATTTGACAGCAGTATTCTATTCATAGTTGAGTTTACGATTGATCTTGTAAGACTTAGTTAAC
2326
AGTTTATTTTCAACGTGGACGCTCCCATTTGTTCTTGTACTGAGCAGTAACCTTGGCTA
I Polyadenylation
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1197 they overlap. The open reading frame that was identified in the c D N A sequence extends a further 259 bps in the 5' direction in the genomic sequence. The derived amino acid sequence of this ORF from the first possible intron 3' splice site is shown in Fig. 1, however, there are no experimental data to confirm that this is the actual intron splice site. Subsequent northern blot analysis showed that the transcript of the B32E gene is approximately 3 kb long, indicating that there must be other ORFs further upstream and that gB32E does not contain the whole gene. The longest ORF upstream of the one indicated runs from position 809 to 1063. The function of the protein encoded by B32E remains unknown. Searches of the PIR protein sequence database and the EMBL and G E N B A N K nucleic acid databases failed to find significant similarities to any other sequences. The amino acid sequence of 255 residues is rich in serine (14.9~o) and proline (13.7~o) and is relatively hydrophilic. The sequence Asp-Asp-PheVal/Ile-Asn-Pro-Thr-Ala occurs three times, at positions 59, 95 and 130. The chromosomal location of the B32E gene was determined by Southern blot analysis of genomic D N A isolated from six wheat-barley disomic addition lines and ditelosomic chromosome 4S and 4L addition lines [4, 3]. The results showed clearly that the B32E gene is located on the short arm of chromosome 4. Southern blot analysis of seven barley cultivars (Andrea, Betzes, CarlsbergII, Hiproly, Sundance, Teo and Vada) showed that they all contain the 5.4 kb Barn HI fragment corresponding to that of pgB 32E. Single hybridising fragments were also detected after digestion with Eco RI (5.2 kb), Kpn I (7.0 kb) and Sst I (approximately 23 kb). The only restriction fragment length polymorphism detected resulted from a Hind III digest. This gave fragments of 2.2 and 2.7 kb in all the cultivars except Teo, which contained fragments of 2.2 kb and 4 kb. Reconstruction experiments suggested that B32E is a single-copy gene.
Northern blot analyses indicated that the B32E gene is expressed predominantly in the developing endosperm, although transcripts were detectable in aleurone, shoot, root and leafRNA after amplification by the polymerase chain reaction. The length of the transcript is approximately 3 kb. It was also expressed in embryos cultured in the dark at 25 ° C on medium containing 1 ~o sucrose, 1 mg/ml thiamine, half-strength MS [10] solidified with 0.5~o agarose, but this expression was reduced to undetectable levels when ABA was added at 50 #M. The repression of expression of B32E by ABA is similar to that reported for a second cDNA clone, B22E, isolated from the same library [11]. B22E m R N A accumulates in the developing seed but is not detectable in the mature seed, it then accumulates again upon germination. As with B32E, its expression in cultured embryos is severely repressed by ABA treatment. However, unlike B32E, B22E transcripts are only detectable in the embryo and aleurone. References 1. Barrels D, Engelhardt K, Roncarati R, Schneider K, Rotter M, Salamini F: An ABA and AG modulated gene expressed in the barley embryo encodes an aldose reductase related protein. EMBO J 10:1037-1043 (1991). 2. Garcia-Maya M, Chapman JM, Black M: Regulation of e-amylase formation and gene expression in the developing wheat embryo. Planta 181:296-303 (1990). 3. Islam AKMR: Ditelosomic additions of barley chromosomes to wheat. In: Proceedings of the 6th International Wheat Genetics Symposium, Kyoto, Japan, pp. 233-238 (1983). 4. Islam AKMR, Shepherd KW: Wheat-barley addition lines: their use in genetic and evolutionary studies of barley. In: Proceedings of the 4th International Barley Genetics Symposium, Edinburgh, Scotland, pp. 729-739 (1981). 5. Jakobsen K, Klemsdal SS, Aalen RB, Bosnes M, Alexander D, Olsen O-A: Barley aleurone cell development: molecular cloning of aleurone-specific cDNAs from immature grains. Plant Mol Biol 12:285-293 (1989). 6. Karssen CM, Lacka E: A revision of the hormone balance theory of seed dormancy: studies on gibberellin and/
Fig. 1. The nucleotide sequence of gB32E and the derived amino acid sequence of the longest ORF. The region corresponding to cB32E is indicated.
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9.
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11.
or ABA-deficient mutants of Arabidopsis thaliana. In: Bopp M (ed) Plant Growth Substances, pp. 315-323. Springer-Verlag, Berlin (1986). Karssen CM, Brinkhorst-van der Swan DLC, Breekland AE, Koorneef M: Induction of dormancy during seed development by endogenous abscisic acid: studies on abscisic acid deficient genotypes ofArabidopsis thaliana (L.) Heynh. Planta 157:158-165 (1983). Kermode AR, Bewley JD: Regulatory processes involved in the switch from seed development to germination: possible roles for desiccation and ABA. In; Monti L, Porceddu E (eds) Drought Resistance in Plants; Physiological and Genetic Aspects, pp. 59-76. EEC (1987). King RW: Abscisic acid in developing wheat grains and its relationship to grain growth and maturation. Planta 132:43-51 (1976). Murashige T, Skoog F: A revised medium for rapid growth and bioassay of tobacco tissue cultures. Physiol Plant 15:473-497 (1962). Olsen O-A, Jakobsen KS, Schmelzer E: Development of barley aleurone cells: temporal and spatial patterns of accumulation of cell-specific mRNAs. Planta 181: 462466 (1990).
12. Quatrano BS, Ballo BL, Williamson JD, Hamblin MT, Mansfield M: ABA controlled expression of embryospecific genes during wheat grain development. In: Goldberg R (ed) Plant Molecular Biology, pp. 343-353. Alan R. Liss, New York (1983). 13. Rogers JC, Dean D, Heck G: Aleurain: a barley thiol protease closely related to mammalian cathepsin H. Proc Natl Acad Sci USA 82:6512-6516 (1985). 14. Skriver K, Mundy J: Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503-512 (1990). 15. Williamson D, Quatrano RS, Cuming AC: Em polypeptide and its messenger RNA levels are modulated by abscisic acid during embryogenesis in wheat. Eur J Biochem 152:501-507 (1985). 16. Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mpl8 and pUC19 vectors. Gene 33: 103-119 (1985). 17. Yomo H, Varner JE: Control of the formation of amylases and proteases in the cotyledons of germinating peas. Plant Physiol 51:708-713 (1973).
