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WO 2013/065046 Al 10 May 2013 (10.05.2013) W P O P C T (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 WO 2013/065046 Al 10 May 2013 (10.05.2013) W P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 15/113 (2010.01) A01H 5/00 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C12N 15/82 (2006.01) DO, 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, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/IL20 12/05043 1 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (22) International Filing Date: NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, 3 1 October 2012 (3 1.10.2012) RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (25) Filing Language: English ZM, ZW. (26) Publication Language: English 4 Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 61/553,355 31 October 201 1 (3 1. 10.201 1) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 61/553,340 31 October 201 1 (3 1. 10.201 1) us TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (71) Applicants: ROSETTA GREEN LTD. [IL/IL]; 13 Gad EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Feinstein Street, 7612101 Rechvot (IL). YANAI-AZU- MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, LAY, Osnat; 24A 9 Hadugit Street, 7543571 Ris- TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, hon-Lezion (IL). ML, MR, NE, SN, TD, TG). (72) Inventors: MAOR, Rudy; 11 Kronnenberg Street, Declarations under Rule 4.17 : 7666206 Rechovot (IL). NESHER, Iris; 4 Kitsis Street, — of inventorship (Rule 4.17(iv)) 6948 103 Tel Aviv (IL). NOIVIRT, Orly; 9 HaTaAs Street, 533971 1 Givataim (IL). Published: (74) Agents: G.E EHRLICH (1995) LTD. et al; 11 Mena- — with international search report (Art. 21(3)) chem Begin Road, 5268 1 Ramat Gan (IL). — with sequence listing part of description (Rule 5.2(a)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: ISOLATED POLYNUCLEOTIDES AND POLYPEPTIDES, TRANSGENIC PLANTS COMPRISING SAME AND USES THEREOF o IN IMPROVINGABIOTIC STRESS TOLERANCE, NITROGEN USE EFFICIENCY, BIOMASS, VIGOR OR YIELD OF PLANTS (57) Abstract: Methods of improving abiotic stress tolerance, nitrogen use efficiency, biomass, vigor or yield of a plant are © provided. According to an aspect the method comprising expressing within the plant an exogenous polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 103, 101-102, 104-216, 223-227, 264-416, wherein the nucleic acid sequence is capable of regulating abiotic stress tolerance of the plant, thereby improving abiotic stress tolerance, nitrogen use efficiency, bio - mass, vigor or yield of the plant. Alternatively, the method comprises, expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a gene encoding an RNAi molecule having a nucleic acid sequence at least 90 % identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-100, 615- 626 and 639, thereby improving abiotic stress tolerance, nitrogen use efficiency, biomass, vigor or yield of a plant. ISOLATED POLYNUCLEOTIDES AND POLYPEPTIDES, TRANSGENIC PLANTS COMPRISING SAME AND USES THEREOF IN IMPROVING ABIOTIC STRESS TOLERANCE, NITROGEN USE EFFICIENCY, BIOMASS, VIGOR OR YIELD OF PLANTS FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to isolated polynucleotides and polypeptides, transgenic plants comprising same and uses thereof in improving abiotic stress tolerance, nitrogen use efficiency, biomass, vigor or yield of plants Abiotic stress (ABST) is a collective term for numerous extreme environmental parameters such as drought, high or low salinity, high or low temperature/light, and nutrient imbalances. The major agricultural crops (corn, rice, wheat, canola and soybean) account for over half of total human caloric intake, giving their overall yield and quality vast importance. Abiotic stress causes more than 50 % yield loss of the above mentioned major crops. Among the various abiotic stresses, drought is the major factor that limits crop productivity worldwide. Furthermore, drought is associated with increase susceptibility to various diseases. Abiotic-stress-induced dehydration or osmotic stress, in the form of reduced availability of water and disruption of turgor pressure, causes irreversible cellular damage. A water-limiting environment at various plant developmental stages may activate various physiological changes. Water deficit, salinity and low/high temperatures are stresses that cause plant cellular dehydration, due to transpiration rate that exceeds water uptake. Drought is known to elicit a response in the plant that mainly affects root architecture, causing activation of plant metabolic pathways driven to maximize water assimilation. Improvement of root architecture, i.e. making branched and longer roots, allows the plant to reach water and nutrient/fertilizer deposits located deeper in the soil by an increase in soil coverage. Thus, genes governing enhancement of root architecture may be used to improve drought tolerance. High salt levels, or salinity, of the soil acts similarly to drought; it prevents roots from extracting water and nutrients and thus reduces the availability of arable land and crop production worldwide, since none of the top five food crops can tolerate excessive salt. Salinity causes a water deficit which leads to osmotic stress (similar to freezing and drought stress) and critically damages biochemical processes. Large land areas throughout the world naturally have high salt levels and thus are currently uncultivable. In regions that rely heavily on agricultural production, soil salinity is a significant problem expected to worsen due to growing population and extreme climatic changes. Since salt accumulates in the upper soil layer where seeds are placed, and may interfere with their germination, salt tolerance is of particular importance early in a plant's lifecycle. Temperature is a critical factor in germination of many crops. Seedlings as well as mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs when transpiration is insufficient to overcome heat stress. Heat shock damages cellular structures and impairs membrane function and overall protein synthesis (except that of heat shock proteins). Heat stress often accompanies conditions of low water availability, such as drought, and the combined stress can fatally alter plant metabolism. Dehydration invokes survival strategies in plants that include structural (lower surface area) as well as cellular content (increase in oil and soluble material) modifications to prevent evaporation and water loss caused by heat, drought, or salinity. There is great variability in responses to abiotic stress among different plant species, but differences also exist among varieties and cultivars within the same plant species. Certain plants are inherently more tolerant to abiotic stress than others, making their genotypes an attractive research subject for potential identification of drought associated genes. The response to any stress may involve both stress specific and common stress pathways, and drains energy from the plant, eventually resulting in lowered yield. Thus, distinguishing between the genes activated in each pathway and subsequent manipulation of only specific relevant genes could lead to a partial stress response without the parallel loss in yield. With a growing world population, increasing demand for food, fuel and fiber, and a changing climate, agriculture faces unprecedented challenges. In general, shortage in water supply is one of the most severe global agricultural problems affecting plant growth and crop yield. To illustrate, large areas of cornfields in the United States may be affected by at least moderate drought in any given year. Excessive efforts are made to alleviate the harmful effects of desertification of the world's arable land. Farmers are seeking advanced, biotechnology-based solutions to enable them to obtain stable high yields and give them the potential to reduce irrigation costs or to grow crops in areas where potable water is a limiting factor. It should be noted that improved ABST will confer plants with improved vigor also under non-stress conditions, resulting in crops having improved biomass and/or yield. Two major small RNA molecules, microRNAs (miRNAs) or small interfering RNAs (siRNAs), potently regulate specific down-regulation/silencing of a target gene, through RNA interference (RNAi). Both miRNAs and siRNAs are oligonucleotides (20-24 bps) processed from longer RNA precursors by Dicer-like ribonucleases, although the source of their precursors is different (i.e., local single RNA molecules with imperfect stem-loop structures for miRNA, and long, double- stranded precursors potentially from bimolecular duplexes for siRNA). Additional characteristics that differentiate miRNAs from siRNAs are their sequence conservation level between related organisms (high in miRNAs, low to non-existent in siRNAs), regulation of genes unrelated to their locus of origin (typical for miRNAs, infrequent in siRNAs) and the genetic requirements for their respective functions are somewhat dissimilar in many organisms. Despite all their differences, miRNAs and siRNAs are overall chemically and functionally similar and both are incorporated into silencing complexes, wherein they can guide post-transcriptional repression of multiple target genes, and thus function catalytically. In contrast to the abundance of genes involved in the responses to abiotic stress in plants, there is limited information on small RNA molecules involved in plant response and adaptation to abiotic stress.
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