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Ep 2038300 B1 (19) TZZ Z¥¥ZZ_T (11) EP 2 038 300 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 14/415 (2006.01) C12N 15/82 (2006.01) 19.02.2014 Bulletin 2014/08 C12N 15/29 (2006.01) A01H 5/00 (2006.01) A01H 5/04 (2006.01) (21) Application number: 07796664.6 (86) International application number: (22) Date of filing: 05.07.2007 PCT/US2007/015437 (87) International publication number: WO 2008/005474 (10.01.2008 Gazette 2008/02) (54) HIGH LEVEL ANTIOXIDANT-CONTAINING FOODS LEBENSMITTEL MIT HOHEM ANTIOXIDATIONSMITTELGEHALT ALIMENTS CONTENANT DE HAUTS NIVEAUX D’ANTIOXYDANTS (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A2-2008/096354 WO-A2-2008/134372 HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR • NIGGEWEG RICARDA ET AL: "Engineering plants with increased levels of the antioxidant (30) Priority: 06.07.2006 US 818516 P chlorogenic acid" NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, (43) Date of publication of application: US, vol. 22, no. 6, June 2004 (2004-06), pages 25.03.2009 Bulletin 2009/13 746-754, XP009087941 ISSN: 1087-0156 cited in the application (73) Proprietor: J.R. Simplot Company • MATHEWS, H. ET AL.: "Activation tagging in Boise, ID 83706 (US) tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and (72) Inventor: ROMMENS, Caius transport" THE PLANT CELL, vol. 15, 14 July 2003 Boise, ID 83712 (US) (2003-07-14), pages 1689-1703, XP002467412 • BUSHMAN BRADLEY S ET AL: "Two loci exert (74) Representative: Murphy, Colm Damien et al major effects on chlorogenic acid synthesis in Venner Shipley LLP maize silks." CROP SCIENCE, vol. 42, no. 5, 200 Aldersgate September 2002 (2002-09), pages 1669-1678, London EC1A 4HD (GB) XP002467413 ISSN: 0011-183X • CHUN SUK JUNG ET AL: "The potato P locus codes for flavonoid 3’,5’-hydroxylase" THEORETICAL AND APPLIED GENETICS ; INTERNATIONAL JOURNAL OF PLANT BREEDING RESEARCH, SPRINGER-VERLAG, BE, vol. 110, no. 2, 1 January 2005 (2005-01-01), pages 269-275, XP019321798 ISSN: 1432-2242 • DIXON RICHARD A: "A two-for-one in tomato nutritional enhancement." NATURE BIOTECHNOLOGY JUL 2005, vol. 23, no. 7, July 2005 (2005-07), pages 825-826, XP002467414 ISSN: 1087-0156 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 2 038 300 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 038 300 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention relates to modifying the level of at least one antioxidant in a crop or plant or any product of a crop or plant. Particularly, the present invention modifies the level of at least one polyphenolic compound in a plant or product thereof by modifying the expression of a chlorogenic acid-inducing (Cai) gene. Furthermore, the present invention additionally boosts antioxidant levels by modifying the expression of at least one of the flavanoid- 3’,5’-hydrox- ylase (F3’5’H) gene, a flavanone- 3’-hydroxylase ( F3h), a dihydroflavonol reductase ( Dfr) gene, and a chalcone isomerase 10 (Chi) gene, in addition to expression of the Cai gene. BACKGROUND [0002] Oxygen is a highly reactive molecular species. Unchecked, it can damage tissues, cells, proteins, and DNA 15 and trigger a cascade of dangerous downstream chain reactions that can culminate in cell death and increased oxidative stress in particular tissues. [0003] But reactive oxygen species do not go unchecked. Living organisms have evolved a vast network of complex antioxidants that counterbalance oxidative- induced molecular destruction by, for example, decreasing localized concen- trations of un-reactive oxygen or by scavenging free radicals that would otherwise promote formation of highly reactive 20 species. [0004] Many green vegetables, fruits, plants, and crops are known to produce a myriad different antioxidants. Accord- ingly, to push the balance of competing oxidative interests more toward the antioxidant side of the equation, and thereby promote a healthy condition, it is thought to be desirable to eat antioxidant-rich plant products or to augment in vivo antioxidant levels by regularly taking appropriate dietary supplements. 25 [0005] Since oxidative stress typically is associated with the pathogenesis of a variety of diseases, such as Parkinson’s disease, Alzheimer’s disease, diabetes, rheumatoid arthritis, cardiovascular diseases, and neurodegeneration, it also is thought desirable to ingest antioxidants to combat or minimize the damaging effects of those diseases on the body. [0006] A problem is that there do not exist many plants, vegetables, fruits, or products thereof that are naturally highly rich in antioxidants. Furthermore, when those plants and products are processed and cooked, the levels of antioxidants 30 that ultimately remain in the foodstuff is severely undercut. Moreover, what little antioxidant remains available in the food still has to be timely absorbed into the body. Accordingly, the absence of a large reservoir of antioxidants in the starting plant material is detrimental to the overall concept of antioxidant- induced health and disease resistance vigor. [0007] The present invention provides a genetic approach to solving this problem. Genetically modified potato plants of the present invention produce tubers with highly increased levels of at least one antioxidant. 35 SUMMARY OF THE INVENTION [0008] Accordingly, the invention provides a method for increasing the level of an antioxidant in a plant, comprising transforming a plant to overexpress: 40 (i) a chlorogenic acid-inducing (Cai) polynucleotide of the Cai gene of SEQ ID NO: 1 or 2; or (ii) a polynucleotide that encodes a Cai protein that shares at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 5; wherein the level of the antioxidant, chlorogenic acid, is increased in the transformed plant or its product compared to the level of chlorogenic acid in a non- transformed plant or product thereof. 45 [0009] In the method of (ii) above, the plant may be transformed with a polynucleotide that encodes a Cai protein that shares at least 90% identity with the amino acid sequence of SEQ ID NO: 5. [0010] Other embodiments of the present invention include co-expressing the Cai or Cai-related gene with a nucleic acid that brings about the downregulation or inhibition of an endogenous gene involved in a biosynthetic pathway. That 50 then results in accumulation of a particular antioxidant because the protein or enzyme that would normally convert that substance to the next one in the pathway is no longer expressed. Accordingly, depending on which gene in a particular pathway is downregulated or switched off, different antioxidants can be made to accumulate in the plant. [0011] Accordingly, with that in mind, in one embodiment this method further comprises co-transforming the Cai- expressing plant with a construct that expresses a polynucleotide that brings about downregulation or inhibition of 55 endogenous F35h gene expression in the transformed plant or in its product, wherein the plant or product that is co- transformed with the F35h gene has increased levels of the antioxidant, kaempferol, after the F35h gene is downregulated or inhibited. [0012] In another embodiment, the method further comprises co- transforming the Cai- expressing plant with a construct 2 EP 2 038 300 B1 that expresses a polynucleotide that brings about downregulation or inhibition of endogenous Chi gene expression in the transformed plant or its product, wherein the plant or product that is co- transformed with the Chi gene has increased levels of the antioxidant, chalcone, after the Chi gene is downregulated or inhibited. [0013] The method of the invention may further comprise downregulating or inhibiting the endogenous expression of 5 at least one of (i) the F3’5’H gene, (ii) the F3h gene, (iii) the Dfr gene, and (iv) the Chi gene. [0014] In another embodiment, the method further comprises co- transforming the Cai- expressing plant with a construct that expresses a polynucleotide that modifies the expression of any endogenous flavonoid pathway gene in the trans- formed plant or its product, wherein the transformed plant or its product has increased antioxidant levels. [0015] In one embodiment of the present invention, a plant that may be transformed and co- transformed according to 10 any of the present methods is a solanaceous crop plant. In one embodiment, the solanaceous crop plant is a potato plant, a tobacco plant, a tomato plant, a capsicum plant, or an eggplant. [0016] In another embodiment, the plant is a cruciferous vegetable. In one embodiment, the cruciferous vegetable is kale, collards, chinese broccoli (gai laan), cabbage, brussel sprout, kohlrabi, cauliflower, wild broccoli, broccoli, bok choy, mizuna, flowering cabbage, chinese cabbage, napa cabbage, turnip root, rutabaga, siberian kale, canola/rape 15 seeds, wrapped heart mustard cabbage, mustard seeds, tatsoi, ethiopian mustard, radish, daikon, horseradish, Japanese horseradish (wasabi), arugula, watercress, or cress. [0017] In another embodiment, the plant is a tea plant. In one embodiment, the tea plant is a black tea plant, a green
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