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Sugar Beet Genes Involved in Stress Tolerance in Stresstoleranz Involvierte Zuckerrüben Gene Genes De Betterave a Sucre Impliques Dans La Tolerance Aux Stress

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Sugar Beet Genes Involved in Stress Tolerance in Stresstoleranz Involvierte Zuckerrüben Gene Genes De Betterave a Sucre Impliques Dans La Tolerance Aux Stress (19) TZZ_¥¥_T (11) EP 1 343 875 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 9/12 (2006.01) C12N 15/29 (2006.01) 29.02.2012 Bulletin 2012/09 C12N 15/54 (2006.01) C12N 15/63 (2006.01) C12N 15/80 (2006.01) C12N 15/85 (2006.01) (2006.01) (2006.01) (21) Application number: 01986428.9 C12N 15/81 C07K 14/415 C12N 9/86 (2006.01) C12N 15/55 (2006.01) C12N 1/19 (2006.01) C12N 1/21 (2006.01) (22) Date of filing: 20.12.2001 C12N 5/10 (2006.01) C07K 16/16 (2006.01) C07K 16/40 (2006.01) A01H 15/00 (2006.01) A01H 5/10 (2006.01) G01N 33/50 (2006.01) G01N 33/53 (2006.01) C12Q 1/68 (2006.01) (86) International application number: PCT/EP2001/015093 (87) International publication number: WO 2002/052012 (04.07.2002 Gazette 2002/27) (54) SUGAR BEET GENES INVOLVED IN STRESS TOLERANCE IN STRESSTOLERANZ INVOLVIERTE ZUCKERRÜBEN GENE GENES DE BETTERAVE A SUCRE IMPLIQUES DANS LA TOLERANCE AUX STRESS (84) Designated Contracting States: (56) References cited: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU WO-A-01/07603 MC NL PT SE TR Designated Extension States: • WEI, C-L. ET AL: "characterization of yeast AL LT LV MK translation initiation factor 1a and cloning of its essential gene" THE JOURNAL OF BIOLOGICAL (30) Priority: 22.12.2000 EP 00870319 CHEMISTRY, vol. 270, no. 39, 1995, pages 26.02.2001 US 271656 P 22788-22794, XP002294578 • RAUSSELL A. ET AL: "the translation initiation (43) Date of publication of application: factor elf1a is an important determinant in the 17.09.2003 Bulletin 2003/38 tolerance to NaCl stress in yeast and plants" THE PLANT JOURNAL, vol. 34, 2003, pages 257-267, (73) Proprietor: CropDesign N.V. XP002294579 9052 Zwijnaarde-Gent (BE) • G. PERACCHIA ET AL: "CHARACTERISATION, SUBCELLULAR LOCALIZATION AND NUCLEAR (72) Inventors: TARGETING OF CASEIN KINASE 2 FROM ZEA • KANHONOU, Rodolphe, Arthur MAYS" PLANT MOLECULAR BIOLOGY, vol. 10, E-46020 Valencia (ES) 1999, pages 199-211, XP002181563 • SERRANO SALOM, Ramon • MI-JEONG ET AL: "Isolation and characterisation E-46023 Valencia (ES) of the gene encoding glyceraldehyde-3- • ROS PALAU, Roque phosphate dehydrogenase" BIOCHEMICAL AND E-46006 Valencia (ES) BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 278, no. 1, 2000, pages 192-196, XP000216272 (74) Representative: De Clercq, Ann G. Y. et al De Clercq & Partners cvba Edgard Gevaertdreef 10 a 9830 Sint-Martens-Latem (BE) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 343 875 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 1 343 875 B1 • HAI LAN PIAO ET AL: "An Arabidopsis • DATABASE EMBL [Online] Acc. No Y11526, 2 GSK3/shaggy-like gene that complements yeast November 1998 (1998-11-02) XP002181566 salt stress-sensitve mutants is induced by NaCL • I. WINICOV: "New molecular approaches to and Abscisic Acid" PLANT PHYSIOLOGY, vol. improving salt tolerance in crop plants" ANNALS 119, April 1999 (1999-04), pages 1527-1534, OF BOTANY, vol. 82, 1998, pages 703-710, XP002181564 XP001007288 • TATJANA KLEINOW ET AL: "Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressor of snf4 deficiency in yeast" THE PLANT JOURNAL, vol. 23, no. 1, 2000, pages 115-122, XP002181565 2 EP 1 343 875 B1 Description FIELD OF INVENTION 5 [0001] The present specification relates to the field of plant molecular biology, more particularly to the use of sugar beet genes and proteins, able to confer a phenotype in eukaryotic cells or organisms of tolerance to stress situations for example mineral salt toxicity caused by ions such as Na+ or Li+. BACKGROUND 10 [0002] Soil salinity is one of the most significant abiotic stresses for plant agriculture and therefore it would be useful to identify and isolate stress tolerance genes for the practical goal of genetically improving the salt tolerance of crop plants. [0003] Two other major abiotic stresses, drought and cold, are intimately linked with salt stress. Many genes that are regulated by salt stress are also responsive to drought or cold stress (Zhu, 1997), therefore these genes are particularly 15 interesting for genetically improving of stress tolerance. [0004] The molecular mechanisms by which plants respond to salt stress are starting to be elucidated (Hasegawa et al. 2000a). The sodium transporters at the vacuole and plasma membrane, identified as the products of the Arabidopsis NHX1 (Gaxiola et al. 1992, Apse et al. 1999) and SOS1 (Shi et al. 2000) genes respectively, have been described as important determinants of salt tolerance. 20 [0005] For the goal of genetically improving stress tolerance in plants it is important to use stress tolerance genes that when introduced can immediately confer stress tolerance. The action of these genes cannot be dependent on other pathway-related events or other components that are necessary for the molecular mechanism of stress tolerance. One can identify important stress factors in the stressed organism, but the question remains whether these genes will also contribute in enhancing stress tolerance in a heterologous host when isolated and transfected herein. 25 Sugar beet (genes) and stress [0006] It is known that Beta vulgaris L. (Chenopodaceae, sugar beet), is rather a stress resistant plant when compared to other plants e. g. Arabidopsis thaliana. Sugar beets are relatively spoken rather stress resistant to salt- and drought. 30 The genes that are responsible for the ability of sugar beet to grow in more difficult conditions are started to be elucidated. A first indication that a gene under investigation might be involved in the induction of resistance, is the increase of its expression under stress conditions. As an example, Matthias et al. (1996) showed that salt stress induces the increased expression of V-type H+ATPase in mature sugar beet leaves. Also betaine, an osmoprotectant, is accumulated by many beet plants in response to salinity and drought. Furthermore in sugar beet, the expression of Choline monooxygenase, 35 which catalyzes the committing step in the synthesis of glycine betaine of sugar beet, is also induced by osmotic stress in Chenopodaceae. The mRNA levels in leaves increased 3- to 7- fold at 400mM salt and returned to uninduced levels (Russel, 1998). As mentioned above there are several alternative pathways to respond to stress situations and therefore many different genes are probably involved in stress responses. 40 SUMMARY OF THE INVENTION [0007] The present invention provides subject matter as set forth in any one and all of (i) to (xxxiii) below: (i) Use of a Beta vulgaris nucleic acid for enhancing osmotic stress tolerance in a plant comprising expression of 45 said Beta vulgaris nucleic acid, characterized in that it confers osmotic stress tolerance to yeast cells, and wherein said Beta vulgaris nucleic acid is chosen from: (a) a nucleic acid comprising a DNA sequence as given in SEQ ID NO 3 or the complement thereof, (b) a nucleic acid comprising the RNA sequence corresponding to SEQ ID NO 3 or the complement thereof, 50 (c) a nucleic acid specifically hybridizing to the nucleic acid of (a) or (b) under high stringency conditions, (d) a nucleic acid encoding a protein with an amino acid sequence which is at least 89% identical to the amino acid sequence as given in SEQ ID NO 8, (e) a nucleic acid encoding a protein comprising the amino acid sequence as given in SEQ ID NO 8, (f) a nucleic acid which is degenerated to a nucleic acid as given in SEQ ID NO 3, or which is degenerated to 55 a nucleic acid as defined in any of (a) to (e) as a result of the genetic code, (g) a nucleic acid which is diverging from a nudeic acid encoding a protein as given in SEQ ID NO 8, or which is diverging from a nucleic acid as defined in any of (a) to (e) as a result of differences in codon usage between organisms, 3 EP 1 343 875 B1 (h) a nucleic acid which is diverging from a nucleic acid encoding a protein as given in SEQ ID NO 8, or which is diverging from a nucleic acid as defined in any of (a) to (e) as a result of differences between alleles, and (i) a nucleic acid as defined in any one of (a) to (h) characterized in that said nucleic acid is DNA, cDNA, genomic DNA or synthetic DNA. 5 (ii) Use of a Beta vulgaris nucleic acid as set forth in (i) above wherein said yeast cells are derived from the Na+ sensitive yeast strain JM26. (iii) A method for enhancing osmotic stress tolerance in a plant comprising the expression or altering the expression 10 of any nucleic acid as set forth in (i) above in cells, tissues or parts of said plant. (iv) A method as set forth in (iii) above wherein said expression of said nucleic acid occurs under the control of a promoter. 15 (v) A method as set forth in any one of (iii) or (iv) above wherein said osmotic stress is caused by salt. (vi) A method as set forth in any one of (iii) or (iv) above wherein said osmotic stress is caused by drought.
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