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Wo 2010/046471 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 29 April 2010 (29.04.2010) WO 2010/046471 A2 (51) International Patent Classification: 1, 14482 Potsdam (DE). PUZIO, Piotr [DE/BE]; Rene v. C12N 15/82 (2006.01) AOlH 5/00 (2006.01) d. Puttestraat 1, B-9030 Mariakerke (Gent) (BE). SCHAUWECKER, Florian [DE/DE]; Herderstrasse 35, (21) International Application Number: 12163 Berlin (DE). SCHON, Hardy [DE/DE]; Eichen- PCT/EP2009/063979 strasse 26, 13 156 Berlin (DE). THIMM, Oliver (22) International Filing Date: [DE/DE]; Prinzregentenstrasse 93, 1071 7 Berlin (DE). 23 October 2009 (23.10.2009) WENDEL, Birgit [DE/DE]; Alt-Wittenau 67, 13437 Berlin (DE). (25) Filing Language: English (74) Agent: FITZNER, Uwe; Hauser Ring 10, 40878 Ratin- (26) Publication Language: English gen (DE). (30) Priority Data: (81) Designated States (unless otherwise indicated, for every 08167450.9 23 October 2008 (23.10.2008) EP kind of national protection available): AE, AG, AL, AM, (71) Applicant (for all designated States except US): BASF AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, PLANT SCIENCE GMBH [-/DE]; 671 17 Limburger- CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, hof(DE). DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (72) Inventors; and KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (71) Applicants : BLAU, Astrid [OEfDE]; Bahnhofstrasse ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 110, 14532 Stahnsdorf (DE). HAAKE, Volker [DE/DE]; NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, Hennigsdorfer Strasse 143 A, 13503 Berlin (DE). HEN- SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, DRIKS, Janneke [NIVDE]; Fliederweg 5, 14548 TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. Schwielowsee (DE). HEROLD, Michael Manfred [DE/ DE]; Quitzowstrasse 87, 1055 1 Berlin (DE). KAM- (84) Designated States (unless otherwise indicated, for every LAGE, Beate [DE/DE]; Varziner Strasse 13/14, 12161 kind of regional protection available): ARIPO (BW, GH, Berlin (DE). PLESCH, Gunnar [-/DE]; Plantagenhof GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, [Continued on next page] (54) Title: A METHOD FOR PRODUCING A TRANSGENIC CELL WITH INCREASED GAMMA-AMINOBUTYRIC ACID (GABA) CONTENT (57) Abstract: This invention relates gen erally to a method for producing a trans Plasmid Figures genic cell with increased gamma-aminobu- tyric acid (GABA) content as compared to a corresponding non-transformed wild type cell. Fig. 1 Vector VC-MME220-1qcz (SEQ ID NO: 35)used for cloning gene of interest for non-targeted expression. TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, Published: ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, — without international search report and to be republished MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, upon receipt of that report (Rule 48.2(g)) TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). — with sequence listing part of description (Rule 5.2(a)) A method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content [0001.1.1.1] This invention relates generally to a method for producing a trans genic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell. [0002.1.1.1] In particular, this invention relates to plant cells and plants with in creased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type. [0003.1.1.1] The invention also deals with methods of producing and screening for and breeding such plant cells or plants. [0004.1.1.1] Gamma-aminobutyric acid is used to enhance growth of specified plants, prevent development of powdery mildew on grapes, and suppress certain other plant diseases. Humans and animals normally ingest and metabolize gamma- aminobutyric acid in variable amounts. Gamma-aminobutyric acid was registered (li censed for sale) as growth enhancing pesticidal active ingredient in 1998. Gamma- aminobutyric acid is an important signal which helps to regulate mineral availability in plants. Minerals support the biochemical pathways governing growth and reproduction as well as the pathways that direct plant's response to a variety of biotic and abiotic stresses. Mineral needs are especially high during times of stress and at certain stages of plant growth. Gamma-aminobutyric acid levels in plants naturally increase at these times. [0005.1.1.1] Gamma-Aminobutyric acid (GABA), a nonprotein amino acid, is often accumulated in plants following environmental stimuli that can also cause ethylene production. Exogenous GABA causes up to a 14-fold increase in the ethylene produc tion rate after about 12 h. GABA causes increases in ACC synthase mRNA accumula tion, ACC levels, ACC oxidase mRNA levels and in vitro ACC oxidase activity. Possible roles of GABA as a signal transducer are suggested, see Plant Physiol.1 15(1 ):129- 35(1997). [0006.1.1.1] Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. In plants, stress initiates a signal-transduction pathway, in which increased cytosolic Ca2+ activates Ca27calmodulin-dependent glutamate decarboxy- lase activity and GABA synthesis. Elevated H+ and substrate levels can also stimulate glutamate decarboxylase activity. GABA accumulation probably is mediated primarily by glutamate decarboxylase. Experimental evidence supports the involvement of GABA synthesis in pH regulation, nitrogen storage, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization, see Trends Plant Sci. 4(1 1):446-452(1 999). [0007.1.1.1] Rapid GABA accumulation in response to wounding may play a role in plant defense against insects (Ramputh and Brown, Plant Physiol. 111(1996): 1349- 1352). The development of gamma aminobutyrate (GABA) as a potential control agent in plant - invertebrate pest systems has been reviewed in Shelp et al., Canadien Jour nal of Botany (2003) 8 1, 11, 1045-1 048. The authors describe that available evidence indicates that GABA accumulation in plants in response to biotic and abiotic stresses is mediated via the activation of glutamate decarboxylase. More applied research, based on the fact that GABA acts as an inhibitory neurotransmitter in invertebrate pests, ind i cates that ingested GABA disrupts nerve functioning and causes damage to oblique- banded leafroller larvae, and that walking or herbivory by tobacco budworm and oblique-banded leafroller larvae stimulate GABA accumulation in soybean and tobacco, respectively. In addition, elevated levels of endogenous GABA in genetically engi- neered tobacco deter feeding by tobacco budworm larvae and infestation by the north ern root-knot nematode. Therefore the author concluded that genetically engineered crop species having high GABA-producing potential may be an alternative strategy to chemical pesticides for the management of invertebrate pests. [0008.1.1.1] During angiosperm reproduction, pollen grains form a tube that navi- gates through female tissues to the micropyle, delivering sperm to the egg. In vitro, GABA stimulates pollen tube growth. [0009.1.1.1] Much of the recent work on GABA in plants has concentrated on its metabolic role (Fait et al., Trends in Plant Sci., Vol. 13, Nr. 1, pp 14-19, 2007) and on stress/pest-associated and signalling roles (Bouche et al., Trends in Plant Sci., Vol. 9, Nr. 3 , pp 110-1 15, 2004). Accumulation of GABA in plant tissues and transport fluids are responses to many abiotic stresses (Allan et al., J Exp Bot, Vol. 59, No. 9 , pp. 2555-2564, 2008). Beuve et al. (in PCE, 27, 1035-1046, 2004) found that nitrate influx and GABA were positively correlated in short- and long-term experiments and that exogenous GABA supply to the roots induced a significant increase of BnNrt2 (Nitrate transporter) mRNA expression. A further approach was the use of GABA for stimulation of plant growth by applying GABA to plants foliage, stems and/ or roots in a 1 to 5000 ppm GABA solution, prefer- rably together with a readily metabolized carbon source (organic acids, amino acids, simple carbohydrates, and mixtures of organic acids amino acids and simple carbohy- drates). [0010.1.1.1] Even though the role of GABA in the cell is not yet understood and the action mechanisms not yet clarified, due to these physiological roles and agrobio- technological potential of GABA there is a need to identify genes of enzymes and other proteins involved in GABA metabolism. Especially there is a need to generate mutants or transgenic plant lines with which to modify the GABA content in plants in order to enhace the plant yield traits. [0011.1.1.1] Accordingly, in a first embodiment, the invention relates to a method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content as compared to a corresponding non-transformed wild type cell by increasing or gener ating one or more activities selected from the group consisting of: 60S ribosomal pro tein, ABC transporter permease protein, acetyltransferase, acyl-carrier protein, At4g32480-protein, At5g16650-protein, ATP-binding protein, Autophagy-related protein , auxin response factor, auxin transcription factor, b1003-protein, b1522-protein, b2739-protein, b3646-protein, B4029-protein, Branched-chain amino acid permease , calcium-dependent protein kinase, cytochrome c oxidase subunit VIII, elongation factor Tu, Factor arrest protein , fumarylacetoacetate hydrolase, geranylgeranyl pyrophos phate synthase, glucose dehydrogenase, glycosyl transferase, harpin-induced family protein, homocitrate synthase, hydrolase, isochorismate synthase, MFS-type trans- porter protein, microsomal beta-keto-reductase, polygalacturonase, protein phos phatase, pyruvate kinase, Sec-independent protein translocase subunit, serine prote ase, thioredoxin, thioredoxin family protein, transcriptional regulator, ubiquinone bio synthesis monooxygenase , and YHR213W-protein.
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