WO2012056401A1

WO2012056401A1 - ISOLATED POLYNUCLEOTIDES EXPRESSING OR MODULATING MICRORNAs OR TARGETS OF SAME, TRANSGENIC PLANTS COMPRISING SAME AND USES THEREOF IN IMPROVING NITROGEN USE EFFICIENCY, ABIOTIC STRESS TOLERANCE, BIOMASS, VIGOR OR YIELD OF A PLANT

Info

Publication number: WO2012056401A1
Application number: PCT/IB2011/054763
Authority: WO
Inventors: Rudy Maor; Iris Nesher
Original assignee: Rosetta Green Ltd.
Priority date: 2010-10-25
Filing date: 2011-10-25
Publication date: 2012-05-03
Also published as: EP2633056A1; CA2815769A1; AU2011322146A1; US20140013469A1; IL225964A0

Abstract

Isolated polynucleotides expressing or modulating microRNAs or targets of same are provided. Also provided are transgenic plants comprising same and uses thereof in improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant

Description

ISOLATED POLYNUCLEOTIDES EXPRESSING OR MODULATING microRNAs OR TARGETS OF SAME, TRANSGENIC PLANTS COMPRISING SAME AND USES THEREOF IN IMPROVING NITROGEN USE EFFICIENCY, ABIOTIC STRESS TOLERANCE, BIOMASS, VIGOR OR YIELD OF A PLANT

RELATED APPLICATION/S

This Application claims priority from U.S. Provisional Patent Application No. 61/406,184 filed on October 25, 2010, the contents of which are hereby incorporated by reference in its entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to isolated polynucleotides expressing or modulating microRNAs or targets of same, transgenic plants comprising same and uses thereof in improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant.

Plant growth is reliant on a number of basic factors: light, air, water, nutrients, and physical support. All these factors, with the exception of light, are controlled by soil to some extent, which integrates non-living substances (minerals, organic matter, gases and liquids) and living organisms (bacteria, fungi, insects, worms, etc.). The soil's volume is almost equally divided between solids and water/gases. An adequate nutrition in the form of natural as well as synthetic fertilizers, may affect crop yield and quality, and its response to stress factors such as disease and adverse weather. The great importance of fertilizers can best be appreciated when considering the direct increase in crop yields over the last 40 years, and the fact that they account for most of the overhead expense in agriculture. Sixteen natural nutrients are essential for plant growth, three of which, carbon, hydrogen and oxygen, are retrieved from air and water. The soil provides the remaining 13 nutrients.

Nutrients are naturally recycled within a self-sufficient environment, such as a rainforest. However, when grown in a commercial situation, plants consume nutrients for their growth and these nutrients need to be replenished in the system. Several nutrients are consumed by plants in large quantities and are referred to as macronutrients. Three macronutrients are considered the basic building blocks of plant growth, and are provided as main fertilizers; Nitrogen (N), Phosphate (P) and Potassium (K). Yet, only nitrogen needs to be replenished every year since plants only absorb approximately half of the nitrogen fertilizer applied. A proper balance of nutrients is crucial; when too much of an essential nutrient is available, it may become toxic to plant growth. Utilization efficiencies of macronutrients directly correlate with yield and general plant tolerance, and increasing them will benefit the plants themselves and the environment by decreasing seepage to ground water.

Nitrogen is responsible for biosynthesis of amino and nucleic acids, prosthetic groups, plant hormones, plant chemical defenses, etc, and thus is utterly essential for the plant. For this reason, plants store nitrogen throughout their developmental stages, in the specific case of corn during the period of grain germination, mostly in the leaves and stalk. However, due to the low nitrogen use efficiency (NUE) of the main crops (e.g., in the range of only 30-70 %), nitrogen supply needs to be replenished at least twice during the growing season. This requirement for fertilizer refill may become the rate-limiting element in plant growth and increase fertilizer expenses for the farmer. Limited land resources combined with rapid population growth will inevitably lead to added increase in fertilizer use. In light of this prediction, advanced, biotechnology- based solutions to allow stable high yields with an added potential to reduce fertilizer costs are highly desirable. Subsequently, developing plants with increased NUE will lower fertilizer input in crop cultivation, and allow growth on lower-quality soils.

The major agricultural crops (corn, rice, wheat, canola and soybean) account for over half of total human caloric intake, giving their yield and quality vast importance. They can be consumed either directly (eating their seeds which are also used as a source of sugars, oils and metabolites), or indirectly (eating meat products raised on processed seeds or forage). Various factors may influence a crop's yield, including but not limited to, quantity and size of the plant organs, plant architecture , vigor (e.g. seedling), growth rate, root development, utilization of water and nutrients (e.g., nitrogen), and stress tolerance. Plant yield may be amplified through multiple approaches; (1) enhancement of innate traits (e.g., dry matter accumulation rate, cellulose/lignin composition), (2) improvement of structural features (e.g., stalk strength, meristem size, plant branching pattern), and (3) amplification of seed yield and quality (e.g., fertilization efficiency, seed development, seed filling or content of oil, starch or protein). Increasing plant yield through any of the above methods would ultimately have many applications in agriculture and additional fields such as in the biotechnology industry.

Two main adverse environmental conditions, malnutrition (nutrient deficiency) and drought, elicit a response in the plant that mainly affects root architecture (Jiang and Huang (2001), Crop Sci 41 : 1168-1173; Lopez-Bucio et al. (2003), Curr Opin Plant Biol, 6:280-287; Morgan and Condon (1986), Aust J Plant Physiol 13:523-532), causing activation of plant metabolic pathways 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. Root morphogenesis has already shown to increase tolerance to low phosphorus availability in soybean (Miller et al, (2003), Funct Plant Biol 30:973-985) and maize (Zhu and Lynch (2004), Funct Plant Biol 31 :949-958). Thus, genes governing enhancement of root architecture may be used to improve NUE and drought tolerance. An example for a gene associated with root developmental changes is ANR1, a putative transcription factor with a role in nitrate (N03 ) signaling. When expression of ANR1 is down-regulated, the resulting transgenic lines are defective in their root response to localized supplies of nitrate (Zhang and Forde (1998), Science 270:407). Enhanced root system and/or increased storage capabilities, which are seen in responses to different environmental stresses, are strongly favorable at normal or optimal growing conditions as well.

Abiotic stress refers to a range of suboptimal conditions as water deficit or drought, extreme temperatures and salt levels, and high or low light levels. High or low nutrient level also falls into the category of abiotic stress. The response to any stress may involve both stress specific and common stress pathways (Pastori and Foyer (2002), Plant Physiol, 129: 460-468), 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. Contrary to the complex polygenic nature of plant traits responsible for adaptations to adverse environmental stresses, information on miRNAs involved in these responses is very limited. The most common approach for crop and horticultural improvements is through cross breeding, which is relatively slow, inefficient, and limited in the degree of variability achieved because it can only manipulate the naturally existing genetic diversity. Taken together with the limited genetic resources (i.e., compatible plant species) for crop improvement, conventional breeding is evidently unfavorable. By creating a pool of genetically modified plants, one broadens the possibilities for producing crops with improved economic or horticultural traits.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 10, 6-9, 21, 22, 23-37, 38-52, 1209, 1211, 1212, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

According to an aspect of some embodiments of the present invention there is provided a transgenic plant exogenously expressing a polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 10, 6-9, 23-37, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant.

According to some embodiments of the invention, said exogenous polynucleotide encodes a precursor of said nucleic acid sequence.

According to some embodiments of the invention, said precursor of said nucleic acid sequence is at least 60 % identical to SEQ ID NO: 21, 22, 38-52, 1209, 1211, 1212.

According to some embodiments of the invention, said exogenous polynucleotide encodes a miRNA or a precursor thereof.

According to some embodiments of the invention, said exogenous polynucleotide encodes a siRNA or a precursor thereof.

According to some embodiments of the invention, said exogenous polynucleotide is selected from the group consisting of SEQ ID NO: 10, 6-9, 21, 22, 23- 37, 38-52, 1209, 1211, 1212. According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NO: 6, 7 and 9, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of a plant.

According to some embodiments of the invention, said nucleic acid sequence is selected from the group consisting of SEQ ID NO: 6, 7 and 9.

According to some embodiments of the invention, said polynucleotide encodes a precursor of said nucleic acid sequence.

According to some embodiments of the invention, said polynucleotide encodes a miRNA or a precursor thereof.

According to some embodiments of the invention, said polynucleotide encodes a siRNA or a precursor thereof.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising the isolated polynucleotide above under the regulation of a cis-acting regulatory element.

According to some embodiments of the invention, said cis-acting regulatory element comprises a promoter.

According to some embodiments of the invention, said promoter comprises a tissue-specific promoter.

According to some embodiments of the invention, said tissue-specific promoter comprises a root specific promoter.

According to an aspect of some embodiments of the present invention there is provided a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising 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 SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant.

According to an aspect of some embodiments of the present invention there is provided a transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a gene encoding an RNAi molecule having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209.

According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide which downregulates an activity or expression of a gene encoding an RNAi molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209.

According to some embodiments of the invention, said polynucleotide encodes a miRNA-Resistant Target as set forth in SEQ ID NO 1104-1124.

According to some embodiments of the invention, said isolated polynucleotide encodes a target mimic as set forth in SEQ ID NO: 18 or 19.

According to some embodiments of the invention, the method further comprises growing the plant under limiting nitrogen conditions.

According to some embodiments of the invention, the method further comprises growing the plant under abiotic stress.

According to some embodiments of the invention, said abiotic stress is selected from the group consisting of salinity, drought, water deprivation, flood, etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution and UV irradiation.

According to some embodiments of the invention, the plant is a monocotyledon.

According to some embodiments of the invention, the plant is a dicotyledon. According to an aspect of some embodiments of the present invention there is provided a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

According to an aspect of some embodiments of the present invention there is provided a transgenic plant exogenously expressing a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, and wherein said polynucleotide is under a transcriptional control of a cis-acting regulatory element.

According to some embodiments of the invention, said polynucleotide is selected from the group consisting of SEQ ID NO: 1022-1090.

According to some embodiments of the invention, said polypeptide is selected from the group consisting of SEQ ID NO: 927-1021.

According to some embodiments of the invention, the plant is a monocotyledon.

According to some embodiments of the invention, the plant is a dicotyledon. According to an aspect of some embodiments of the present invention there is provided a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

According to an aspect of some embodiments of the present invention there is provided a transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of a plant, said nucleic acid sequence being under the regulation of a cis-acting regulatory element.

According to some embodiments of the invention, said polynucleotide acts by a mechanism selected from the group consisting of sense suppression, antisense suppresion, ribozyme inhibition, gene disruption.

According to some embodiments of the invention, said tissue-specific promoter comprises a root specific promoter. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a scheme of a binary vector that can be used according to some embodiments of the invention;

FIGs. 2 A- J are schematic illustrations of some of the miRNA sequences which may be used in accordance with the present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The doubling of agricultural food production worldwide over the past four decades has been associated with a 7-fold increase in the use of nitrogen (N) fertilizers. As a consequence, both the recent and future intensification of the use of nitrogen fertilizers in agriculture already has and will continue to have major detrimental impacts on the diversity and functioning of the non-agricultural neighbouring bacterial, animal, and plant ecosystems. The most typical examples of such an impact are the eutrophication of freshwater and marine ecosystems as a result of leaching when high rates of nitrogen fertilizers are applied to agricultural fields. In addition, there can be gaseous emission of nitrogen oxides reacting with the stratospheric ozone and the emission of toxic ammonia into the atmosphere. Furthermore, farmers are facing increasing economic pressures with the rising fossil fuels costs required for production of nitrogen fertilizers.

It is therefore of major importance to identify the critical steps controlling plant nitrogen use efficiency (NUE). Such studies can be harnessed towards generating new energy crop species that have a larger capacity to produce biomass with the minimal amount of nitrogen fertilizer.

While reducing the present invention to practice, the present inventors have uncovered microRNA (miR A) sequences that are differentially expressed in maize plants grown under nitrogen limiting conditions versus maize plants grown under conditions wherein nitrogen is a non-limiting factor. Following extensive experimentation and screening the present inventors have identified miRNA sequences that are upregulated or downregulated in roots and leaves, and suggest using same or sequences controlling same in the generation of transgenic plants having improved nitrogen use efficiency. While further reducing the present invention to practice, the present inventors have analyzed the level of expression of the identified miRNA sequences under optima, and nitrogen deficient conditions by quantitiative RT-PCR and validated the correlation between miRNA expression nitrogen availability. These findings support the use of the miRNA sequences or sequences controlling same or targets thereof in the generation of transgenic plants characterized by improved nitrogen use efficiency and abiotic stress tolerance.

According to some embodiments, the newly uncovered miRNA sequences relay their effect by affecting at least one of:

root architecture so as to increase nutrient uptake; activation of plant metabolic pathways so as to maximize nitrogen absorption or localization; or alternatively or additionally

modulating plant surface permeability.

Each of the above mechanisms may affect water uptake as well as salt absorption and therefore embodiments of the invention further relate to enhancement of abiotic stress tolerance, biomass, vigor or yield of the plant.

Thus, according to an aspect of the invention there is provided a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide having a nucleic acid sequence at least 80 %, 85 %, 90 % or 95 % identical to SEQ ID NOs: 10, 6-9 and 23-37 wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

According to a specific embodiment the exogenous polynucleotide has a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 10, 6-9, 23-37.

According to a specific embodiment the exogenous polynucleotide has a nucleic acid sequence at least 95 % identical to SEQ ID NOs: 10, 6-9, 23-37.

According to a specific embodiment the exogenous polynucleotide has a nucleic acid sequence as set forth in SEQ ID NOs: 10, 6-9, 23-37.

As used herein the phrase "nitrogen use efficiency (NUE)" refers to a measure of crop production per unit of nitrogen fertilizer input. Fertilizer use efficiency (FUE) is a measure of NUE. Crop production can be measured by biomass, vigor or yield. The plant's nitrogen use efficiency is typically a result of an alteration in at least one of the uptake, spread, absorbance, accumulation, relocation (within the plant) and use of nitrogen absorbed by the plant. Improved NUE is with respect to that of a non- transgenic plant (i.e., lacking the transgene of the transgenic plant) of the same species and of the same developmental stage and grown under the same conditions.

As used herein the phrase "nitrogen-limiting conditions" refers to growth conditions which include a level (e.g., concentration) of nitrogen (e.g., ammonium or nitrate) applied which is below the level needed for optimal plant metabolism, growth, reproduction and/or viability. The phrase "abiotic stress" as used herein refers to any adverse effect on metabolism, growth, viability and/or reproduction of a plant. Abiotic stress can be induced by any of suboptimal environmental growth conditions such as, for example, water deficit or drought, flooding, freezing, low or high temperature, strong winds, heavy metal toxicity, anaerobiosis, high or low nutrient levels (e.g. nutrient deficiency), high or low salt levels (e.g. salinity), atmospheric pollution, high or low light intensities (e.g. insufficient light) or UV irradiation. Abiotic stress may be a short term effect (e.g. acute effect, e.g. lasting for about a week) or alternatively may be persistent (e.g. chronic effect, e.g. lasting for example 10 days or more). The present invention contemplates situations in which there is a single abiotic stress condition or alternatively situations in which two or more abiotic stresses occur.

According to an exemplary embodiment the abiotic stress refers to salinity.

According to another exemplary embodiment the abiotic stress refers to drought.

As used herein the phrase "abiotic stress tolerance" refers to the ability of a plant to endure an abiotic stress without exhibiting substantial physiological or physical damage (e.g. alteration in metabolism, growth, viability and/or reproductivity of the plant).

As used herein the term/phrase "biomass", "biomass of a plant" or "plant biomass" refers to the amount (e.g., measured in grams of air-dry tissue) of a tissue produced from the plant in a growing season. An increase in plant biomass can be in the whole plant or in parts thereof such as aboveground (e.g. harvestable) parts, vegetative biomass, roots and/or seeds.

As used herein the term/phrase "vigor", "vigor of a plant" or "plant vigor" refers to the amount (e.g., measured by weight) of tissue produced by the plant in a given time. Increased vigor could determine or affect the plant yield or the yield per growing time or growing area. In addition, early vigor (e.g. seed and/or seedling) results in improved field stand.

As used herein the term/phrase "yield", "yield of a plant" or "plant yield" refers to the amount (e.g., as determined by weight or size) or quantity (e.g., numbers) of tissues or organs produced per plant or per growing season. Increased yield of a plant can affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time. According to an exemplary embodiment the yield is measured by cellulose content.

According to another exemplary embodiment the yield is measured by oil content.

According to another exemplary embodiment the yield is measured by protein content.

According to another exemplary embodiment, the yield is measured by seed number per plant or part thereof (e.g., kernel).

A plant yield can be affected by various parameters including, but not limited to, plant biomass; plant vigor; plant growth rate; seed yield; seed or grain quantity; seed or grain quality; oil yield; content of oil, starch and/or protein in harvested organs (e.g., seeds or vegetative parts of the plant); number of flowers (e.g. florets) per panicle (e.g. expressed as a ratio of number of filled seeds over number of primary panicles); harvest index; number of plants grown per area; number and size of harvested organs per plant and per area; number of plants per growing area (e.g. density); number of harvested organs in field; total leaf area; carbon assimilation and carbon partitioning (e.g. the distribution/allocation of carbon within the plant); resistance to shade; number of harvestable organs (e.g. seeds), seeds per pod, weight per seed; and modified architecture [such as increase stalk diameter, thickness or improvement of physical properties (e.g. elasticity)] .

As used herein the term "improving" or "increasing" refers to at least about 2 %, at least about 3 %, at least about 4 %, at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 % or greater increase in NUE, in tolerance to abiotic stress, in yield, in biomass or in vigor of a plant, as compared to a native or wild-type plants [i.e., plants not genetically modified to express the biomolecules (polynucleotides) of the invention, e.g., a non-transformed plant of the same species and of the same developmental stage which is grown under the same growth conditions as the transformed plant].

Improved plant NUE is translated in the field into either harvesting similar quantities of yield, while implementing less fertilizers, or increased yields gained by implementing the same levels of fertilizers. Thus, improved NUE or FUE has a direct effect on plant yield in the field.

The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and isolated plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.

As used herein the phrase "plant cell" refers to plant cells which are derived and isolated from disintegrated plant cell tissue or plant cell cultures.

As used herein the phrase "plant cell culture" refers to any type of native (naturally occurring) plant cells, plant cell lines and genetically modified plant cells, which are not assembled to form a complete plant, such that at least one biological structure of a plant is not present. Optionally, the plant cell culture of this aspect of the present invention may comprise a particular type of a plant cell or a plurality of different types of plant cells. It should be noted that optionally plant cultures featuring a particular type of plant cell may be originally derived from a plurality of different types of such plant cells.

Any commercially or scientifically valuable plant is envisaged in accordance with these embodiments of the invention. Plants that are particularly useful in the methods of the invention include all plants which belong to the super family Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fieckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barely, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively algae and other non-Viridiplantae can be used for the methods of the present invention.

According to some embodiments of the invention, the plant used by the method of the invention is a crop plant including, but not limited to, cotton, Brassica vegetables, oilseed rape, sesame, olive tree, palm oil, banana, wheat, corn or maize, barley, alfalfa, peanuts, sunflowers, rice, oats, sugarcane, soybean, turf grasses, barley, rye, sorghum, sugar cane, chicory, lettuce, tomato, zucchini, bell pepper, eggplant, cucumber, melon, watermelon, beans, hibiscus, okra, apple, rose, strawberry, chile, garlic, pea, lentil , canola, mums, arabidopsis, broccoli, cabbage, beet, quinoa, spinach, squash, onion, leek, tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, and also plants used in horticulture, floriculture or forestry, such as, but not limited to, poplar, fir, eucalyptus, pine, an ornamental plant, a perennial grass and a forage crop, coniferous plants, moss, algae, as well as other plants listed in World Wide Web (dot) nationmaster (dot) com/encyclopedia/Plantae.

According to a specific embodiment of the present invention, the plant comprises corn.

According to a specific embodiment of the present invention, the plant comprises sorghum.

As used herein, the phrase "exogenous polynucleotide" refers to a heterologous nucleic acid sequence which may not be naturally expressed within the plant or which overexpression in the plant is desired. The exogenous polynucleotide may be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the plant.

As mentioned the present teachings are based on the identification of miRNA sequences which modulate nitrogen use efficiency of plants.

According to some embodiments the exogenous polynucleotide encodes a miRNA or a precursor thereof.

As used herein, the phrase "microRNA (also referred to herein interchangeably as "miRNA" or "miR") or a precursor thereof refers to a microRNA (miRNA) molecule acting as a post-transcriptional regulator. Typically, the miRNA molecules are RNA molecules of about 20 to 22 nucleotides in length which can be loaded into a RISC complex and which direct the cleavage of another RNA molecule, wherein the other RNA molecule comprises a nucleotide sequence essentially complementary to the nucleotide sequence of the miRNA molecule. Typically, a miRNA molecule is processed from a "pre-miRNA" or as used herein a precursor of a pre-miRNA molecule by proteins, such as DCL proteins, present in any plant cell and loaded onto a RISC complex where it can guide the cleavage of the target RNA molecules.

Pre-microRNA molecules are typically processed from pri-microRNA molecules (primary transcripts). The single stranded RNA segments flanking the pre- microRNA are important for processing of the pri-miRNA into the pre-miRNA. The cleavage site appears to be determined by the distance from the stem-ssRNA junction (Han et al. 2006, Cell 125, 887-901, 887-901).

As used herein, a "pre-miRNA" molecule is an RNA molecule of about 100 to about 200 nucleotides, preferably about 100 to about 130 nucleotides which can adopt a secondary structure comprising a double stranded RNA stem and a single stranded RNA loop (also referred to as "hairpin") and further comprising the nucleotide sequence of the miRNA (and its complement sequence) in the double stranded RNA stem. According to a specific embodiment, the miRNA and its complement are located about 10 to about 20 nucleotides from the free ends of the miRNA double stranded RNA stem. The length and sequence of the single stranded loop region are not critical and may vary considerably, e.g. between 30 and 50 nt in length. The complementarity between the miRNA and its complement need not be perfect and about 1 to 3 bulges of unpaired nucleotides can be tolerated. The secondary structure adopted by an RNA molecule can be predicted by computer algorithms conventional in the art such as mFOLD. The particular strand of the double stranded RNA stem from the pre-miRNA which is released by DCL activity and loaded onto the RISC complex is determined by the degree of complementarity at the 5' end, whereby the strand which at its 5' end is the least involved in hydrogen bounding between the nucleotides of the different strands of the cleaved dsRNA stem is loaded onto the RISC complex and will determine the sequence specificity of the target RNA molecule degradation. However, if empirically the miRNA molecule from a particular synthetic pre-miRNA molecule is not functional (because the "wrong" strand is loaded on the RISC complex), it will be immediately evident that this problem can be solved by exchanging the position of the miRNA molecule and its complement on the respective strands of the dsRNA stem of the pre- miRNA molecule. As is known in the art, binding between A and U involving two hydrogen bounds, or G and U involving two hydrogen bounds is less strong that between G and C involving three hydrogen bounds. Exemplary hairpin sequences are provided in Tables 1, 3 and 4, below.

Naturally occurring miRNA molecules may be comprised within their naturally occurring pre-miRNA molecules but they can also be introduced into existing pre- miRNA molecule scaffolds by exchanging the nucleotide sequence of the miRNA molecule normally processed from such existing pre-miRNA molecule for the nucleotide sequence of another miRNA of interest. The scaffold of the pre-miRNA can also be completely synthetic. Likewise, synthetic miRNA molecules may be comprised within, and processed from, existing pre-miRNA molecule scaffolds or synthetic pre- miRNA scaffolds. Some pre-miRNA scaffolds may be preferred over others for their efficiency to be correctly processed into the designed microRNAs, particularly when expressed as a chimeric gene wherein other DNA regions, such as untranslated leader sequences or transcription termination and polyadenylation regions are incorporated in the primary transcript in addition to the pre-microRNA.

According to the present teachings, the miRNA molecules may be naturally occurring or synthetic.

Thus, the present teachings contemplate expressing an exogenous polynucleotide having a nucleic acid sequence at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % 99 % or 100 % identical to SEQ ID NOsl-10, 23-37, 57-449, provided that they regulate nitrogen use efficiency.

Alternatively or additionally, the present teachings contemplate expressing an exogenous polynucleotide having a nucleic acid sequence at least 65%, 50 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % 99 % or 100 % identical to SEQ ID NOs. 1-10, 21 and 22 (mature and precursors Tables 1 and 3, and Figures 2A-H representing the core maize genes), provided that they regulate nitrogen use efficiency.

Tables 1 and 3 below illustrates exemplary miRNA sequences and precursors thereof which over expression are associated with modulation of nitrogen use efficiency.

The present invention envisages the use of homologous and orthologous sequences of the above miRNA molecules. At the precursor level use of homologous sequences can be done to a much broader extend. Thus, in such precursor sequences the degree of homology may be lower in all those sequences not including the mature miRNA segment therein.

As used herein, the phrase "stem-loop precursor" refers to stem loop precursor RNA structure from which the miRNA can be processed.

As used herein, a "pre -miRNA" molecule is an RNA molecule of about 100 to about 200 nucleotides, preferably about 100 to about 130 nucleotides which can adopt a secondary structure comprising a double stranded RNA stem and a single stranded RNA loop (also referred to as "hairpin") and further comprising the nucleotide sequence of the miRNA (and its complement sequence) in the double stranded RNA stem. According to a specific embodiment, the miRNA and its complement are located about 10 to about 20 nucleotides from the free ends of the miRNA double stranded RNA stem. The length and sequence of the single stranded loop region are not critical and may vary considerably, e.g. between 30 and 50 nt in length. The complementarity between the miRNA and its complement need not be perfect and about 1 to 3 bulges of unpaired nucleotides can be tolerated. The secondary structure adopted by an RNA molecule can be predicted by computer algorithms conventional in the art such as mFOLD. The particular strand of the double stranded RNA stem from the pre -miRNA which is released by DCL activity and loaded onto the RISC complex is determined by the degree of complementarity at the 5' end, whereby the strand which at its 5' end is the least involved in hydrogen bounding between the nucleotides of the different strands of the cleaved dsRNA stem is loaded onto the RISC complex and will determine the sequence specificity of the target RNA molecule degradation. However, if empirically the miRNA molecule from a particular synthetic pre -miRNA molecule is not functional (because the "wrong" strand is loaded on the RISC complex), it will be immediately evident that this problem can be solved by exchanging the position of the miRNA molecule and its complement on the respective strands of the dsRNA stem of the pre- miRNA molecule. As is known in the art, binding between A and U involving two hydrogen bounds, or G and U involving two hydrogen bounds is less strong that between G and C involving three hydrogen bounds.

Thus, according to a specific embodiment, the exogenous polynucleotide encodes a stem-loop precursor of the nucleic acid sequence. Such a stem-loop precursor can be at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 % or more identical to SEQ ID NOs: 21-22, 38-52, 1209, 1211, 1212, 454-846, 53- 56, 1209 (homologs precursor Tables 1 and 3 and Figures 2A-H), provided that it regulates nitrogen use efficiency.

Identity (e.g., percent identity) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

Homology (e.g., percent homology, identity + similarity) can be determined using any homology comparison software, including for example, the TBLASTN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

According to some embodiments of the invention, the term "homology" or "homologous" refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences.

Homologous sequences include both orthologous and paralogous sequences. The term "paralogous" relates to gene-duplications within the genome of a species leading to paralogous genes. The term "orthologous" relates to homologous genes in different organisms due to ancestral relationship.

One option to identify orthologues in monocot plant species is by performing a reciprocal blast search. This may be done by a first blast involving blasting the sequence-of-interest against any sequence database, such as the publicly available NCBI database which may be found at: Hypertext Transfer Protocol ://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov. The blast results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence-of- interest is derived. The results of the first and second blasts are then compared. An orthologue is identified when the sequence resulting in the highest score (best hit) in the first blast identifies in the second blast the query sequence (the original sequence-of- interest) as the best hit. Using the same rational a paralogue (homolog to a gene in the same organism) is found. In case of large sequence families, the ClustalW program may be used [Hypertext Transfer Protocol ://World Wide Web (dot) ebi (dot) ac (dot) uk/Tools/clustalw2/index (dot) html], followed by a neighbor-joining tree (Hypertext Transfer Protocol://en (dot) wikipedia (dot) org/wiki/Neighbor-joining) which helps visualizing the clustering.

Interestingly, while screening for R Ai regulatory sequences, the present inventors have identified a number of miR A sequences which have never been described before.

Thus, according to an aspect of the invention there is provided an isolated polynucleotide having a nucleic acid sequence at least 80 %, 85 % or preferably 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % 99 % or 100 % identical to SEQ ID NO: 6, 7, 9, 1209, 1210, 1211, 1212 (Table 1 predicted both upregulated and downregulated), wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of a plant.

According to a specific embodiment, the isolated polynucleotide encodes a stem-loop precursor of the nucleic acid sequence.

According to a specific embodiment, the stem-loop precursor is at least about 60

%, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 % or more identical to the precursor sequence of SEQ ID NOs: 21, 22, 38-52, 1209, 1211, 1212, 454-846 and 53- 56, 1209 (predicted stem and loop), provided that it regulates nitrogen use efficiency.

As mentioned, the present inventors have also identified RNAi sequences which are down regulated under nitrogen limiting conditions.

Thus, according to an aspect of the invention there is provided a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a gene encoding a miRNA molecule having a nucleic acid sequence at least 80 %, 85 % or preferably 90 %, 95 % or even 100 % identical to the sequence selected from the group consisting of SEQ ID NOs: 4, 1-3, 5, 53-56, 1209, 57-449, 454-846 (Tables 1 and 4 down-regulated), thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant

There are various approaches to down regulate miRNA sequences.

As used herein the term "down-regulation" refers to reduced activity or expression of the miRNA (at least 10 %, 20 %, 30 %, 50 %, 60 %, 70 %, 80 %, 90 % or 100 % reduction in activity or expression) as compared to its activity or expression in a plant of the same species and the same developmental stage not expressing the exogenous polynucleotide.

Nucleic acid agents that down-regulate miR activity include, but are not limited to, a target mimic, a micro-RNA resistant gene and a miRNA inhibitor.

The target mimic or micro-RNA resistant target is essentially complementary to the microRNA provided that one or more of following mismatches are allowed:

(a) a mismatch between the nucleotide at the 5' end of the microRNA and the corresponding nucleotide sequence in the target mimic or micro-RNA resistant target;

(b) a mismatch between any one of the nucleotides in position 1 to position 9 of the microRNA and the corresponding nucleotide sequence in the target mimic or micro-RNA resistant target; or

(c) three mismatches between any one of the nucleotides in position 12 to position 21 of the microRNA and the corresponding nucleotide sequence in the target mimic or micro-RNA resistant target provided that there are no more than two consecutive mismatches.

The target mimic RNA is essentially similar to the target RNA modified to render it resistant to miRNA induced cleavage, e.g. by modifying the sequence thereof such that a variation is introduced in the nucleotide of the target sequence complementary to the nucleotides 10 or 11 of the miRNA resulting in a mismatch.

Alternatively, a microRNA-resistant target may be implemented. Thus, a silent mutation may be introduced in the microRNA binding site of the target gene so that the DNA and resulting RNA sequences are changed in a way that prevents microRNA binding, but the amino acid sequence of the protein is unchanged. Thus, a new sequence can be synthesized instead of the existing binding site, in which the DNA sequence is changed, resulting in lack of miRNA binding to its target.

Tables 10 and 11 below provide non-limiting examples of target mimics and target resistant sequences that can be used to down-regulate the activity of the miRs of the invention.

According to a specific embodiment, the target mimic or micro-RNA resistant target is linked to the promoter naturally associated with the pre-miRNA recognizing the target gene and introduced into the plant cell. In this way, the miRNA target mimic or micro-RNA resistant target RNA will be expressed under the same circumstances as the miRNA and the target mimic or micro-RNA resistant target RNA will substitute for the non-target mimic/micro-RNA resistant target RNA degraded by the miRNA induced cleavage.

Non-functional miRNA alleles or miRNA resistant target genes may also be introduced by homologous recombination to substitute the miRNA encoding alleles or miRNA sensitive target genes.

Recombinant expression is effected by cloning the nucleic acid of interest (e.g., miRNA, target gene, silencing agent etc) into a nucleic acid expression construct under the expression of a plant promoter, as further described hereinbelow.

In other embodiments of the invention, synthetic single stranded nucleic acids are used as miRNA inhibitors. A miRNA inhibitor is typically between about 17 to 25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90 % complementary to the 5' to 3' sequence of a mature miRNA. In certain embodiments, a miRNA inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, a miRNA inhibitor has a sequence (from 5' to 3^*) that is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100 % complementary, or any range derivable therein, to the 5' to 3' sequence of a mature miRNA, particularly a mature, naturally occurring miRNA.

While further reducing the present invention to practice, the present inventors have identified gene targets for the differentially expressed miRNA molecules. It is therefore contemplated, that gene targets of those miRNAs that are down regulated during stress should be overexpressed in order to confer tolerance, while gene targets of those miR As that are up regulated during stress should be downregulated in the plant in order to confer tolerance.

Thus, according to an aspect of the invention there is provided a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide having an amino acid sequence at least 80 %, 82 %, 84 %, 85 %, 86 %, 88 %, 90 %, 92 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or 100 % homologous to SEQ ID NOs: 927-1021 (gene targets of down regulated miRNAs, see Table 6), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

Nucleic acid sequences (also referred to herein as polynucleotides) of the polypeptides of some embodiments of the invention may be optimized for expression in a specific plant host. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.

The phrase "codon optimization" refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approaches codon usage within the plant of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant. The nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681). In this method, the standard deviation of codon usage, a measure of codon usage bias, may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation. The formula used is: 1 SDCU = n = 1 N [ ( Xn - Yn ) / Yn ] 2 / N, where Xn refers to the frequency of usage of codon n in highly expressed plant genes, where Yn to the frequency of usage of codon n in the gene of interest and N refers to the total number of codons in the gene of interest. A table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).

One method of optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular plant cell type is based on the direct use, without performing any extra statistical calculations, of codon optimization tables such as those provided on-line at the Codon Usage Database through the NIAS (National Institute of Agrobiological Sciences) DNA bank in Japan (www.kazusa.or.jp/codon/). The Codon Usage Database contains codon usage tables for a number of different species, with each codon usage table having been statistically determined based on the data present in Genbank.

By using the above tables to determine the most preferred or most favored codons for each amino acid in a particular species (for example, rice), a naturally- occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored. However, one or more less- favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5' and 3' ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively effect mRNA stability or expression.

The naturally-occurring encoding nucleotide sequence may already, in advance of any modification, contain a number of codons that correspond to a statistically- favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative. A modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.

Target genes which are contemplated according to the present teachings are provided in the polynucleotide sequences which comprise nucleic acid sequences as set forth in the maize polynucleotides listed in Tables 5 and 6). However the present teachings also relate to orthologs or homologs at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, or at least about 95 % or more identical or similar to SEQ ID NO: 895-926 or 1022-1090 (polynucleotides listed in Tables 5 and 6). Parameters for determining the level of identity are provided hereinbelow.

Alternatively or additionally, target genes which are contemplated according to the present teachings are provided in the polypeptide sequences which comprise amino acid sequences as set forth the maize polypeptides of Tables 5 and 6). However the present teachings also relate to of orthologs or homologs at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, or at least about 95 % or more identical or similar to SEQ ID NO: 854-894 or 927-1021 (Tables 5 and 6).

Homology (e.g., percent homology, identity + similarity) can be determined using any homology comparison software, including for example, the TBLASTN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters, when starting from a polypeptide sequence; or the tBLASTX algorithm (available via the NCBI) such as by using default parameters, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.

According to some embodiments of the invention, the term "homology" or

"homologous" refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences.

Homologous sequences include both orthologous and paralogous sequences. The term "paralogous" relates to gene-duplications within the genome of a species leading to paralogous genes. The term "orthologous" relates to homologous genes in different organisms due to ancestral relationship. One option to identify orthologues in monocot plant species is by performing a reciprocal blast search. This may be done by a first blast involving blasting the sequence-of-interest against any sequence database, such as the publicly available NCBI database which may be found at: Hypertext Transfer Protocol ://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov. The blast results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence-of- interest is derived. The results of the first and second blasts are then compared. An orthologue is identified when the sequence resulting in the highest score (best hit) in the first blast identifies in the second blast the query sequence (the original sequence-of- interest) as the best hit. Using the same rational a paralogue (homolog to a gene in the same organism) is found. In case of large sequence families, the ClustalW program may be used [Hypertext Transfer Protocol ://World Wide Web (dot) ebi (dot) ac (dot) uk/Tools/clustalw2/index (dot) html], followed by a neighbor-joining tree (Hypertext Transfer Protocol://en (dot) wikipedia (dot) org/wiki/Neighbor-joining) which helps visualizing the clustering.

As mentioned the present inventors have also identified genes which down- regulation may be done in order to improve their NUE, biomass, vigor, yield and abiotic stress tolerance.

Thus, according to an aspect of the invention there is provided a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 %, 85 %, 90 %, 95 %, or 100 % homologous to SEQ ID NOs: 854- 894 (polypeptides of Table 5), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

Down regulation of activity or expression is by at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or even complete (100 %) loss of activity or expression. Assays for measuring gene expression can be effected at the protein level (e.g,. Western blot, ELISA) or at the mRNA level such as by RT-PCR. According to a specific embodiment the amino acid sequence of the target gene is as set forth in SEQ ID NOs: 854-894 of Table 5.

Alternatively or additionally, the amino acid sequence of the target gene is encoded by a polynucleotide sequence as set forth in SEQ ID NOs: 895-926 of Table 5.

Examples of polynucleotide downregulating agents that inhibit (also referred to herein as inhibitors or nucleic acid agents) the expression of a target gene are given below.

1. Polynucleotide -Based Inhibition of Gene Expression.

It will be appreciated, that any of these methods when specifically referring to downregulating expression/activity of the target genes can be used, at least in part, to downregulate expression or activity of endogenous RNA molecules,

i. Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression of target gene may be obtained by sense suppression or cosuppression. For cosuppression, an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding a target gene in the "sense" orientation. Over-expression of the RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the cosuppression expression cassette are screened to identify those that show the greatest inhibition of target gene expression.

The polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the target gene, all or part of the 5' and/or 3' untranslated region of a target transcript, or all or part of both the coding sequence and the untranslated regions of a transcript encoding the target gene. In some embodiments where the polynucleotide comprises all or part of the coding region for the target gene, the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be transcribed.

Cosuppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. See, for example, Broin, et al, (2002) Plant Cell 15: 1517-1532. Cosuppression may also be used to inhibit the expression of multiple proteins in the same plant. Methods for using cosuppression to inhibit the expression of endogenous genes in plants are described in Flavell, et al, (1995) Proc. Natl. Acad. Sci. USA 91 :3590-3596; Jorgensen, et al, (1996) Plant Mol. Biol. 31 :957-973; Johansen and Carrington, (2001) Plant Physiol. 126:930-938; Broin, et al, (2002) Plant Cell 15: 1517-1532; Stoutjesdijk, et al, (2002) Plant Physiol. 129: 1723-1731; Yu, et al, (2003) Phytochemistry 63:753-763; and U.S. Pat. Nos. 5,035,323, 5,283,185 and 5,952,657; each of which is herein incorporated by reference. The efficiency of cosuppression may be increased by including a poly-dt region in the expression cassette at a position 3' to the sense sequence and 5' of the polyadenylation signal. See, US Patent Publication Number 20020058815, herein incorporated by reference. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65 % sequence identity, more optimally greater than about 85 % sequence identity, most optimally greater than about 95 % sequence identity. See, U.S. Pat. Nos. 5,283,185 and 5,035,323; herein incorporated by reference.

Transcriptional gene silencing (TGS) may be accomplished through use of hpRNA constructs wherein the inverted repeat of the hairpin shares sequence identity with the promoter region of a gene to be silenced. Processing of the hpRNA into short RNAs which can interact with the homologous promoter region may trigger degradation or methylation to result in silencing. (Aufsatz, et al, (2002) PNAS 99(4): 16499-16506; Mette, et al, (2000) EMBO J. 19(19):5194-5201)

ii. Antisense Suppression

In some embodiments of the invention, inhibition of the expression of the target gene may be obtained by antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the target gene. Over-expression of the antisense RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the greatest inhibition of target gene expression.

The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the target gene, all or part of the complement of the 5' and/or 3' untranslated region of the target gene transcript, or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the target gene. In addition, the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 500, 550, 500, 550 or greater may be used. Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu, et al, (2002) Plant Physiol. 129: 1732-1753 and U.S. Pat. No. 5,759,829, which is herein incorporated by reference. Efficiency of antisense suppression may be increased by including a poly-dt region in the expression cassette at a position 3' to the antisense sequence and 5' of the polyadenylation signal. See, US Patent Publication Number 20020058815.

iii. Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of a target gene may be obtained by double-stranded RNA (dsRNA) interference. For dsRNA interference, a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the greatest inhibition of target gene expression. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse, et al, (1998) Proc. Natl. Acad. Sci. USA 95: 13959-13965, Liu, et al, (2002) Plant Physiol. 129: 1732-1753, and WO 99/59029, WO 99/53050, WO 99/61631, and WO 00/59035;

iv. Hairpin RNA Interference and Intron-Containing Hairpin RNA Interference

In some embodiments of the invention, inhibition of the expression of one or more target gene may be obtained by hairpin RNA (hpRNA) interference or intron- containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at downregulating the expression of endogenous genes. See, Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single- stranded loop region and a base-paired stem. The base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:5985- 5990; Stoutjesdijk, et al, (2002) Plant Physiol. 129:1723-1731; and Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, for example, in Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:5985-5990; Stoutjesdijk, et al, (2002) Plant Physiol. 129: 1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38; Pandolfmi, et al, BMC Biotechnology 3:7, and US Patent Publication Number 20030175965; each of which is herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga, et al, (2003) Mol. Biol. Rep. 30: 135-150, herein incorporated by reference.

For ihpRNA, the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in the cell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith, et al, (2000) Nature 507:319-320. In fact, Smith, et al, show 100 % suppression of endogenous gene expression using ihpRNA-mediated interference. Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in Smith, et al, (2000) Nature 507:319-320; Wesley, et al, (2001) Plant J. 27:584, 1-3, 590; Wang and Waterhouse, (2001) Curr. Opin. Plant Biol. 5: 156-150; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38; Helliwell and Waterhouse, (2003) Methods 30:289- 295, and US Patent Publication Number 20030180955, each of which is herein incorporated by reference.

The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, it is the loop region that determines the specificity of the RNA interference. See, for example, WO 02/00905, herein incorporated by reference.

v. Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for target gene). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe, (1997) EMBO J. 16:3675-3685, Angell and Baulcombe, (1999) Plant J. 20:357-362, and U.S. Pat. No. 6,656,805, each of which is herein incorporated by reference.

vi. Ribozymes

In some embodiments, the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of target gene. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the target gene. This method is described, for example, in U.S. Pat. No. 5,987,071, herein incorporated by reference.

2. Gene Disruption

In some embodiments of the present invention, the activity of a miRNA or a target gene is reduced or eliminated by disrupting the gene encoding the target polypeptide. The gene encoding the target polypeptide may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing plants using random or targeted mutagenesis, and selecting for plants that have reduced response regulator activity.

Any of the nucleic acid agents described herein (for overexpression or downregulation of either the target gene of the miR A) can be provided to the plant as naked RNA or expressed from a nucleic acid expression construct, where it is operaly linked to a regulatory sequence.

According to a specific embodiment of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a the nucleic acid agent (e.g., miRNA or a precursor thereof as described herein, gene targetm or silencing agent), said nucleic acid sequence being under a transcriptional control of a regulatory sequence such as a tissue specific promoter.

An exemplary nucleic acid construct which can be used for plant transformation include, the pORE E2 binary vector (Figure 1) in which the relevant nucleic acid sequence is ligated under the transcriptional control of a promoter.

A coding nucleic acid sequence is "operably linked" or "transcriptionally linked to a regulatory sequence (e.g., promoter)" if the regulatory sequence is capable of exerting a regulatory effect on the coding sequence linked thereto. Thus, the regulatory sequence controls the transcription of the miRNA or precursor thereof, gene target or silencing agent.

The term "regulatory sequence", as used herein, means any DNA, that is involved in driving transcription and controlling (i.e., regulating) the timing and level of transcription of a given DNA sequence, such as a DNA coding for a miRNA, precursor or inhibitor of same. For example, a 5' regulatory region (or "promoter region") is a DNA sequence located upstream (i.e., 5') of a coding sequence and which comprises the promoter and the 5 '-untranslated leader sequence. A 3' regulatory region is a DNA sequence located downstream (i.e., 3') of the coding sequence and which comprises suitable transcription termination (and/or regulation) signals, including one or more polyadenylation signals.

For the purpose of the invention, the promoter is a plant-expressible promoter.

As used herein, the term "plant-expressible promoter" means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin Thus, any suitable promoter sequence can be used by the nucleic acid construct of the present invention. According to some embodiments of the invention, the promoter is a constitutive promoter, a tissue-specific promoter or an inducible promoter (e.g. an abiotic stress-inducible promoter).

Suitable constitutive promoters include, for example, hydroperoxide lyase (HPL) promoter, CaMV 35S promoter (Odell et al, Nature 313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No. WO04081173A2); Arabidopsis new At6669 promoter; maize Ubi 1 (Christensen et al., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al, Plant Cell 2:163-171, 1990); pEMU (Last et al, Theor. Appl. Genet. 81 :584, 1-3, 588, 1991); CaMV 19S (Nilsson et al, Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al, Plant J Nov;2(6):837-44, 1992); ubiquitin (Christensen et al, Plant Mol. Biol. 18: 675-689, 1992); Rice cyclophilin (Bucholz et al, Plant Mol Biol. 25(5):837-43, 1994); Maize H3 histone (Lepetit et al, Mol. Gen. Genet. 231 : 276-285, 1992); Actin 2 (An et al, Plant J. 10(1); 107- 121, 1996) and Synthetic Super MAS (Ni et al, The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5.608,144; 5,604,121; 5.569,597: 5.466,785; 5,399,680; 5,268,463; and 5,608,142.

Suitable tissue-specific promoters include, but not limited to, leaf-specific promoters [such as described, for example, by Yamamoto et al, Plant J. 12:255-265, 1997; Kwon et al, Plant Physiol. 105:357-67, 1994; Yamamoto et al, Plant Cell Physiol. 35:773-778, 1994; Gotor et al, Plant J. 3:509-18, 1993; Orozco et al, Plant Mol. Biol. 23: 1129-1138, 1993; and Matsuoka et al, Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993], seed-preferred promoters [e.g., from seed specific genes (Simon, et al, Plant Mol. Biol. 5. 191, 1985; Scofield, et al, J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al., Plant Mol. Biol. 18: 235- 245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203- 214, 1988), Glutelin (rice) (Takaiwa, et al, Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al, FEBS Letts. 221 : 43-47, 1987), Zein (Matzke et al, Plant Mol Biol, 143)323-32 1990), napA (Stalberg, et al, Planta 199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171- 184, 1997), sunflower oleosin (Cummins, etal, Plant Mol. Biol. 19: 873- 876, 1992)], endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMB03: 1409-15, 1984), Barley ltrl promoter, barley Bl, C, D hordein (Theor Appl Gen 98: 1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750- 60, 1996), Barley DOF (Mena et al, The Plant Journal, 116(1): 53- 62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al, Plant J. 13: 629-640, 1998), rice prolamin NRP33, rice -globulin GIb-I (Wu et al, Plant Cell Physiology 39(8) 885- 889, 1998), rice alpha-globulin REB/OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6: 157-68, 1997), maize ESR gene family (Plant J 12:235-46, 1997), sorghum gamma- kafirin (PMB 32:1029-35, 1996); e.g., the Napin promoter], embryo specific promoters [e.g., rice OSH1 (Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma et al, Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)], and flower- specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al, Mol. Gen Genet. 217:240-245; 1989), apetala- 3]. Also contemplated are root-specific promoters such as the ROOTP promoter described in Vissenberg K, et al. Plant Cell Physiol. 2005 January; 46(1): 192- 200.

The nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication.

The nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells. In stable transformation, the exogenous polynucleotide is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.

When naked RNA or DNA is introduced into a cell, the polynucleotides may be synthesized using any method known in the art, including either enzymatic syntheses or solid-phase syntheses. These are especially useful in the case of short polynucleotide sequences with or without modifications as explained above. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example: Sambrook, J. and Russell, D. W. (2001), "Molecular Cloning: A Laboratory Manual"; Ausubel, R. M. et al, eds. (1994, 1989), "Current Protocols in Molecular Biology," Volumes I-III, John Wiley & Sons, Baltimore, Maryland; Perbal, B. (1988), "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York; and Gait, M. J., ed. (1984), "Oligonucleotide Synthesis"; utilizing solid-phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting, and purification by, for example, an automated trityl-on method or HPLC.

There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, L, Annu. Rev. Plant. Physiol, Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al, Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer (e.g., T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes); see for example, Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2- 25; Gatenby, in Plant Biotechnology, eds. Kung, S, and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6: 1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923- 926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al, Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.

According to a specific embodiment of the present invention, the exogenous polynucleotide is introduced into the plant by infecting the plant with a bacteria, such as using a floral dip transformation method (as described in further detail in Example 5, of the Examples section which follows).

There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. For this reason it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.

Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant- free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.

Although stable transformation is presently preferred, transient transformation of leaf cells, meristematic cells or the whole plant is also envisaged by the present invention.

Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.

Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants are described in WO 87/06261. According to some embodiments of the invention, the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting. A suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus. Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259- 269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).

Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tatlor, Eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.

Construction of plant RNA viruses for the introduction and expression of non- viral exogenous polynucleotide sequences in plants is demonstrated by the above references as well as by Dawson, W. O. et al, Virology (1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al. Science (1986) 231 : 1294-1297; Takamatsu et al. FEBS Letters (1990) 269:73-76; and U.S. Pat. No. 5,316,931.

When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat proteins which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.

In one embodiment, a plant viral nucleic acid is provided in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native nucleic acid sequence within it, such that a protein is produced. The recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign) nucleic acid sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included. The non-native nucleic acid sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non- native coat protein coding sequence.

In a third embodiment, a recombinant plant viral nucleic acid is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native nucleic acid sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.

In a fourth embodiment, a recombinant plant viral nucleic acid is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. The recombinant plant viral nucleic acid or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral nucleic acid is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (isolated nucleic acid) in the host to produce the desired sequence.

In addition to the above, the nucleic acid molecule of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.

A technique for introducing exogenous nucleic acid sequences to the genome of the chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous nucleic acid is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous nucleic acid molecule into the chloroplasts. The exogenous nucleic acid is selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast. To this end, the exogenous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid stretch which is derived from the chloroplast's genome. In addition, the exogenous nucleic acid includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous nucleic acid. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference.

Regardless of the method of transformation, propagation or regeneration, the present invention also contemplates a transgenic plant exogenous ly expressing the polynucleotide/nucleic acid agent of the invention. According to a specific embodiment, the transgenic plant exogenously expresses a polynucleotide having a nucleic acid sequence at least , 80 %, 85 %, 90 %, 95 % or even 100 % identical to SEQ ID NOs: 2-20, 23-37, 57-449, 21-22, 38-52, 1209, 1211, 1212, 454-846 and 53-56, 1209 (Tables 1, 3 and 4), wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant.

According to further embodiments, the exogenous polynucleotide encodes a precursor of said nucleic acid sequence.

According to yet further embodiments, the stem-loop precursor is at least 60 %, 65 % , 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or even 100 % identical to SEQ ID NOs: 21-22, 38-52, 1209, 121 1, 1212, 454-846, 53-56, 1209 (Tables 1, 3 and 4) identical to SEQ ID NO: 21-22, 38-52, 1209, 1211, 1212, 54-846 and 53-56, 1209 (precursor sequences of Tables 1, 3 and 4). More specifically the exogenous polynucleotide is selected from the group consisting of SEQ ID NO: 21-22 and 38-52, 1209, 1211, 1212 (precursor and mature sequences of upregulated Tables 1 and 3).

Alternatively, there is provided a transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a gene encoding a miRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209 (downregulated Tables 1 and 4) or homologs thereof which are at least at least 80 %, 85 %, 90 % or 95 % identical to SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209 (downregulated Tables 1 and 4)·

More specifically, the transgenic plant expresses the nucleic acid agent of Tables

8-11.

More specifically, the transgenic plant expresses the nucleic acid agent of Tables 8 and 11.

Alternatively or additionally there is provided a transgenic plant exogenously expressing a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 %, 85 %, 90 %, 95 % or even 100 % homologous to SEQ ID NOs: 854-894 (polypeptides of Table 5), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.

Alternatively or additionally there is provided a transgenic plant exogenously expressing a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 %, 85 %, 90 %, 95 % or even 100 % homologous to SEQ ID NOs: 927-1021 (polypeptides of Table 6), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.

Alternatively or additionally there is provided a transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 %, 85 %, 90 %, 95 % or even 100 % homologous to SEQ ID NOs: 854-894, 927-1021 (targets of Tables 5 and 6), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.

Also contemplated are hybrids of the above described transgenic plants. A "hybrid plant" refers to a plant or a part thereof resulting from a cross between two parent plants, wherein one parent is a genetically engineered plant of the invention (transgenic plant expressing an exogenous miRNA sequence or a precursor thereof). Such a cross can occur naturally by, for example, sexual reproduction, or artificially by, for example, in vitro nuclear fusion. Methods of plant breeding are well-known and within the level of one of ordinary skill in the art of plant biology.

Since nitrogen use efficiency, abiotic stress tolerance as well as yield, vigor or biomass of the plant can involve multiple genes acting additively or in synergy (see, for example, in Quesda et al., Plant Physiol. 130:951-063, 2002), the invention also envisages expressing a plurality of exogenous polynucleotides in a single host plant to thereby achieve superior effect on the efficiency of nitrogen use, yield, vigor and biomass of the plant.

Expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell. The transformed cell can then be regenerated into a mature plant using the methods described hereinabove. Alternatively, expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing into a single plant-cell a single nucleic-acid construct including a plurality of different exogenous polynucleotides. Such a construct can be designed with a single promoter sequence which can transcribe a polycistronic messenger RNA including all the different exogenous polynucleotide sequences. Alternatively, the construct can include several promoter sequences each linked to a different exogenous polynucleotide sequence. The plant cell transformed with the construct including a plurality of different exogenous polynucleotides can be regenerated into a mature plant, using the methods described hereinabove.

Alternatively, expressing a plurality of exogenous polynucleotides can be effected by introducing different nucleic acid constructs, including different exogenous polynucleotides, into a plurality of plants. The regenerated transformed plants can then be cross-bred and resultant progeny selected for superior yield or tolerance traits as described above, using conventional plant breeding techniques.

Expression of the miR As of the present invention or precursors thereof can be qualified using methods which are well known in the art such as those involving gene amplification e.g., PCR or RT-PCR or Northern blot or in-situ hybrdization.

According to some embodiments of the invention, the plant expressing the exogenous polynucleotide(s) is grown under stress (nitrogen or abiotic) or normal conditions (e.g., biotic conditions and/or conditions with sufficient water, nutrients such as nitrogen and fertilizer). Such conditions, which depend on the plant being grown, are known to those skilled in the art of agriculture, and are further, described above.

According to some embodiments of the invention, the method further comprises growing the plant expressing the exogenous polynucleotide(s) under abiotic stress or nitrogen limiting conditions. Non-limiting examples of abiotic stress conditions include, water deprivation, drought, excess of water (e.g., flood, waterlogging), freezing, low temperature, high temperature, strong winds, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, salinity, atmospheric pollution, intense light, insufficient light, or UV irradiation, etiolation and atmospheric pollution.

Thus, the invention encompasses plants exogenously expressing the polynucleotide(s), the nucleic acid constructs of the invention.

Methods of determining the level in the plant of the RNA transcribed from the exogenous polynucleotide are well known in the art and include, for example, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-m situ hybridization.

The sequence information and annotations uncovered by the present teachings can be harnessed in favor of classical breeding. Thus, sub-sequence data of those polynucleotides described above, can be used as markers for marker assisted selection (MAS), in which a marker is used for indirect selection of a genetic determinant or determinants of a trait of interest (e.g., tolerance to abiotic stress). Nucleic acid data of the present teachings (DNA or RNA sequence) may contain or be linked to polymorphic sites or genetic markers on the genome such as restriction fragment length polymorphism (RFLP), microsatellites and single nucleotide polymorphism (SNP), DNA fingerprinting (DFP), amplified fragment length polymorphism (AFLP), expression level polymorphism, and any other polymorphism at the DNA or RNA sequence.

Examples of marker assisted selections include, but are not limited to, selection for a morphological trait (e.g., a gene that affects form, coloration, male sterility or resistance such as the presence or absence of awn, leaf sheath coloration, height, grain color, aroma of rice); selection for a biochemical trait (e.g., a gene that encodes a protein that can be extracted and observed; for example, isozymes and storage proteins); selection for a biological trait (e.g., pathogen races or insect biotypes based on host pathogen or host parasite interaction can be used as a marker since the genetic constitution of an organism can affect its susceptibility to pathogens or parasites).

The polynucleotides described hereinabove can be used in a wide range of economical plants, in a safe and cost effective manner.

Plant lines exogenously expressing the polynucleotide of the invention can be screened to identify those that show the greatest increase of the desired plant trait.

Thus, according to an additional embodiment of the present invention, there is provided a method of evaluating a trait of a plant, the method comprising: (a) expressing in a plant or a portion thereof the nucleic acid construct; and (b) evaluating a trait of a plant as compared to a wild type plant of the same type; thereby evaluating the trait of the plant.

Thus, the effect of the transgene (the exogenous polynucleotide) on different plant characteristics may be determined any method known to one of ordinary skill in the art.

Thus, for example, tolerance to limiting nitrogen conditions may be compared in transformed plants {i.e., expressing the transgene) compared to non-transformed (wild type) plants exposed to the same stress conditions ( other stress conditions are contemplated as well, e.g. water deprivation, salt stress e.g. salinity, suboptimal temperatureosmotic stress, and the like), using the following assays.

Methods of qualifying plants as being tolerant or having improved tolerance to abiotic stress or limiting nitrogen levels are well known in the art and are further described hereinbelow.

Fertilizer use efficiency - To analyze whether the transgenic plants are more responsive to fertilizers, plants are grown in agar plates or pots with a limited amount of fertilizer, as described, for example, in Yanagisawa et al (Proc Natl Acad Sci U S A. 2004; 101 :7833-8). The plants are analyzed for their overall size, time to flowering, yield, protein content of shoot and/or grain. The parameters checked are the overall size of the mature plant, its wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Other parameters that may be tested are: the chlorophyll content of leaves (as nitrogen plant status and the degree of leaf verdure is highly correlated), amino acid and the total protein content of the seeds or other plant parts such as leaves or shoots, oil content, etc. Similarly, instead of providing nitrogen at limiting amounts, phosphate or potassium can be added at increasing concentrations. Again, the same parameters measured are the same as listed above. In this way, nitrogen use efficiency (NUE), phosphate use efficiency (PUE) and potassium use efficiency (KUE) are assessed, checking the ability of the transgenic plants to thrive under nutrient restraining conditions.

Nitrogen use efficiency - To analyze whether the transgenic plants (e.g., Arabidopsis plants) are more responsive to nitrogen, plant are grown in 0.75-3 millimolar (mM, nitrogen deficient conditions) or 10, 6-9 mM (optimal nitrogen concentration). Plants are allowed to grow for additional 25 days or until seed production. The plants are then analyzed for their overall size, time to flowering, yield, protein content of shoot and/or grain/ seed production. The parameters checked can be the overall size of the plant, wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Other parameters that may be tested are: the chlorophyll content of leaves (as nitrogen plant status and the degree of leaf greenness is highly correlated), amino acid and the total protein content of the seeds or other plant parts such as leaves or shoots and oil content. Transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher measured parameters levels than wild-type plants, are identified as nitrogen use efficient plants.

Nitrogen Use efficiency assay using plantlets - The assay is done according to Yanagisawa-S. et al. with minor modifications ("Metabolic engineering with Dofl transcription factor in plants: Improved nitrogen assimilation and growth under low- nitrogen conditions" Proc. Natl. Acad. Sci. USA 101, 7833-7838). Briefly, transgenic plants which are grown for 7-10 days in 0.5 x MS [Murashige-Skoog] supplemented with a selection agent are transferred to two nitrogen-limiting conditions: MS media in which the combined nitrogen concentration (NH₄NO3 and KNO3) was 0.75 mM (nitrogen deficient conditions) or 6-15 mM (optimal nitrogen concentration). Plants are allowed to grow for additional 30-40 days and then photographed, individually removed from the Agar (the shoot without the roots) and immediately weighed (fresh weight) for later statistical analysis. Constructs for which only Tl seeds are available are sown on selective media and at least 20 seedlings (each one representing an independent transformation event) are carefully transferred to the nitrogen- limiting media. For constructs for which T2 seeds are available, different transformation events are analyzed. Usually, 20 randomly selected plants from each event are transferred to the nitrogen- limiting media allowed to grow for 3-4 additional weeks and individually weighed at the end of that period. Transgenic plants are compared to control plants grown in parallel under the same conditions. Mock- transgenic plants expressing the uidA reporter gene (GUS) under the same promoter or transgenic plants carrying the same promoter but lacking a reporter gene are used as control.

Nitrogen determination - The procedure for N (nitrogen) concentration determination in the structural parts of the plants involves the potassium persulfate digestion method to convert organic N to NO3^" (Purcell and King 1996 Argon. J. 88: 111-113, the modified Cd^" mediated reduction of N0₃ to N0₂ (Vodovotz 1996 Biotechniques 20:390-394) and the measurement of nitrite by the Griess assay (Vodovotz 1996, supra). The absorbance values are measured at 550 nm against a standard curve of NaN0₂. The procedure is described in details in Samonte et al. 2006 Agron. J. 98: 168-176.

Tolerance to abiotic stress (e.g. tolerance to drought or salinity) can be evaluated by determining the differences in physiological and/or physical condition, including but not limited to, vigor, growth, size, or root length, or specifically, leaf color or leaf area size of the transgenic plant compared to a non-modified plant of the same species grown under the same conditions. Other techniques for evaluating tolerance to abiotic stress include, but are not limited to, measuring chlorophyll fluorescence, photosynthetic rates and gas exchange rates. Further assays for evaluating tolerance to abiotic stress are provided hereinbelow and in the Examples section which follows.

Drought tolerance assay - Soil-based drought screens are performed with plants overexpressing the polynucleotides detailed above. Seeds from control Arabidopsis plants, or other transgenic plants overexpressing nucleic acid of the invention are germinated and transferred to pots. Drought stress is obtained after irrigation is ceased. Transgenic and control plants are compared to each other when the majority of the control plants develop severe wilting. Plants are re-watered after obtaining a significant fraction of the control plants displaying a severe wilting. Plants are ranked comparing to controls for each of two criteria: tolerance to the drought conditions and recovery (survival) following re-watering.

Quantitative parameters of tolerance measured include, but are not limited to, the average wet and dry weight, growth rate, leaf size, leaf coverage (overall leaf area), the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher biomass than wild-type plants, are identified as drought stress tolerant plants

Salinity tolerance assay - Transgenic plants with tolerance to high salt concentrations are expected to exhibit better germination, seedling vigor or growth in high salt. Salt stress can be effected in many ways such as, for example, by irrigating the plants with a hyperosmotic solution, by cultivating the plants hydroponically in a hyperosmotic growth solution (e.g., Hoagland solution with added salt), or by culturing the plants in a hyperosmotic growth medium [e.g., 50 % Murashige-Skoog medium (MS medium) with added salt]. Since different plants vary considerably in their tolerance to salinity, the salt concentration in the irrigation water, growth solution, or growth medium can be adjusted according to the specific characteristics of the specific plant cultivar or variety, so as to inflict a mild or moderate effect on the physiology and/or morphology of the plants (for guidelines as to appropriate concentration see, Bernstein and Kafkafi, Root Growth Under Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002, and reference therein).

For example, a salinity tolerance test can be performed by irrigating plants at different developmental stages with increasing concentrations of sodium chloride (for example 50 mM, 150 mM, 300 mM NaCl) applied from the bottom and from above to ensure even dispersal of salt. Following exposure to the stress condition the plants are frequently monitored until substantial physiological and/or morphological effects appear in wild type plants. Thus, the external phenotypic appearance, degree of chlorosis and overall success to reach maturity and yield progeny are compared between control and transgenic plants. Quantitative parameters of tolerance measured include, but are not limited to, the average wet and dry weight, growth rate, leaf size, leaf coverage (overall leaf area), the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher biomass than wild-type plants, are identified as abiotic stress tolerant plants.

Osmotic tolerance test - Osmotic stress assays (including sodium chloride and PEG assays) are conducted to determine if an osmotic stress phenotype was sodium chloride-specific or if it was a general osmotic stress related phenotype. Plants which are tolerant to osmotic stress may have more tolerance to drought and/or freezing. For salt and osmotic stress experiments, the medium is supplemented for example with 50 mM, 100 mM, 200 mM NaCl or 15 %, 20 % or 25 % PEG.

Cold stress tolerance - One way to analyze cold stress is as follows. Mature (25 day old) plants are transferred to 4 °C chambers for 1 or 2 weeks, with constitutive light. Later on plants are moved back to greenhouse. Two weeks later damages from chilling period, resulting in growth retardation and other phenotypes, are compared between control and transgenic plants, by measuring plant weight (wet and dry), and by comparing growth rates measured as time to flowering, plant size, yield, and the like.

Heat stress tolerance - One way to measure heat stress tolerance is by exposing the plants to temperatures above 34 °C for a certain period. Plant tolerance is examined after transferring the plants back to 22 °C for recovery and evaluation after 5 days relative to internal controls (non-transgenic plants) or plants not exposed to neither cold or heat stress.

The biomass, vigor and yield of the plant can also be evaluated using any method known to one of ordinary skill in the art. Thus, for example, plant vigor can be calculated by the increase in growth parameters such as leaf area, fiber length, rosette diameter, plant fresh weight, oil content, seed yield and the like per time.

As mentioned, the increase of plant yield can be determined by various parameters. For example, increased yield of rice may be manifested by an increase in one or more of the following: number of plants per growing area, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in the seed filling rate, increase in thousand kernel weight (1000-weight), increase oil content per seed, increase starch content per seed, among others. An increase in yield may also result in modified architecture, or may occur because of modified architecture. Similarly, increased yield of soybean may be manifested by an increase in one or more of the following: number of plants per growing area, number of pods per plant, number of seeds per pod, increase in the seed filling rate, increase in thousand seed weight (1000-weight), reduce pod shattering, increase oil content per seed, increase protein content per seed, among others. An increase in yield may also result in modified architecture, or may occur because of modified architecture.

Thus, the present invention is of high agricultural value for increasing tolerance of plants to nitrogen deficiency or abiotic stress as well as promoting the yield, biomass and vigor of commercially desired crops.

According to another embodiment of the present invention, there is provided a food or feed comprising the plants or a portion thereof of the present invention.

In a further aspect the invention, the transgenic plants of the present invention or parts thereof are comprised in a food or feed product (e.g., dry, liquid, paste). A food or feed product is any ingestible preparation containing the transgenic plants, or parts thereof, of the present invention, or preparations made from these plants. Thus, the plants or preparations are suitable for human (or animal) consumption, i.e. the transgenic plants or parts thereof are more readily digested. Feed products of the present invention further include a oil or a beverage adapted for animal consumption. It will be appreciated that the transgenic plants, or parts thereof, of the present invention may be used directly as feed products or alternatively may be incorporated or mixed with feed products for consumption. Furthermore, the food or feed products may be processed or used as is. Exemplary feed products comprising the transgenic plants, or parts thereof, include, but are not limited to, grains, cereals, such as oats, e.g. black oats, barley, wheat, rye, sorghum, corn, vegetables, leguminous plants, especially soybeans, root vegetables and cabbage, or green forage, such as grass or hay.

It is expected that during the life of a patent maturing from this application many relevant homolog/ortholog sequences will be developed and the scope of the term polynucleotide/nucleic acid agent is intended to include all such new technologies a priori.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or

"at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al, "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al, "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. EXAMPLE 1

Differential Expression of miRNAs in Maize Plant under Optimal Versus limited

Nitrogen

Experimental Procedures

Plant Material

Corn seeds were obtained from Galil seeds (Israel). Corn variety 5605 was used in all experiments. Plants were grown at 28 °C under a 16 hr light:8 hr dark regime.

Stress Induction

Corn seeds were germinated and grown on defined growth media containing either sufficient (100% N₂) or insufficient nitrogen levels (1 % or 10 % N₂). Seedlings aged one or two weeks were used for tissue samples for RNA analysis, as described below.

Total RNA extraction

Total RNA of leaf or root samples from four to eight biological repeats were extracted using the mirVana™ kit (Ambion, Austin, TX) by pooling 3-4 plants to one biological repeat.

Microarray design

Custom microarrays were manufactured by Agilent Technologies by in situ synthesis. The first generation microarray consisted of a total of 13619 non-redundant DNA probes, the majority of which arose from deep sequencing data and includes different small RNA molecules (i.e. miRNAs, siRNA and predicted small RNA sequences), with each probe being printed once. An in-depth analysis of the first generation microarray, which included hybridization experiments as well as structure and orientation verifications on all its small RNAs, resulted in the formation of an improved, second generation, microarray. The second generation microarray consisted of a total 4721 non-redundant DNA 45 -nucleotide long probes for all known plant small RNAs, with 912 sequences (19.32 %) from Sanger version 15 and the rest (3809), encompassing miRNAs (968=20.5%), siRNAs (1626=34.44%) and predicted small RNA sequences (1215=25.74%)), from deep sequencing data accumulated by the inventors, with each probe being printed in triplicate. Results

Wild type maize plants were allowed to grow at standard, optimal conditions or nitrogen deficient conditions for one or two weeks, at the end of which they were evaluated for NUE. Three to four plants from each group were used for reproducibility. Four to eight repeats were obtained for each group, and RNA was extracted from leaf or root tissue. The expression level of the maize miRNAs was analyzed by high throughput microarray to identify miRNAs that were differentially expressed between the experimental groups.

Tables 1-2 below presents sequences that were found to be differentially expressed in corn grown in various nitrogen levels. To clarify, the sequence of an up- regulated miRNA is induced under nitrogen limiting conditions and the sequence of a down-regulated miRNA is repressed under nitrogen limiting conditions compared to optimal conditions. Table 1: Differentially Expressed miRNAs in Leaf of Plants Growing under

Nitrogen Deficient Versus Optimal Conditions.

Fold

P value - Change Up/Down

Leaf Leaf Sequence/SEQ ID NO: regulated Small RNA name

3.90E-03 1.66 AGAAGAGAGAGAGTACAGCCT/1 Down Zma-miR529

3.30E-06 3.35 TAGCCAGGGATGATTTGCCTG/2 Down Zma-miR1691

ND ND GGAATCTTGATGATGCTGCAT/3 Down Zma-miR172e

ND ND GTGAAGTGTTTGGGGGAACTC/4 Down Zma-miR395b

Predicted zma mir

2.20E-07 2.51 TAGCCAAGCATGATTTGCCCG/5 Down 50601

Predicted zma mir

ND ND AGGATGTGAGGCTATTGGGGAC/6 Up 48492

Predicted zma mir

ND ND CCAAGTCGAGGGCAGACCAGGC/7 Up 48879

ND ND ATTCACGGGGACGAACCTCCT/8 Up Mtr-miR2647a

Predicted zma mir

1.80E-02 1.72 AGGATGCTGACGCAATGGGAT/9 Up 48486

9.80E-03 1.61 TTAGATGACCATCAGCAAACA/10 Up Zma-miR827 Table 2: Differentially Expressed miRNAs in Roots of Plants Growing under Nitrogen Deficient Versus Optimal Conditions.

EXAMPLE 2

Identification of Homologous and Orthologous Sequences of Differential Small

RNAs Associated with Increased NUE

The small RNA sequences of the invention that were either down- or upregulated under nitrogen limiting conditions were examined for homologous and orthologous sequences using the miRBase database (wwwdotmirbasedotorg ) and the Plant MicroRNA Database (PMRD, wwwdotbioinformaticsdotcaudotedudotcn/PMRD). The mature miRNA sequences that are homologous or orthologous to the miRNAs of the invention (listed in Table 1) are found using miRNA public databases, having at least 90% identity of the entire small RNA length, and are summarized in Table 3 below. Of note, if homo logs of only 90 % are uncovered, they are subject for family members search and are listed with a cutoff of 80 % identity to the homolog sequence, not to the original maize miR. Table 3: Summary of Homologs/Orthologs to NUE small RNA Probes

(upregulated)

Homolog Stem- Stem- loop

loop seq % Homolog Homolog seq id MiR Mature MiR id no: Identity length sequence names no: length sequence Name

ATTCAC

ATTCACGG GGGGAC GGACGAAC mtr- GAACCT mtr-

38 1 21 CTCCT/23 miR2647b 21 21 CCT/8 miR2647a

ATTCACGG GGACGAAC mtr-

39 1 21 CTCCT/24 miR2647c

TTAGAT

TTAGATGA GACCAT CCATCAAC aly- CAGCAA zma-

40 0.9 21 AAACG/25 miR827 22 21 AC A/10 miR827

TTAGATGA CCATCAAC ath-

41 0.9 21 AAACT/26 miR827

TTAGATGA CCATCAGC bdi-

42 1 21 AAACA/27 miR827

TTAGATGA CCATCAAC csi-

43 0.95 21 AAACA/28 miR827

TTAGATGA CCATCAAC ghr-

44 0.95 21 AAACA/29 miR827a

TTAGATGA CCATCAAC ghr-

45 0.95 21 AAACA/30 miR827b

TTAGATGA CCATCAAC ghr-

46 0.95 21 AAACA/31 miR827c

TAAGATGA CCATCAGC osa-

47 0.86 21 GAAAA/32 miR827

TTAGATGA CCATCAGC osa-

48 1 21 AAACA/33 miR827a

TTAGATGA CCATCAGC osa-

49 1 21 AAACA/34 miR827b

TTAGATGA CCATCAAC ptc-

50 0.86 21 GAAAA/35 miR827 TTAGATGA

CCATCAGC ssp-

51 1 21 AAACA/36 miR827

TTAGATGA CCATCAAC tcc-

52 0.95 21 AAACA/37 miR827

Table 4: Summary of Homologs/Orthologs to NUE small RNA Probes

(Downregulated)

Stem

-loop

Homolog seq

Stem-loop % Homolog Homolog id MiR Mature MiR seq id no: Identity length sequence names no: length sequence Name

TAGCCA

CAGCCAAG GGGATG GATGACTT aly- ATTTGCC zma-

454 0.81 21 GCCGG/57 miR169b 53 21 TG/2 miR1691

CAGCCAAG GATGACTT aly-

455 0.81 21 GCCGG/58 miR169c

TAGCCAAG GATGACTT aly-

456 0.9 21 GCCTG/59 miR169h

TAGCCAAG GATGACTT aly-

457 0.9 21 GCCTG/60 miR169i

TAGCCAAG GATGACTT aly-

458 0.9 21 GCCTG/61 miR169j

TAGCCAAG GATGACTT aly-

459 0.9 21 GCCTG/62 miR169k

TAGCCAAG GATGACTT aly-

460 0.9 21 GCCTG/63 miR1691

TAGCCAAG GATGACTT aly-

461 0.9 21 GCCTG/64 miR169m

TAGCCAAA GATGACTT aly-

462 0.86 21 GCCTG/65 miR169n

TAGCCAAG GATGACTT aqc-

463 0.86 21 GCCTA/66 miR169a

TAGCCAAG GATGACTT aqc-

464 0.9 21 GCCTG/67 miR169b

465 0.81 21 CAGCCAAG aqc- GATGACTT miR169c GCCGG/68

TAGCCAAG GATGAATT ata-

466 0.86 21 GCCAG/69 miR169

CAGCCAAG GATGACTT ath-

467 0.81 21 GCCGG/70 miR169b

CAGCCAAG GATGACTT ath-

468 0.81 21 GCCGG/71 miR169c

TAGCCAAG GATGACTT ath-

469 0.9 21 GCCTG/72 miR169h

TAGCCAAG GATGACTT ath-

470 0.9 21 GCCTG/73 miR169i

TAGCCAAG GATGACTT ath-

471 0.9 21 GCCTG/74 miR169j

TAGCCAAG GATGACTT ath-

472 0.9 21 GCCTG/75 miR169k

TAGCCAAG GATGACTT ath-

473 0.9 21 GCCTG/76 miR1691

TAGCCAAG GATGACTT ath-

474 0.9 21 GCCTG/77 miR169m

TAGCCAAG GATGACTT ath-

475 0.9 21 GCCTG/78 miR169n

TAGCCAAG GATGACTT bdi-

476 0.86 21 GCCGG/79 miR169b

CAGCCAAG GATGACTT bdi-

477 0.81 21 GCCGG/80 miR169c

TAGCCAAG AATGACTT bdi-

478 0.81 21 GCCTA/81 miR169d

TAGCCAAG GATGACTT bdi-

479 0.9 21 GCCTG/82 miR169e

CAGCCAAG GATGACTT bdi-

480 0.81 21 GCCGG/83 miR169f

TAGCCAAG GATGACTT bdi-

481 0.9 21 GCCTG/84 miR169g

TAGCCAAG GATGACTT bdi-

482 0.86 21 GCCTA/85 miR169h

TAGCCAGG AATGGCTT bdi-

483 0.81 21 GCCTA/86 miR169j TAGCCAAG

GATGATTT bdi-

484 0.95 22 GCCTGT/87 miR169k

TAGCCAAG GATGACTT bna-

485 0.86 21 GCCTA/88 miR169c

TAGCCAAG GATGACTT bna-

486 0.86 21 GCCTA/89 miR169d

TAGCCAAG GATGACTT bna-

487 0.86 21 GCCTA/90 miR169e

TAGCCAAG GATGACTT bna-

488 0.86 21 GCCTA/91 miR169f

TAGCCAAG GATGACTT bna-

489 0.9 22 GCCTGC/92 miR169g

TAGCCAAG GATGACTT bna-

490 0.9 22 GCCTGC/93 miR169h

TAGCCAAG GATGACTT bna-

491 0.9 22 GCCTGC/94 miR169i

TAGCCAAG GATGACTT bna-

492 0.9 22 GCCTGC/95 miR169j

TAGCCAAG GATGACTT bna-

493 0.9 22 GCCTGC/96 miR169k

TAGCCAAG GATGACTT bna-

494 0.9 22 GCCTGC/97 miR1691

TAGCCAAG GATGACTT far-

495 0.86 21 GCCTA/98 miR169

TAGCCAAG GATGACTT ghb-

496 0.9 21 GCCTG/99 miR169a

CAGCCAAG GATGACTT gma-

497 0.81 21 GCCGG/100 miR169a

TGAGCCAA GGATGACT TGCCGGT/10 gma-

498 0.81 23 1 miR169d

AGCCAAGG

ATGACTTG gma-

499 0.81 20 CCGG/102 miR169e

AAGCCAAG GATGAGTT hvu-

500 0.86 21 GCCTG/103 miR169

CAGCCAAG

GGTGATTT mtr-

501 0.81 21 GCCGG/104 miR169c

502 0.81 21 AAGCCAAG mtr- GATGACTT miR169d GCCGG/105

AAGCCAAG

GATGACTT mtr-

503 0.81 21 GCCTA/106 miR169f

CAGCCAAG GATGACTT mtr-

504 0.81 21 GCCGG/107 miR169g

CAGCCAAG GATGACTT mtr-

505 0.81 21 GCCGG/108 miR169j

CAGCCAAG

GGTGATTT mtr-

506 0.81 21 GCCGG/109 miR169k

AAGCCAAG

GATGACTT mtr-

507 0.81 21 GCCGG/110 miR1691

GAGCCAAG

GATGACTT mtr-

508 0.81 21 GCCGG/111 miR169m

CAGCCAAG GATGACTT osa-

509 0.81 21 GCCGG/112 miR169b

CAGCCAAG GATGACTT osa-

510 0.81 21 GCCGG/113 miR169c

TAGCCAAG GATGAATT osa-

511 0.86 21 GCCGG/114 miR169d

TAGCCAAG GATGACTT osa-

512 0.86 21 GCCGG/115 miR169e

TAGCCAAG GATGACTT osa-

513 0.86 21 GCCTA/116 miR169f

TAGCCAAG GATGACTT osa-

514 0.86 21 GCCTA/117 miR169g

TAGCCAAG GATGACTT osa-

515 0.9 21 GCCTG/118 miR169h

TAGCCAAG GATGACTT osa-

516 0.9 21 GCCTG/119 miR169i

TAGCCAAG GATGACTT osa-

517 0.9 21 GCCTG/120 miR169j

TAGCCAAG GATGACTT osa-

518 0.9 21 GCCTG/121 miR169k

TAGCCAAG GATGACTT osa-

519 0.9 21 GCCTG/122 miR1691

TAGCCAAG GATGACTT osa-

520 0.9 21 GCCTG/123 miR169m TAGCCAAG

AATGACTT osa-

521 0.81 21 GCCTA/124 miR169n

TAGCCAAG AATGACTT osa-

522 0.81 21 GCCTA/125 miR169o

CAGCCAAG GATGACTT ptc-

523 0.81 21 GCCGG/126 miR169d

CAGCCAAG GATGACTT ptc-

524 0.81 21 GCCGG/127 miR169e

CAGCCAAG GATGACTT ptc-

525 0.81 21 GCCGG/128 miR169f

CAGCCAAG GATGACTT ptc-

526 0.81 21 GCCGG/129 miR169g

CAGCCAAG GATGACTT ptc-

527 0.81 21 GCCGG/130 miR169h

TAGCCAAG GATGACTT ptc-

528 0.9 21 GCCTG/131 miR169i

TAGCCAAG GATGACTT ptc-

529 0.9 21 GCCTG/132 miR169j

TAGCCAAG GATGACTT ptc-

530 0.9 21 GCCTG/133 miR169k

TAGCCAAG GATGACTT ptc-

531 0.9 21 GCCTG/134 miR1691

TAGCCAAG GATGACTT ptc-

532 0.9 21 GCCTG/135 miR169m

AAGCCAAG

GATGACTT ptc-

533 0.86 21 GCCTG/136 miR169o

AAGCCAAG

GATGACTT ptc-

534 0.86 21 GCCTG/137 miR169p

TAGCCAAG GACGACTT ptc-

535 0.86 21 GCCTG/138 miR169q

TAGCCAAG GATGACTT ptc-

536 0.86 21 GCCTA/139 miR169r

TAGCCAAG GACGACTT ptc-

537 0.81 21 GCCTA/140 miR169u

TAGCCAAG GATGACTT ptc-

538 0.81 21 GCCCA/141 miR169v

TAGCCAAG ptc-

539 0.81 21 GATGACTT miR169w GCCCA/142

TAGCCAAG GATGACTT ptc-

540 0.81 21 GCTCG/143 miR169x

TAGCCATG GATGAATT ptc-

541 0.9 21 GCCTG/144 miR169y

CAGCCAAG

AATGATTT ptc-

542 0.81 21 GCCGG/145 miR169z

CAGCCAAG GATGACTT rco-

543 0.81 21 GCCGG/146 miR169a

CAGCCAAG GATGACTT rco-

544 0.81 21 GCCGG/147 miR169b

CAGCCAAG GATGACTT sbi-

545 0.81 21 GCCGG/148 miR169b

TAGCCAAG GATGACTT sbi-

546 0.86 21 GCCTA/149 miR169c

TAGCCAAG GATGACTT sbi-

547 0.86 21 GCCTA/150 miR169d

TAGCCAAG GATGACTT sbi-

548 0.86 21 GCCGG/151 miR169e

TAGCCAAG GATGACTT sbi-

549 0.9 21 GCCTG/152 miR169f

TAGCCAAG GATGACTT sbi-

550 0.9 21 GCCTG/153 miR169g

TAGCCAAG GATGACTT sbi-

551 0.86 21 GCCTA/154 miR169h

TAGCCAAG AATGACTT sbi-

552 0.81 21 GCCTA/155 miR169i

TAGCCAAG GATGACTT sbi-

553 0.86 21 GCCGG/156 miR169j

CAGCCAAG GATGACTT sbi-

554 0.81 21 GCCGG/157 miR169k

TAGCCAAG GATGACTT sbi-

555 0.9 21 GCCTG/158 miR1691

TAGCCAAG GATGACTT sbi-

556 0.86 21 GCCTA/159 miR169m

TAGCCAAG GATGACTT sbi-

557 0.86 21 GCCTA/160 miR169n

558 0.95 21 TAGCCAAG sbi- GATGATTT miR169o GCCTG/161

CAGCCAAG GATGACTT sly-

559 0.81 21 GCCGG/162 miR169a

TAGCCAAG GATGACTT sly-

560 0.9 21 GCCTG/163 miR169b

TAGCCAAG GATGACTT sly-

561 0.86 21 GCCTA/164 miR169d

TAGCCAAG GATGACTT ssp-

562 0.86 21 GCCGG/165 miR169

CAGCCAAG GATGACTT tcc-

563 0.81 21 GCCGG/166 miR169b

TAGCCAAG GATGACTT tcc-

564 0.86 21 GCCTA/167 miR169d

AAGCCAAG

AATGACTT tcc-

565 0.81 21 GCCTG/168 miR169f

TAGCCAGG GATGACTT tcc-

566 0.9 21 GCCTA/169 miR169g

TAGCCAAG GATGACTT tcc-

567 0.9 21 GCCTG/170 miR169h

TAGCCAAG GATGAGTT tcc-

568 0.9 21 GCCTG/171 miR169i

TAGCCAAG GATGACTT tcc-

569 0.9 21 GCCTG/172 miR169j

CAGCCAAG GATGACTT tcc-

570 0.81 21 GCCGG/173 miR169k

CAGCCAAG GATGACTT tcc-

571 0.81 21 GCCGG/174 miR1691

CAGCCAAG GATGACTT vvi-

572 0.81 21 GCCGG/175 miR169a

CAGCCAAG GATGACTT vvi-

573 0.81 21 GCCGG/176 miR169c

CAGCCAAG

AATGATTT vvi-

574 0.81 21 GCCGG/177 miR169d

TAGCCAAG GATGACTT vvi-

575 0.9 22 GCCTGC/178 miR169e

CAGCCAAG GATGACTT vvi-

576 0.81 21 GCCGG/179 miR169j CAGCCAAG

GATGACTT vvi-

577 0.81 21 GCCGG/180 miR169k

GAGCCAAG

GATGACTT vvi-

578 0.81 21 GCCGG/181 miR169m

GAGCCAAG

GATGACTT vvi-

579 0.81 21 GCCGG/182 miR169n

GAGCCAAG

GATGACTT vvi-

580 0.81 21 GCCGG/183 miR169p

GAGCCAAG

GATGACTT vvi-

581 0.81 21 GCCGG/184 miR169q

CAGCCAAG GATGACTT vvi-

582 0.81 21 GCCGG/185 miR169s

AAGCCAAG GATGAATT vvi-

583 0.81 21 GCCGG/186 miR169v

CAGCCAAG GATGACTT vvi-

584 0.81 21 GCCGG/187 miR169w

TAGCCAAG GATGACTT vvi-

585 0.86 21 GCCTA/188 miR169x

TAGCGAAG GATGACTT vvi-

586 0.81 21 GCCTA/189 miR169y

CAGCCAAG GATGACTT zma-

587 0.81 21 GCCGG/190 miR169c

TAGCCAAG GATGACTT zma-

588 0.86 21 GCCTA/191 miR169f

TAGCCAAG GATGACTT zma-

589 0.86 21 GCCTA/192 miR169g

TAGCCAAG GATGACTT zma-

590 0.86 21 GCCTA/193 miR169h

TAGCCAAG GATGACTT zma-

591 0.9 21 GCCTG/194 miR169i

TAGCCAAG GATGACTT zma-

592 0.9 21 GCCTG/195 miR169j

TAGCCAAG GATGACTT zma-

593 0.9 21 GCCTG/196 miR169k

TAGCCAAG AATGACTT zma-

594 0.81 21 GCCTA/197 miR169o

TAGCCAAG zma-

595 0.86 21 GATGACTT miR169p GCCGG/198

CAGCCAAG GATGACTT zma-

596 0.81 21 GCCGG/199 miR169r

GGAATC

AGAATCTT TTGATG GATGATGC aly- ATGCTG zma-

597 0.95 21 TGCAT/200 miR172a 54 21 CAT/3 miR172e

AGAATCTT GATGATGC aly-

598 0.95 21 TGC AT/201 miR172b

AGAATCTT GATGATGC aly-

599 0.9 21 TGCAG/202 miR172c

AGAATCTT GATGATGC aly-

600 0.9 21 TGCAG/203 miR172d

GAATCTTG ATGATGCT aly-

601 0.95 20 GCAT/204 miR172e

AGAATCTT GATGATGC aqc-

602 0.95 21 TGCAT/205 miR172a

GGAATCTT GATGATGC aqc-

603 1 21 TGCAT/206 miR172b

AGGATCTT GATGATGC asp-

604 0.86 21 TGCAG/207 miR172

TGAGAATC TTGATGAT GCTGCAT/20 ata-

605 0.95 23 8 miR172

AGAATCTT GATGATGC ath-

606 0.95 21 TGCAT/209 miR172a

AGAATCTT GATGATGC ath-

607 0.95 21 TGC AT/210 miR172b

AGAATCTT GATGATGC ath-

608 0.9 21 TGC AG/211 miR172c

AGAATCTT GATGATGC ath-

609 0.9 21 TGCAG/212 miR172d

GGAATCTT GATGATGC ath-

610 1 21 TGCAT/213 miR172e

AGAATCCT GATGATGC ath-

611 0.86 21 TGCAG/214 miR172m

AGAATCTT GATGATGC bdi-

612 0.95 21 TGCAT/215 miR172a

GGAATCTT bdi-

613 1 21 GATGATGC miR172b TGCAT/216

AGAATCCT GATGATGC bdi-

614 0.86 21 TGCAG/217 miR172d

AGAATCTT GATGATGC bol-

615 0.95 21 TGCAT/218 miR172a

AGAATCTT GATGATGC bol-

616 0.95 21 TGCAT/219 miR172b

AGAATCTT GATGATGC bra-

617 0.95 21 TGCAT/220 miR172a

AGAATCTT GATGATGC bra-

618 0.95 21 TGC AT/221 miR172b

AGAATCTT GATGATGC csi-

619 0.9 20 TGC A/222 miR172

AGAATCTT GATGATGC csi-

620 0.9 20 TGC A/223 miR172a

AGAATCTT GATGATGC csi-

621 0.86 21 GGCAA/224 miR172b

TGGAATCTT

GATGATGC csi-

622 0.95 22 TGCAG/225 miR172c

AGAATCCT GATGATGC ghr-

623 0.86 21 TGCAG/226 miR172

AGAATCTT GATGATGC gma-

624 0.95 21 TGCAT/227 miR172a

AGAATCTT GATGATGC gma-

625 0.95 21 TGCAT/228 miR172b

GGAATCTT GATGATGC gma-

626 0.95 21 TGCAG/229 miR172c

GGAATCTT GATGATGC TGCAGCAG/ gma-

627 0.95 24 230 miR172d

GGAATCTT GATGATGC TGCAGCAG/ gma-

628 0.95 24 231 miR172e

AGAATCTT GATGATGC gma-

629 0.9 20 TGC A/232 miR172f

AGAATCTT GATGATGC gra-

630 0.95 21 TGC AT/233 miR172a

AAAATCTT gra-

631 0.9 21 GATGATGC miR172b TGCAT/234

AGAATCCT GATGATGC hvv-

632 0.86 21 TGCAG/235 miR172a

AGAATCCT GATGATGC hvv-

633 0.86 21 TGCAG/236 miR172b

AGAATCCT GATGATGC hvv-

634 0.86 21 TGCAG/237 miR172c

AGAATCCT GATGATGC hvv-

635 0.86 21 TGCAG/238 miR172d

AGAATCTT GATGATGC mes-

636 0.95 21 TGCAT/239 miR172

AGAATCCT GATGATGC mtr-

637 0.86 21 TGCAG/240 miR172

GGAATCTT GATGATTCT mtr-

638 0.9 21 GC AC/241 miR172a

AGAATCTT GATGATGC osa-

639 0.95 21 TGCAT/242 miR172a

GGAATCTT GATGATGC osa-

640 1 21 TGCAT/243 miR172b

TGAATCTTG

ATGATGCT osa-

641 0.9 21 GCAC/244 miR172c

AGAATCTT GATGATGC osa-

642 0.95 21 TGCAT/245 miR172d

AGAATCCT GATGATGC osa-

643 0.86 21 TGCAG/246 miR172m

AGAATCCT GATGATGC osa-

644 0.86 21 TGCAG/247 miR172n

AGAATCCT GATGATGC osa-

645 0.86 21 TGCAG/248 miR172o

AGAATCCT GATGATGC osa-

646 0.86 21 TGCAG/249 miR172p

AGAATCCT GATGATGC pga-

647 0.86 21 TGCAC/250 miR172

AGAATCTT GATGATGC ppd-

648 0.95 21 TGCAT/251 miR172a

TGAATCTTG

ATGATGCT ppd-

649 0.86 21 CCAC/252 miR172b

650 0.86 21 AGAATCCT psi- GATGATGC miR172 TGCAC/253

AGAATCTT GATGATGC ptc-

651 0.95 21 TGCAT/254 miR172a

AGAATCTT GATGATGC ptc-

652 0.95 21 TGCAT/255 miR172b

AGAATCTT GATGATGC ptc-

653 0.95 21 TGCAT/256 miR172c

GGAATCTT GATGATGC ptc-

654 1 21 TGCAT/257 miR172d

GGAATCTT GATGATGC ptc-

655 1 21 TGCAT/258 miR172e

AGAATCTT GATGATGC ptc-

656 0.95 21 TGCAT/259 miR172f

GGAATCTT GATGATGC ptc-

657 0.95 21 TGCAG/260 miR172g

GGAATCTT GATGATGC ptc-

658 0.95 21 TGC AG/261 miR172h

AGAATCCT GATGATGC ptc-

659 0.86 21 TGCAA/262 miR172i

GGAATCTT GATGATGC rco-

660 0.95 21 TGCAG/263 miR172

AGAATCTT GATGATGC sbi-

661 0.9 20 TGC A/264 miR172a

GGAATCTT GATGATGC sbi-

662 0.95 20 TGC A/265 miR172b

AGAATCTT GATGATGC sbi-

663 0.9 20 TGC A/266 miR172c

AGAATCTT GATGATGC sbi-

0.9 20 TGC A/267 miR172d

TGAATCTTG

ATGATGCT sbi-

665 0.9 21 GCAC/268 miR172e

AGAATCCT GATGATGC sbi-

666 0.86 21 TGCAC/269 miR172f

AGAATCTT GATGATGC sly-

667 0.95 21 TGCAT/270 miR172a

AGAATCTT GATGATGC sly-

668 0.95 21 TGC AT/271 miR172b AGAATCCT

GATGATGC sof-

669 0.86 21 TGCAG/272 miR172a

AGAATCTT GATGATGC stu-

670 0.95 21 TGCAT/273 miR172

AGAATCCT GATGATGC tae-

671 0.86 21 TGCAG/274 miR172a

AGAATCCT GATGATGC tae-

672 0.86 21 TGCAG/275 miR172b

AGGATCTT GATGATGC tae-

673 0.86 21 TGCAG/276 miR172c

AGAATCCT GATGATGC tca-

674 0.86 21 TGCAG/277 miR172

GGAATCTT GATGATGC tcc-

675 0.95 20 TGC A/278 miR172a

AGAATCTT GATGATGC tcc-

676 0.95 21 TGCAT/279 miR172b

GGAATCTT GATGATGC tcc-

677 1 21 TGCAT/280 miR172c

AGAATCCT GATGATGC tcc-

678 0.9 21 TGCAT/281 miR172d

AGAATCTT GATGATGC tcc-

679 0.95 21 TGCAT/282 miR172e

TGAATCTTG

ATGATGCT vvi-

680 0.9 21 ACAT/283 miR172a

TGAATCTTG

ATGATGCT vvi-

681 0.86 21 ACAC/284 miR172b

GGAATCTT GATGATGC vvi-

682 0.95 21 TGCAG/285 miR172c

TGAGAATC

TTGATGAT

GCTGCAT/28

6/AGAATCT

TGATGATG vvi-

683 0.95 23/21 CTGCAT/450 miR172d

AGAATCTT GATGATGC zma-

0.9 20 TGC A/287 miR172a

AGAATCTT GATGATGC zma-

685 0.9 20 TGC A/288 miR172b

AGAATCTT zma-

686 0.9 20 GATGATGC miR172c TGC A/289

AGAATCTT GATGATGC zma-

687 0.9 20 TGC A/290 miR172d

GGAATCTT GATGATGC zma-

688 1 21 TGC AT/291 miR172f

AGAATCCT GATGATGC zma-

689 0.86 21 TGCAG/292 miR172m

AGAATCCT GATGATGC zma-

690 0.9 21 TGCAT/293 miR172n

GTGAAG

CTGAAGTG TGTTTGG TTTGGGGG aly- GGGAAC zma-

691 0.9 21 GACTC/294 miR395b 55 21 TC/4 miR395b

CTGAAGTG TTTGGGGG aly-

692 0.86 21 GACTT/295 miR395c

CTGAAGTG TTTGGGGG aly-

693 0.95 21 AACTC/296 miR395d

CTGAAGTG TTTGGGGG aly-

694 0.95 21 AACTC/297 miR395e

CTGAAGTG TTTGGGGG aly-

695 0.9 21 GACTC/298 miR395f

CTGAAGTG TTTGGGGG aly-

696 0.95 21 AACTC/299 miR395g

CTGAAGTG TTTGGGGG aly-

697 0.9 21 GACTC/300 miR395h

CTGAAGTG TTTGGAGG aly-

698 0.9 21 AACTC/301 miR395i

CTGAAGGG TTTGGAGG aqc-

699 0.86 21 AACTC/302 miR395a

CTGAAGGG TTTGGAGG aqc-

700 0.86 21 AACTC/303 miR395b

CTGAAGTG TTTGGGGG ath-

701 0.95 21 AACTC/304 miR395a

CTGAAGTG TTTGGGGG ath-

702 0.9 21 GACTC/305 miR395b

CTGAAGTG TTTGGGGG ath-

703 0.9 21 GACTC/306 miR395c

CTGAAGTG TTTGGGGG ath-

704 0.95 21 AACTC/307 miR395d CTGAAGTG

TTTGGGGG ath-

705 0.95 21 AACTC/308 miR395e

CTGAAGTG TTTGGGGG ath-

706 0.9 21 GACTC/309 miR395f

TGAAGTGT TTGGGGGA bdi-

707 0.95 20 ACTC/310 miR395a

TGAAGTGT TTGGGGGA bdi-

708 0.95 20 ACTC/311 miR395b

TGAAGTGT TTGGGGGA bdi-

709 0.95 20 ACTC/312 miR395c

AAGTGTTT GGGGAACT bdi-

710 0.81 21 CTAGG/313 miR395d

TGAAGTGT TTGGGGGA bdi-

711 0.95 20 ACTC/314 miR395e

TGAAGTGT TTGGGGGA bdi-

712 0.95 20 ACTC/315 miR395f

TGAAGTGT TTGGGGGA bdi-

713 0.95 20 ACTC/316 miR395g

TGAAGTGT TTGGGGGA bdi-

714 0.95 20 ACTC/317 miR395h

TGAAGTGT TTGGGGGA bdi-

715 0.95 20 ACTC/318 miR395i

TGAAGTGT TTGGGGGA bdi-

716 0.95 20 ACTC/319 miR395j

TGAAGTGT TTGGGGGA bdi-

717 0.95 20 ACTC/320 miR395k

TGAAGTGT TTGGGGGA bdi-

718 0.95 20 ACTC/321 miR3951

TGAAGTGT TTGGGGGA bdi-

719 0.95 20 ACTC/322 miR395m

TGAAGTGT TTGGGGGA bdi-

720 0.95 20 ACTC/323 miR395n

CTGAAGTG TTTGGGGG csi-

721 0.95 21 AACTC/324 miR395

TTGAAGTG TTTGGGGG ghr-

722 0.9 21 AACTT/325 miR395a

CTAAAGTG ghr-

723 0.86 21 TTTAGGGG miR395c AACTC/326

CTGAAGTG TTTGGGGG ghr-

724 0.95 21 AACTC/327 miR395d

ATGAAGTG TTTGGGGG gma-

725 0.95 21 AACTC/328 miR395

ATGAAGTG TTTGGGGG mtr-

726 0.95 21 AACTC/329 miR395a

ATGAAGTA TTTGGGGG mtr-

727 0.9 21 AACTC/330 miR395b

ATGAAGTG TTTGGGGG mtr-

728 0.95 21 AACTC/331 miR395c

ATGAAGTG TTTGGGGG mtr-

729 0.95 21 AACTC/332 miR395d

ATGAAGTG TTTGGGGG mtr-

730 0.95 21 AACTC/333 miR395e

ATGAAGTG TTTGGGGG mtr-

731 0.95 21 AACTC/334 miR395f

TTGAAGTG TTTGGGGG mtr-

732 0.95 21 AACTC/335 miR395g

ATGAAGTG TTTGGGGG mtr-

733 0.9 21 AACTT/336 miR395h

ATGAAGTG TTTGGGGG mtr-

734 0.95 21 AACTC/337 miR395i

ATGAAGTG TTTGGGGG mtr-

735 0.95 21 AACTC/338 miR395j

ATGAAGTG TTTGGGGG mtr-

736 0.95 21 AACTC/339 miR395k

ATGAAGTG TTTGGGGG mtr-

737 0.95 21 AACTC/340 miR3951

ATGAAGTG TTTGGGGG mtr-

738 0.95 21 AACTC/341 miR395m

ATGAAGTG TTTGGGGG mtr-

739 0.95 21 AACTC/342 miR395n

ATGAAGTG TTTGGGGG mtr-

740 0.95 21 AACTC/343 miR395o

TTGAAGCG TTTGGGGG mtr-

741 0.9 21 AACTC/344 miR395p

742 0.95 21 ATGAAGTG mtr- TTTGGGGG miR395q AACTC/345

ATGAAGTG TTTGGGGG mtr-

743 0.95 21 AACTC/346 miR395r

GTGAAGTG CTTGGGGG osa-

744 0.95 21 AACTC/347 miR395a

TGAAGTGC TTGGGGGA osa-

745 0.9 20 ACTC/348 miR395a.2

GTGAAGTG TTTGGGGG osa-

746 1 21 AACTC/349 miR395b

GTGAAGTG TTTGGAGG osa-

747 0.95 21 AACTC/350 miR395c

GTGAAGTG TTTGGGGG osa-

748 1 21 AACTC/351 miR395d

GTGAAGTG TTTGGGGG osa-

749 1 21 AACTC/352 miR395e

GTGAATTG TTTGGGGG osa-

750 0.95 21 AACTC/353 miR395f

GTGAAGTG TTTGGGGG osa-

751 1 21 AACTC/354 miR395g

GTGAAGTG TTTGGGGG osa-

752 1 21 AACTC/355 miR395h

GTGAAGTG TTTGGGGG osa-

753 1 21 AACTC/356 miR395i

GTGAAGTG TTTGGGGG osa-

754 1 21 AACTC/357 miR395j

GTGAAGTG TTTGGGGG osa-

755 1 21 AACTC/358 miR395k

GTGAAGTG TTTGGGGG osa-

756 1 21 AACTC/359 miR3951

GTGAAGTG TTTGGGGG osa-

757 1 21 AACTC/360 miR395m

GTGAAGTG TTTGGGGG osa-

758 1 21 AACTC/361 miR395n

ATGAAGTG TTTGGAGG osa-

759 0.9 21 AACTC/362 miR395o

GTGAAGTG TTTGGGGG osa-

760 1 21 AACTC/363 miR395p GTGAAGTG

TTTGGGGG osa-

761 1 21 AACTC/364 miR395q

GTGAAGTG TTTGGGGG osa-

762 1 21 AACTC/365 miR395r

GTGAAGTG TTTGGGGG osa-

763 1 21 AACTC/366 miR395s

GTGAAGTG TTTGGGGA osa-

764 0.95 21 AACTC/367 miR395t

GTGAAGCG TTTGGGGG osa-

765 0.9 21 AAATC/368 miR395u

GTGAAGTA TTTGGCGG osa-

766 0.9 21 AACTC/369 miR395v

GTGAAGTG TTTGGGGG osa-

767 0.81 22 ATTCTC/370 miR395w

GTGAAGTG TTTGGAGT osa-

768 0.86 21 AGCTC/371 miR395x

GTGAAGTG TTTGGGGG osa-

769 1 21 AACTC/372 miR395y

CTGAAGTG TTTGGAGG pab-

770 0.86 21 AACTT/373 miR395

CTGAAGGG TTTGGAGG ptc-

771 0.86 21 AACTC/374 miR395a

CTGAAGTG TTTGGGGG ptc-

772 0.95 21 AACTC/375 miR395b

CTGAAGTG TTTGGGGG ptc-

773 0.95 21 AACTC/376 miR395c

CTGAAGTG TTTGGGGG ptc-

774 0.95 21 AACTC/377 miR395d

CTGAAGTG TTTGGGGG ptc-

775 0.95 21 AACTC/378 miR395e

CTGAAGTG TTTGGGGG ptc-

776 0.95 21 AACTC/379 miR395f

CTGAAGTG TTTGGGGG ptc-

777 0.95 21 AACTC/380 miR395g

CTGAAGTG TTTGGGGG ptc-

778 0.95 21 AACTC/381 miR395h

CTGAAGTG ptc-

779 0.95 21 TTTGGGGG miR395i AACTC/382

CTGAAGTG TTTGGGGG ptc-

780 0.95 21 AACTC/383 miR395j

CTGAAGTG TTTGGGGG rco-

781 0.95 21 AACTC/384 miR395a

CTGAAGTG TTTGGGGG rco-

782 0.95 21 AACTC/385 miR395b

CTGAAGTG TTTGGGGG rco-

783 0.95 21 AACTC/386 miR395c

CTGAAGTG TTTGGGGG rco-

784 0.95 21 AACTC/387 miR395d

CTGAAGTG TTTGGGGG rco-

785 0.95 21 AACTC/388 miR395e

GTGAAGTG TTTGGGGG sbi-

786 1 21 AACTC/389 miR395a

GTGAAGTG TTTGGGGG sbi-

787 1 21 AACTC/390 miR395b

GTGAAGTG TTTGGGGG sbi-

788/849 1 21 AACTC/391 miR395c

GTGAAGTG TTTGGGGG sbi-

789/850 1 21 AACTC/392 miR395d

GTGAAGTG TTTGGGGG sbi-

790 1 21 AACTC/393 miR395e

ATGAAGTG TTTGGGGG sbi-

791 0.95 21 AACTC/394 miR395f

GTGAAGTG TTTGGGGG sbi-

792 1 21 AACTC/395 miR395g

GTGAAGTG TTTGGGGG sbi-

793 1 21 AACTC/396 miR395h

GTGAAGTG TTTGGGGG sbi-

794 1 21 AACTC/397 miR395i

GTGAAGTG TTTGGGGG sbi-

795 1 21 AACTC/398 miR395j

GTGAAGTG TTTGGAGG sbi-

796 0.95 21 AACTC/399 miR395k

GTGAAGTG CTTGGGGG sbi-

797 0.95 21 AACTC/400 miR3951

798 0.95 21 CTGAAGTG sde- TTTGGGGG miR395 AACTC/401

CTGAAGTG TTTGGGGG sly-

799 0.95 22 AACTCC/402 miR395a

CTGAAGTG TTTGGGGG sly-

800 0.95 22 AACTCC/403 miR395b

GTGAAGTG TTTGGGGG tae-

801 1 21 AACTC/404 miR395a

TGAAGTGT TTGGGGGA tae-

802 0.95 20 ACTC/405 miR395b

CTGAAGTG TTTGGGGG tcc-

803 0.95 21 AACTC/406 miR395a

CTGAAGTG TTTGGGGG tcc-

804 0.95 21 AACTC/407 miR395b

CTGAAGTG TTTGGGGG vvi-

805 0.95 21 AACTC/408 miR395a

CTGAAGTG TTTGGGGG vvi-

806 0.95 21 AACTC/409 miR395b

CTGAAGTG TTTGGGGG vvi-

807 0.95 21 AACTC/410 miR395c

CTGAAGTG TTTGGGGG vvi-

808 0.95 21 AACTC/411 miR395d

CTGAAGTG TTTGGGGG vvi-

809 0.95 21 AACTC/412 miR395e

CTGAAGTG TTTGGGGG vvi-

810 0.95 21 AACTC/413 miR395f

CTGAAGTG TTTGGGGG vvi-

811 0.95 21 AACTC/414 miR395g

CTGAAGTG TTTGGGGG vvi-

812 0.95 21 AACTC/415 miR395h

CTGAAGTG TTTGGGGG vvi-

813 0.95 21 AACTC/416 miR395i

CTGAAGTG TTTGGGGG vvi-

814 0.95 21 AACTC/417 miR395j

CTGAAGTG TTTGGGGG vvi-

815 0.95 21 AACTC/418 miR395k

CTGAAGTG TTTGGGGG vvi-

816 0.95 21 AACTC/419 miR3951 CTGAAGTG

TTTGGGGG vvi-

817 0.95 21 AACTC/420 miR395m

CTGAAGAG TCTGGAGG vvi-

818 0.81 21 AACTC/421 miR395n

GTGAAGTG TTTGGGGG zma-

819 1 21 AACTC/422 miR395a

GTGAAGTG TTTGGAGG zma-

820 0.95 21 AACTC/423 miR395c

GTGAAGTG TTTGGGGG AACTC/424/

GTGAAGTG TTTGGAGG zma-

821/851 1.00/0.90 21/20 AACT/451 miR395d

GTGAAGTG TTTGGGGG AACTC/425/

GTGAAGTG TTTGGAGG zma-

822/852 1.00/0.95 21 AACTC/452 miR395e

GTGAAGTG TTTGGGGG AACTC/426/

GTGAAGTG TTTGAGGA zma-

823/853 1.00/0.90 21 AACTC/453 miR395f

GTGAAGTG TTTGGGGG zma-

824 1 21 AACTC/427 miR395g

GTGAAGTG TTTGGGGG zma-

825 1 21 AACTC/428 miR395h

GTGAAGTG TTTGGGGG zma-

826 1 21 AACTC/429 miR395i

GTGAAGTG TTTGGGGG zma-

827 1 21 AACTC/430 miR395j

GTGAAGTG TTTGAGGA zma-

828 0.9 21 AACTC/431 miR395k

GTGAAGTG TTTGGAGG zma-

829 0.95 21 AACTC/432 miR3951

GTGAAGTG TTTGGAGG zma-

830 0.95 21 AACTC/433 miR395m

GTGAAGTG TTTGGGGG zma-

831 1 21 AACTC/434 miR395n

GTGAAGTG zma-

832 0.95 21 TTTGGGTG miR395o AACTC/435

GTGAAGTG

TTTGGGGG zma-

833 1 21 AACTC/436 miR395p

AGAAGA

AGAAGAGA GAGAGA

GAGAGCAC aqc- GTACAG zma-

834 0.86 21 AACCC/437 miR529 56 21 CCT/1 miR529

AGAAGAGA

GAGAGTAC bdi-

835 1 21 AGCCT/438 miR529

AGAAGAGA

GAGAGCAC far-

836 0.9 21 AGCTT/439 miR529

AGAAGAGA

GAGAGTAC osa-

837 0.95 21 AGCTT/440 miR529b

CGAAGAGA

GAGAGCAC ppt-

838 0.86 21 AGCCC/441 miR529a

CGAAGAGA

GAGAGCAC ppt-

839 0.86 21 AGCCC/442 miR529b

CGAAGAGA

GAGAGCAC ppt-

840 0.86 21 AGCCC/443 miR529c

AGAAGAGA

GAGAGCAC ppt-

841 0.9 21 AGCCC/444 miR529d

AGAAGAGA

GAGAGTAC ppt-

842 0.95 21 AGCCC/445 miR529e

AGAAGAGA

GAGAGTAC ppt-

843 0.95 21 AGCCC/446 miR529f

CGAAGAGA

GAGAGCAC ppt-

844 0.81 21 AGTCC/447 miR529g

TAGCCA

TAGCCAAG AGCATG Predicted

GATGATTT bdi- ATTTGCC zma mir

845 0.9 22 GCCTGT/448 miR169k 21 CG/5 50601

TAGCCAAG

GATGATTT sbi-

846 0.9 21 GCCTG/449 miR169o

EXAMPLE 3

Identification of miRNAs Associated with Increased NUE and Target Prediction

Using Bioinformatics Tools

miRNAs that are associated with improved NUE and/or abiotic or biotic stress tolerance were identified by computational algorithms that analyze RNA expression profiles alongside publicly available gene and protein databases. A high throughput screening was performed on microarrays loaded with miRNAs that were found to be differentially expressed under multiple stress and optimal environmental conditions and in different plant tissues. The initial trait-associated miRNAs were later validated by 5 quantitative Real Time PCR (qRT-PCR).

Target prediction - orthologous genes to the genes of interest in maize and/or Arabidopsis were found through a bioinformatic tool that analyzes publicly available genomic as well as expression and gene annotation databases from multiple plant species. Homologous as well as orthologous protein and nucleotide sequences of target 10 genes of the small RNA sequences of the invention, were found using BLAST having at

least 70 % identity on at least 60 % of the entire master (maize) gene length, and are summarized in Tables 5-6 below.

Table 5: Target Genes of Small RNA Molecules that are upregulated during NUE.

Protein

Nucleotide Sequence % Nucleotide Homolog miR

Sequence seq id Identit NCBI GI NCBI Binding miR miR seq id no: no: Organism y Anotation number Accession Position sequence name

hypothetical

protein

LOCI 00384

547 [Zea

mays] Predi

>gi|23800 cted 5886|gb|AC AGGATG zma 33978.11 CTGACG mir unknown NP 00117 CAATGG 4848

895 854 Zea mays 1 [Zea mays] 293331460 0533 105-125 GAT/9 6

Predi cted putative gag- AGGATG zma pol TGAGGC mir polyprotein AAN4003 TATTGG 4849

855 Zea mays 1 [Zea mays] 23928433 0 33-54 GGAC/6 2 embryonic

flower 1 TTAGAT protein GACCAT zma-

Eulaliopsis [Eulaliopsis ADU3288 1977- CAGCAA miR8

896 856 binata 1 binata] 315493433 9 1997 AC A/ 10 27

0.9234 EMF-like ABC6915

897 857 Zea mays 45 [Zea mays] 85062576 4 VEF family

protein [Zea

mays]

>gi|29569

l l l |gb|AAO

84022.11

VEF family

protein [Zea

mays]

>gi|60687

422|gb|AAX

35735.11

embryonic

0.9346 flower 2 [Zea NP 00110

898 858 Zea mays 093 mays] 162461707 5530

EMF2

Dendrocal [Dendrocala

amus 0.8054 mus ABB7721

899 859 latiflorus 226 latiflorus] 82469918 0

embryonic

flower 2

Triticum 0.7974 [Triticum AAX7823

900 860 aestivum 482 aestivum] 62275660 2

Os09g03068

00 [Oryza

sativa

Japonica

Group]

>gi|25567

8755|dbj|BA

F24739.2|

Os09g03068

Oryza 00 [Oryza

sativa sativa

Japonica 0.7575 Japonica NP 00106

901 861 Group 758 Group] 115478459 2825 putative VEF

family

Oryza protein

sativa [Oryza sativa

Japonica 0.7575 Japonica BAD3651

862 Group 758 Group] 51091694 0

embryonic

flower 2

protein

Eulaliopsis 0.7575 [Eulaliopsis ADU3289

902 863 binata 758 binata] 315493435 0

predicted

protein

Hordeum [Hordeum

vulgare vulgare

subsp. 0.7687 subsp.

903 864 vulgare 4 vulgare] 326503299 BAJ99275

HvEMF2b

Hordeum 0.7703 [Hordeum BAD9913

904 865 vulgare 349 vulgare] 66796110 1 VEF family

protein [Zea

mays]

>gi|29569

l l l |gb|AAO

84022.11

VEF family

protein [Zea

mays]

>gi|60687

422|gb|AAX

35735.11

embryonic

flower 2 [Zea NP 00110 1748-

905 866 Zea mays 1 mays] 162461707 5530 1768

0.9792 EMF-like ABC6915

906 867 Zea mays 332 [Zea mays] 85062576 4

embryonic

flower 1

protein

Eulaliopsis 0.9361 [Eulaliopsis ADU3288

907 868 binata 022 binata] 315493433 9

EMF2

Dendrocal [Dendrocala

amus 0.8083 mus ABB7721

908 869 latiflorus 067 latiflorus] 82469918 0

embryonic

flower 2

Triticum 0.8019 [Triticum AAX7823

909 870 aestivum 169 aestivum] 62275660 2

Os09g03068

00 [Oryza

sativa

Japonica

Group]

>gi|25567

8755|dbj|BA

F24739.2|

Os09g03068

Oryza 00 [Oryza

sativa sativa

Japonica 0.7571 Japonica NP 00106

910 871 Group 885 Group] 115478459 2825

putative VEF

family

Oryza protein

sativa [Oryza sativa

Japonica 0.7555 Japonica BAD3651

872 Group 911 Group] 51091694 0

embryonic

flower 2

protein

Eulaliopsis 0.7635 [Eulaliopsis ADU3289

911 873 binata 783 binata] 315493435 0

Hordeum predicted

vulgare 0.7747 protein

912 874 subsp. 604 [Hordeum 326503299 BAJ99275 vulgare vulgare

subsp.

vulgare]

HvEMF2b

Hordeum 0.7763 [Hordeum BAD9913

875 vulgare 578 vulgare] 66796110 1

hypothetical

protein

SORBIDRA

FT_04g0319

20 [Sorghum

bicolor]

>gi|24193

4313|gb|EES

07458.11

hypothetical

protein

SORBIDRA

FT_04g0319

Sorghum 20 [Sorghum XP 00245

876 bicolor 1 bicolor] 255761094 4482 580-600

0.9425 unknown ACN3067

877 Zea mays 287 [Zea mays] 223972968 2

hypothetical

protein

LOC100501

893 [Zea

mays]

>gi|23801

1698|gb|ACR

36884.11

0.9410 unknown NP 00118

878 Zea mays 92 [Zea mays] 308044322 3461

RecName:

Full=SPX

domain- containing

membrane

protein

Os02g45520

>gi|30675

6291 |sp|A2X

8A7.2|SPXM l_ORYSI

RecName:

Full=SPX

domain- containing

membrane

protein

Osl_08463

>gi|50252

990|dbj|BAD

29241.11

SPX

(SYGl/Pho8

1 XPRl)

domain- containing

protein-like

[Oryza sativa

Japonica

Group]

>gi|50253

121 |dbj|BAD

29367.11

SPX

(SYGl/Pho8

1/XPRl)

domain- containing

Oryza protein-like

sativa [Oryza sativa

Japonica 0.8706 Japonica

879 Group 897 Group] Q6EPQ3 predicted

protein

Hordeum [Hordeum

vulgare vulgare

subsp. 0.8347 subsp.

880 vulgare 701 vulgare] 326502341 BAJ95234

OSJNBaOOl

9K04.6

[Oryza sativa

Japonica

Group]

>gi| 12559

Oryza 1348|gb|EAZ

sativa 31698.11

Japonica 0.8089 hypothetical CAD4165

881 Group 08 protein 38605939 9 OsJ_15847

[Oryza sativa

Japonica

Group]

Os04g05730

00 [Oryza

sativa

Japonica

Group]

>gi|30675

6012|sp|B8A

T51.1|SPXM

2 0RYSI

RecName:

Full=SPX

domain- containing

membrane

protein

Osl 17046

>gi|30675

6288|sp|Q0J

AW2.2|SPX

M2 0RYSJ

RecName:

Full=SPX

domain- containing

membrane

protein

Os04g05730

00

>gi|21569

4614|dbj|BA

G89805.1 |

unnamed

protein

product

[Oryza sativa

Japonica

Group]

>gi|21819

5403|gb|EEC

77830.11

hypothetical

protein

Osl_17046

[Oryza sativa

Indie a

Oryza Group]

sativa >gi|25567

Japonica 0.8089 5707|dbj|BA NP 00105 Group 08 F15525 115460021 3611 Os04g05730

00 [Oryza

sativa

Japonica

Group]

OSIGBa0147

Oryza H17.5

sativa [Oryza sativa

Indica 0.8060 Indica CAH6695

883 Group 345 Group] 116309919 7

hypothetical protein

SORBIDRA

FT_06g0259

50 [Sorghum bicolor]

>gi|24193

8147|gb|EES

11292.11

hypothetical protein

SORBIDRA

FT_06g0259

Sorghum 0.7844 50 [Sorghum XP 00244

884 bicolor 828 bicolor] 255761094 6964

PREDICTE

D:

hypothetical protein [Vitis

vinifera]

Vitis 0.7212 >gi|29774 XP 00228

885 vinifera 644 2609|emb|CB 225426756 2540 134758.31

unnamed

protein

product

[Vitis

vinifera] hypothetical

protein

SORBIDRA

FT_02g0279

20 [Sorghum

bicolor]

>gi|24192

5925|gb|EER

99069.11

hypothetical

protein

SORBIDRA

FT_02g0279

Sorghum 20 [Sorghum XP 00246

886 bicolor 1 bicolor] 255761094 2548 965-985 hypothetical

protein

LOCI 00279

277 [Zea

mays]

>gi|21988

4365|gb|ACL

52557.11

0.8819 unknown NP 00114

887 Zea mays 188 [Zea mays] 226498793 5770

0.8523 unknown ACN3447

888 Zea mays 985 [Zea mays] 224030802 7

hypothetical

protein

LOC100278

416 [Zea

mays]

>gi| 19565

2339|gb|AC

G45637.1 |

hypothetical

0.8523 protein [Zea NP 00114

889 Zea mays 985 mays] 226530255 5176

hypothetical

protein

LOC100191

388 [Zea

mays]

>gi| 19468

8768|gb|ACF

78468.11

unknown NP 00113 1075-

890 Zea mays 1 [Zea mays] 212274814 0294 1095 Os09g01354

00 [Oryza

sativa

Japonica

Group]

>gi|47848

428|dbj|BAD

22285.11

putative

octicosapepti

de/Phox/Bem

lp (PB1)

domain- containing

protein

[Oryza sativa

Japonica

Group]

>gi|11363

0871 |dbj|BA

F24552.1|

Os09g01354

Oryza 00 [Oryza

sativa sativa

Japonica 0.7869 Japonica NP 00106

891 Group 822 Group] 115478085 2638

hypothetical

protein

SORBIDRA

FT_02g0377

70 [Sorghum

bicolor]

>gi|24192

4313|gb|EER

97457.11

hypothetical

protein

SORBIDRA ATTCAC

FT_02g0377 GGGGAC mtr-

Sorghum 70 [Sorghum XP 00246 GAACCT miR2

892 bicolor 1 bicolor] 255761094 0936 547-567 CCT/8 647a hypothetical

protein

LOCI 00279

098 [Zea

mays]

>gi| 19565

8887|gb|AC

G48911. l l

hypothetical

0.8738 protein [Zea NP 00114

893 Zea mays 462 mays] 226507742 5615 hypothetical

protein

LOC100278

263 [Zea

mays]

>gi| 19565

0593|gb|AC

G44764.1 |

hypothetical

0.8307 protein [Zea NP_00114

926 Zea mays 692 mays] 226495966 5067

Table 6: Target Genes of Small RNA Molecules that are down regulated during

NUE.

Protein Nucleotide Homolog miR

Nucleotide seq id % NCBI GI ue NCBI Binding miR miR seq id no: no: Organism Identity Annotation number Accession Position sequence name

hypothetical

protein

SORBIDRA

FT_01g0084

50 [Sorghum

bicolor]

>gi|24191

7750|gb|EER

90894.11

hypothetical

protein

SORBIDRA GTGAAG

FT_01g0084 TGTTTG zma-

Sorghum 50 [Sorghum XP 00246 GGGGAA miR3

927 bicolor 1 bicolor] 255761094 3896 426-446 CTC/4 95b

0.946721 unknown ACN2860

1022 928 Zea mays 311 [Zea mays] 223949050 9

0.954918 unknown ACN3402

1023 929 Zea mays 033 [Zea mays] 224029894 3

bifunctional

phosphoaden

osine 5- phosphosulfa

0.942622 te synthetase ACG4519

1024 930 Zea mays 951 [Zea mays] 195651448 2 ATP

sulfurylase

[Zea mays]

>gi|27387

50|gb|AAB9

4542.11 ATP

0.946721 sulfurylase NP 00110

1025 931 Zea mays 311 [Zea mays] 162463127 4877 hypothetical

protein

Oryza Osl_13470

sativa [Oryza

Indica 0.799180 sativa Indica EAY9182

932 Group 328 Group] 54362548 5

Os03g07439

00 [Oryza

sativa

Japonica

Group]

>gi|30017

582|gb|AAP

13004.11

putative

ATP

sulfurylase

[Oryza

sativa

Japonica

Group]

>gi| 10871

1024|gb|AB

F98819.1 |

Bifunctional

3'- phosphoaden

osine

5'- phosphosulfa

te

synthethase,

putative,

expressed

[Oryza

sativa

Japonica

Group]

>gi| 11354

9705|dbj|BA

F 13148.1 |

Os03g07439

00 [Oryza

sativa

Japonica

Group]

Oryza >gi|21570

sativa 4581 |dbj|BA

Japonica 0.797131 G94214.1 NP 00105

1026 933 Group 148 unnamed 115455266 1234 protein

product

[Oryza

sativa

Japonica

Group]

predicted

protein

[Hordeum

vulgare

subsp.

vulgare]

>gi|32650

2564|dbj|BA

J95345.ll

predicted

protein

Hordeum [Hordeum

vulgare vulgare

subsp. 0.793032 subsp. BAK0566

1027 934 vulgare 787 vulgare] 326491124 2

plastidic

ATP

Oryza sulfurylase

sativa [Oryza

Indica 0.797131 sativa Indica BAA3627

1028 935 Group 148 Group] 3986152 4

hypothetical

Oryza protein

sativa OsJ_12530

Japonica 0.770491 [Oryza EAZ2854

936 Group 803 sativa 54398660 8 Japonica

Group] hypothetical

protein

SORBIDRA

FT_08g0046

50 [Sorghum

bicolor]

>gi|24194

2597|gb|EES

15742.11

hypothetical

protein

SORBIDRA

FT_08g0046

Sorghum 50 [Sorghum XP 00244 bicolor 1 bicolor] 255761094 1904 352-372

Osl2g01741

00 [Oryza

sativa

Japonica

Group]

>gi|77553

790|gb|ABA

96586.11

Growth

regulator

protein,

putative,

expressed

[Oryza

sativa

Japonica

Group]

>gi|25567

0095|dbj|BA

F29304.2|

Osl2g01741

Oryza 00 [Oryza

sativa sativa

Japonica 0.705440 Japonica NP 00106 Group 901 Group] 115487595 6285

hypothetical

protein

OsJ_35390

Oryza [Oryza

sativa sativa

Japonica 0.705440 Japonica EEE5285

Group 901 Group] 54398660 1

hypothetical

protein

Oryza OsI_37646

sativa [Oryza

Indica 0.701688 sativa Indica EEC6894

Group 555 Group] 54362548 0 unknown ACN3402

1030 941 Zea mays 1 [Zea mays] 224029894 3 616-636

ATP

sulfurylase

[Zea mays]

>gi|27387

50|gb|AAB9

4542.11 ATP

0.983640 sulfurylase NP 00110

1031 942 Zea mays 082 [Zea mays] 162463127 4877

0.940695 unknown ACN2860

1032 943 Zea mays 297 [Zea mays] 223949050 9

bifunctional

3- phosphoaden

osine 5- phosphosulfa

0.936605 te synthetase ACG4519

1033 944 Zea mays 317 [Zea mays] 195651448 2

hypothetical

protein

SORBIDRA

FT_01g0084

50 [Sorghum

bicolor]

>gi|24191

7750|gb|EER

90894.11

hypothetical

protein

SORBIDRA

FT_01g0084

Sorghum 0.938650 50 [Sorghum XP 00246

945 bicolor 307 bicolor] 255761094 3896

predicted

protein

[Hordeum

vulgare

subsp.

vulgare]

>gi|32650

2564|dbj|BA

J95345.ll

predicted

protein

Hordeum [Hordeum

vulgare vulgare

subsp. 0.842535 subsp. BAK0566

1034 946 vulgare 787 vulgare] 326491124 2

hypothetical

protein

Oryza Osl_13470

sativa [Oryza

Indica 0.795501 sativa Indica EAY9182

947 Group 022 Group] 54362548 5 Os03g07439

00 [Oryza

sativa

Japonica

Group]

>gi|30017

582|gb|AAP

13004.11

putative

ATP

sulfurylase

[Oryza

sativa

Japonica

Group]

>gi| 10871

1024|gb|AB

F98819.1 |

Bifunctional

3'- phosphoaden

osine

5'- phosphosulfa

te

synthethase,

putative,

expressed

[Oryza

sativa

Japonica

Group]

>gi| 11354

9705|dbj|BA

F 13148.1 |

Os03g07439

00 [Oryza

sativa

Japonica

Group]

>gi|21570

4581 |dbj|BA

G94214.1

unnamed

protein

product

Oryza [Oryza

sativa sativa

Japonica 0.793456 Japonica NP 00105

1035 948 Group 033 Group] 115455266 1234 plastidic

ATP

Oryza sulfurylase

sativa [Oryza

Indica 0.793456 sativa Indica BAA3627

1036 949 Group 033 Group] 3986152 4 hypothetical

protein

OsJ_12530

Oryza [Oryza

sativa sativa

Japonica 0.764826 Japonica EAZ2854

950 Group 176 Groupl 54398660 8

hypothetical

protein

SORBIDRA

FT_04g0267

10 [Sorghum

bicolor]

>gi|24193

2317|gb|EES

05462.11

hypothetical

protein

SORBIDRA GGAATC

FT_04g0267 TTGATG zma-

Sorghum 10 [Sorghum XP 00245 1000- ATGCTG miRl

951 bicolor 1 bicolor] 255761094 2486 1020 CAT/3 72e

0.880208 unknown ACN3122

1037 952 Zea mays 333 [Zea mays] 223974072 4

hypothetical

protein

LOCI 00276

301 [Zea

mays]

>gi| 19562

3072|gb|AC

G33366.1

hypothetical

0.880208 protein [Zea NP 00114

1038 953 Zea mays 333 mays] 226500051 3596

hypothetical

protein

LOCI 00277

041 [Zea

mays]

>gi| 19563

8130|gb|AC

G38533.1 |

hypothetical

protein [Zea

mays]

>gi|22394

2145|gb|AC

N25156.1

0.864583 unknown NP 00114

1039 954 Zea mays 333 [Zea mays] 226492590 4184 Os02g06310

00 [Oryza

sativa

Japonica

Group]

>gi|49389

184|dbj|BAD

26474.11

unknown

protein

[Oryza

sativa

Japonica

Group]

>gi| 11353

7028|dbj|BA

F09411.l l

Os02g06310

00 [Oryza

sativa

Japonica

Group]

>gi|21569

7023|dbj|BA

G91017.1 | unnamed

protein

product

[Oryza

sativa

Japonica

Group]

>gi|21819

1219|gb|EEC

73646.11

hypothetical protein

Osl_08167

[Oryza

sativa Indica

Group]

>gi|22262

3287|gb|EEE

57419.11

hypothetical protein

OsJ_07614

Oryza [Oryza

sativa sativa

Japonica 0.776041 Japonica NP_00104 Group 667 Group] 115447434 7497 predicted

protein

[Hordeum

vulgare

subsp.

vulgare]

>gi| 32651

9272|dbj|BA

J96635.ll

predicted

protein

Hordeum [Hordeum

vulgare vulgare

subsp. 0.760416 subsp.

1041 956 vulgare 667 vulgare] 326512283 BAJ96123

AP2 domain

transcription

factor [Zea ABR1987

1042 957 Zea mays 1 mays] 148964889 1 869-889

AP2 domain

transcription

0.960439 factor [Zea ABR1987

1043 958 Zea mays 56 mays] 148964859 0

hypothetical

protein

SORBIDRA

FT 02g0070

00 [Sorghum

bicolor]

>gi|24192

2957|gb|EER

96101.11

hypothetical

protein

SORBIDRA

FT_02g0070

Sorghum 00 [Sorghum XP 00245 1539-

959 bicolor 1 bicolor] 255761094 9580 1559 sister of

indeterminat

e spikelet 1

[Zea mays]

>gi|22394

7941 |gb|AC

N28054.i l

0.855287 unknown NP 00113

1044 960 Zea mays 57 [Zea mays] 225703093 9539

sister of

indeterminat

0.844155 e spikelet 1 ACN5822

1045 961 Zea mays 844 [Zea mays] 224579291 4

floral

homeotic

protein [Zea

mays]

0.742115 >gi|23801 ACG4630

1046 962 Zea mays 028 134|gb|AC 195653672 4 R38602.1 |

unknown

[Zea mays]

Os01g08345

00 [Oryza

sativa

Japonica

Group]

>gi| 11545

6215|ref]NP

001051708.1

1

Os03g08184

00 [Oryza

sativa

Japonica

Group]

>gi|29772

0551 |reflNP

001172637.1

1

Os01g08346

01 [Oryza

sativa

Japonica

Group]

>gi|31310

3637|pdb|3I

Z6 L Chain

L,

Localization

Of The

Small

Subunit

Ribosomal

Proteins Into

A 5.5 A

Cryo-Em

Map Of

Triticum

Aestivum

Translating

80s

Ribosome

>gi|20805

266|dbj|BAB

92932.11

putative 40s

ribosomal

protein S23

[Oryza

Oryza sativa

sativa Japonica

Japonica Group] NP 00104 1121- Group 1 >gi|20805 115440880 4720 1141 267|dbj|BAB

92933.11 putative 40s ribosomal protein S23

[Oryza sativa

Japonic a

Group]

>gi|21671

347|dbj|BAC

02683.11 putative 40s ribosomal protein S23

[Oryza sativa

Japonic a

Group]

>gi|21671

348|dbj|BAC

02684.11 putative 40s ribosomal protein S23

[Oryza sativa

Japonic a

Group]

>gi|28876

025|gb|AAO

60034.11 40S ribosomal protein S23

[Oryza sativa

Japonic a

Group]

>gi|29124

115|gb|AAO

65856.11 40S ribosomal protein S23

[Oryza sativa

Japonic a

Group]

>gi| 10871

1771 |gb|AB

F99566.1 |

40S ribosomal protein S23, putative, expressed

[Oryza sativa

Japonic a Group]

>gi| 11353

4251 |dbj|BA

F06634.1 |

Os01g08345

00 [Oryza sativa

Japonic a

Group]

>gi| 11355

0179|dbj|BA

F13622.1 |

Os03g08184

00 [Oryza sativa

Japonic a

Group]

>gi| 12552

8286|gb|EA

Y76400.i l hypothetical protein

Osl_04329

[Oryza sativa Indica

Group]

>gi| 12554

6216|gb|EA

Y92355.i l hypothetical protein

Osl_14082

[Oryza sativa Indica

Group]

>gi|21569

7420|dbj|BA

G91414.1 | unnamed protein product

[Oryza sativa

Japonic a

Group]

>gi|21573

4943|dbj|BA

G95665.1 unnamed protein product

[Oryza sativa

Japonic a

Group]

>gi|25567

3847|dbj|BA

H91367.1 Os01g08346

01 [Oryza sativa

Japonic a

Group]

>gi|32650

1134|dbj|BA

J98798.ll predicted protein

[Hordeum vulgare subsp. vulgare]

>gi|32650

6086|dbj|BA

J91282.ll predicted protein

[Hordeum vulgare subsp. vulgare]

hypothetical

protein

LOCI 00192

600 [Zea

mays]

>gi|24203

2479|reflXP_

002463634.1

I

hypothetical

protein

SORBIDRA

FT_01g0034

10 [Sorghum

bicolor]

>gi|24205

9153|reflXP_

002458722.1

I

hypothetical

protein

SORBIDRA

FT_03g0390

10 [Sorghum

bicolor]

>gi|24209

0801 |reflXP_

002441233.1

I

hypothetical

protein

SORBIDRA

FT_09g0228

40 [Sorghum

bicolor]

>gi| 19469

1088|gb|AC

F79628.1 |

unknown

[Zea mays]

>gi| 19469

7612|gb|AC

F82890.1 |

unknown

[Zea mays]

>gi| 19470

2740|gb|AC

F85454.1 |

unknown

[Zea mays]

>gi| 19560

6082|gb|AC

G24871.1 |

40S

ribosomal

protein S23

0.992957 [Zea mays] NP_00113

1048 964 Zea mays 746 >gi| 19561 212722729 1287 8728|gb|AC

G31194.l l

40S ribosomal protein S23 [Zea mays] >gi| 19561 9636|gb|AC G31648.1 40S ribosomal protein S23 [Zea mays] >gi| 19562 5318|gb|AC G34489.1 40S ribosomal protein S23 [Zea mays] >gi| 19562 8702|gb|AC G36181.1 | 40S ribosomal protein S23 [Zea mays] >gi| 19565 7679|gb|AC G48307.1 | 40S ribosomal protein S23

[Zea mays]

>gi|23801

2290|gb|AC

R37180.1 | unknown

[Zea mays]

>gi|24191

7488|gb|EER

90632.11 hypothetical protein

SORBIDRA

FT_01g0034

10 [Sorghum bicolor]

>gi|24193

0697|gb|EES

03842.11 hypothetical protein

SORBIDRA

FT_03g0390

10 [Sorghum bicolor]

>gi|24194 6518|gb|EES

19663.11

hypothetical

protein

SORBIDRA

FT_09g0228

40 [Sorghum

bicolor]

40S

ribosomal

0.985915 protein S23 ACG3284

1049 965 Zea mays 493 [Zea mays] 195622025 3

40S

ribosomal

protein S23

[Elaeis

guineensis]

>gi| 19291

0894|gb|AC

F06555.1 |

40S

ribosomal

protein S23

Elaeis 0.978873 [Elaeis ACF0651

1050 966 guineensis 239 guineensis] 192910819 8

40S

ribosomal

protein S23

Elaeis 0.971830 [Elaeis ACF0651

1051 967 guineensis 986 guineensis] 192910821 9 unknown

Solatium 0.964788 [Solanum ABB 1699

1052 968 tuberosum 732 tuberosum] 77999292 3

40S

ribosomal

protein S23,

putative

[Ricinus

communis]

>gi|25556

8414|rellXP

002525181.1

1 40S

ribosomal

protein S23,

putative

[Ricinus

communis]

>gi|22353

5478|gb|EEF

37147.11 40S

ribosomal

protein S23,

putative

[Ricinus

communis]

>gi|22353

6832|gb|EEF

38471.11 40S

ribosomal

protein S23,

putative

Ricinus 0.964788 [Ricinus XP 00252

969 communis 732 communis] 255761086 3902

PREDICTE

D:

hypothetical

protein

Vitis 0.964788 [Vitis XP 00227

1053 970 vinifera 732 vinifera] 225439887 9025

unknown

[Zea mays]

>gi|22397

3927|gb|AC

N31151.i l

unknown

[Zea mays]

>gi|32338

8595|gb|AD

X60102.l l

SBP AGAAGA

transcription GAGAGA zma- factor [Zea ACN3057 GTACAG miR5

1054 971 Zea mays 1 mays] 223972764 0 882-902 CCT/1 29 hypothetical

protein

LOCI 00278

824 [Zea

mays]

>gi| 19565

6399|gb|AC

G47667.1 |

hypothetical

0.984615 protein [Zea NP 00114

1055 972 Zea mays 385 mays] 226530074 5445

hypothetical

protein

SORBIDRA

FT_05g0175

10 [Sorghum

bicolor]

>gi|24193

6618|gb|EES

09763.11

hypothetical

protein

SORBIDRA

FT_05g0175

Sorghum 0.870769 10 [Sorghum XP 00245

973 bicolor 231 bicolor] 255761094 0775 hypothetical

protein

SORBIDRA

FT_03g0254

10 [Sorghum

bicolor]

>gi|24192

7774|gb|EES

00919.11

hypothetical

protein

SORBIDRA

FT_03g0254

Sorghum 10 [Sorghum XP 00245

974 bicolor 1 bicolor] 255761094 5799 45-65

0.893939 unknown ACN2752

1056 975 Zea mays 394 [Zea mays] 223946882 5

hypothetical

protein

LOCI 00278

489 [Zea

mays]

>gi| 19565

3155|gb|AC

G46045.1

hypothetical

0.890151 protein [Zea NP 00114

1057 976 Zea mays 515 mays] 226501393 5223 unknown

[Zea mays]

>gi|32338

8573|gb|AD

X60091.ll

SBP

transcription

factor [Zea ACF8678 9910, 6-

1058 977 Zea mays 1 mays] 238908852 2 916 squamosa

promoter- binding-like

0.997354 protein 9 ACG4511

1059 978 Zea mays 497 [Zea mays] 195651290 3

hypothetical

protein

SORBIDRA

FT_02g0284

20 [Sorghum

bicolor]

>gi|24192

5948|gb|EER

99092.11

hypothetical

protein

SORBIDRA

FT_02g0284

Sorghum 0.828042 20 [Sorghum XP 00246

979 bicolor 328 bicolor] 255761094 2571

hypothetical

protein

LOCI 00217

104 [Zea

mays]

>gi| 19469

7718|gb|AC

F82943.1 |

0.756613 unknown NP 00113

1060 980 Zea mays 757 [Zea mays] 219363104 6945

squamosa

promoter- binding-like

protein 11

[Zea mays]

>gi| 19562

7850|gb|AC

G35755.1 |

squamosa

promoter- binding-like

protein 11

[Zea mays]

>gi| 19564

4948|gb|AC

G41942.1 |

squamosa NP 00114 1348-

1061 981 Zea mays 1 promoter- 226529809 9534 1368 binding-like

protein 11

[Zea mays]

hypothetical

protein

SORBIDRA

FT 10g0291

90 [Sorghum

bicolor]

>gi|24191

7194|gb|EER

90338.11

hypothetical

protein

SORBIDRA

FT_10g0291

Sorghum 0.876993 90 [Sorghum XP 00243

982 bicolor 166 bicolor] 255761094 8971

hypothetical

protein

LOCI 00217

104 [Zea

mays]

>gi| 19469

7718|gb|AC

F82943.1 |

unknown NP 00113

983 Zea mays 1 [Zea mays] 219363104 6945 973-993 hypothetical

protein

SORBIDRA

FT_02g0284

20 [Sorghum

bicolor]

>gi|24192

5948|gb|EER

99092.11

hypothetical

protein

SORBIDRA

FT_02g0284

Sorghum 0.817232 20 [Sorghum XP 00246

984 bicolor 376 bicolor] 255761094 2571 unknown

[Zea mays]

>gi|32338

8573|gb|AD

X60091.ll

SBP

transcription

0.759791 factor [Zea ACF8678

1063 985 Zea mays 123 mays] 238908852 2

squamosa

promoter- binding-like

0.757180 protein 9 ACG4511

1064 986 Zea mays 157 [Zea mays] 195651290 3

SBP-domain

protein 5 CAB5663

1065 987 Zea mays 1 [Zea mays] 5931785 1 558-578 hypothetical

protein

SORBIDRA

FT_07g0277

40 [Sorghum

bicolor]

>gi|24194

1121 |gb|EES

14266.11

hypothetical

protein

SORBIDRA

FT_07g0277

Sorghum 0.854103 40 [Sorghum XP 00244

988 bicolor 343 bicolor] 255761094 4771

0.784194 unknown ACL5294

1066 989 Zea mays 529 [Zea mays] 219885132 1

MTA/SAH

nucleosidase

[Zea mays]

>gi| 19565

8647|gb|AC

G48791.ll

MTA/SAH

nucleosidase

[Zea mays]

>gi|22397

3627|gb|AC

N31001.1

unknown NP 00115 1410-

1067 990 Zea mays 1 [Zea mays] 226529725 2658 1430

0.884462 unknown ACF8383

1068 991 Zea mays 151 [Zea mays] 194699507 8

MTA/SAH

0.884462 nucleosidase ACG3959

1069 992 Zea mays 151 [Zea mays] 195640251 4 hypothetical

protein

SORBIDRA

FT 07g0261

90 [Sorghum

bicolor]

>gi|24194

2163|gb|EES

15308.11

hypothetical

protein

SORBIDRA

FT_07g0261

Sorghum 0.884462 90 [Sorghum XP 00244

993 bicolor 151 bicolor] 255761094 5813

0.900398 unknown ACN3148

1070 994 Zea mays 406 [Zea mays] 223974590 3

Os06g01122

00 [Oryza

sativa

Japonica

Group]

>gi|73632

90|dbj|BAA9

3034.11

methylthioad

enosine/S- adenosyl

homocystein

e

nucleosidase

[Oryza

sativa

Japonica

Group]

>gi|32352

128|dbj|BAC

78557.11

hypothetical

protein

[Oryza

sativa

Japonica

Group]

>gi| 11359

4632|dbj|BA

F 18506.1 |

Os06g01122

00 [Oryza

sativa

Japonica

Group]

>gi| 12559

804|gb|EA

Oryza Z35584.l l

sativa hypothetical

Japonica 0.796812 protein NP 00105

1071 995 Group 749 OsJ 19870 115465985 6592 [Oryza

sativa

Japonic a

Group]

>gi|21569

4661 |dbj|BA

G89852.1 |

unnamed

protein

product

[Oryza

sativa

Japonic a

Group]

>gi|21574

0802|dbj|BA

G96958.1 |

unnamed

protein

product

[Oryza

sativa

Japonic a

Group]

methyltbioad

enosine/S- adenosyl

homocystein

e

nucleosidase

Oryza 0.792828 [Oryza AAL5888

1072 996 sativa 685 sativa] 18087496 3

mta/sah

Oryza nucleosidase

sativa [Oryza

Indica 0.792828 sativa Indica AB 2549

1073 997 Group 685 Group] 149390954 5

predicted

protein

[Hordeum

vulgare

subsp.

Hordeum vulgare]

vulgare >gi|32653

subsp. 0.780876 4118|dbj|BA BAK0331

1074 998 vulgare 494 J89409.ll 326512819 7 predicted

protein

[Hordeum

vulgare

subsp.

vulgare] hypothetical

protein

Oryza Osl_21350

sativa [Oryza

Indica 0.784860 sativa Indica EAY9938

999 Group 558 Group] 54362548 2

teosinte

glume

Zea mays architecture

subsp. 1 [Zea mays AAX8387 1197-

1075 1000 mays 1 subsp. mays] 72536147 2 1217 teosinte

glume

Zea mays architecture

subsp. 0.983796 1 [Zea mays AAX8387

1001 mays 296 subsp. mays] 5

teosinte

glume

architecture

1 [Zea mays

subsp. mays]

>gi| 62467

440|gb|AAX

83874.11

teosinte

glume

Zea mays architecture

subsp. 0.990740 1 [Zea mays AAX8387

1076 1002 mays 741 subsp. mays] 62467433 3

hypothetical

protein

SORBIDRA

FT_07g0262

20 [Sorghum

bicolor]

>gi|24194

2165|gb|EES

15310.11

hypothetical

protein

SORBIDRA

FT_07g0262

Sorghum 0.800925 20 [Sorghum XP 00244

1003 bicolor 926 bicolor] 255761094 5815 hypothetical

protein

SORBIDRA

FT_02g0389

60 [Sorghum

bicolor]

>gi|24192

6544|gb|EER

99688.11

hypothetical

protein

SORBIDRA TAGCCA

FT_02g0389 GGGATG zma-

Sorghum 60 [Sorghum XP 00246 1112- ATTTGC miRl

1004 bicolor 1 bicolor] 255761094 3167 1132 CTG/2 691 nuclear

transcription

factor Y

0.897009 subunit A- 3 ACG3682

1077 1005 Zea mays 967 [Zea mays] 195634708 3

hypothetical

protein

LOCI 00194

182 [Zea

mays]

>gi| 19469

5138|gb|AC

F81653.1 |

unknown

[Zea mays]

>gi| 19562

5280|gb|AC

G34470.1 |

nuclear

transcription

factor Y

0.890365 subunit A- 3 NP 00113

1078 1006 Zea mays 449 [Zea mays] 212723473 2701

0.887043 unknown ACN3330

1079 1007 Zea mays 189 [Zea mays] 224028448 0

0.853820 unknown ACF8371

1080 1008 Zea mays 598 [Zea mays] 194699259 4

nuclear

transcription

factor Y

0.853820 subunit A- 3 ACG2673

1081 1009 Zea mays 598 [Zea mays] 195609807 4

nuclear

transcription

factor Y

subunit A- 3

[Zea mays]

>gi| 19560

9780|gb|AC

G26720.1

0.850498 nuclear NP 00114

1082 1010 Zea mays 339 transcription 226499901 7311 factor Y

subunit A- 3

[Zea mays]

hypothetical

protein

LOCI 00194

182 [Zea

mays]

>gi| 19469

5138|gb|AC

F81653.1 |

unknown

[Zea mays]

>gi| 19562

5280|gb|AC

G34470.1 |

nuclear

transcription

factor Y

subunit A- 3 NP 00113 1108-

1083 1011 Zea mays 1 [Zea mays] 212723473 2701 1128

0.996666 unknown ACN3330

1084 1012 Zea mays 667 [Zea mays] 224028448 0

nuclear

transcription

factor Y

subunit A- 3 ACG3682

1085 1013 Zea mays 0.98 [Zea mays] 195634708 3

hypothetical

protein

SORBIDRA

FT_02g0389

60 [Sorghum

bicolor]

>gi|24192

6544|gb|EER

99688.11

hypothetical

protein

SORBIDRA

FT_02g0389

Sorghum 0.893333 60 [Sorghum XP 00246

1014 bicolor 333 bicolor] 255761094 3167

0.853333 unknown ACF8371

1086 1015 Zea mays 333 [Zea mays] 194699259 4

nuclear

transcription

factor Y

0.856666 subunit A- 3 ACG2673

1087 1016 Zea mays 667 [Zea mays] 195609807 4 nuclear

transcription

factor Y

subunit A- 3

[Zea mays]

>gi| 19560

9780|gb|AC

G26720.1 |

nuclear

transcription

factor Y

0.853333 subunit A- 3 NP 00114

1088 1017 Zea mays 333 [Zea mays] 226499901 7311

nuclear

transcription

factor Y

subunit A- 3

[Zea mays]

>gi| 19562

4530|gb|AC

G34095.1 |

nuclear

transcription

factor Y

subunit A- 3 NP 00114

1089 1018 Zea mays 1 [Zea mays] 226502984 9075 979-999 hypothetical

protein

SORBIDRA

FT_04g0347

60 [Sorghum

bicolor]

>gi|24193

4478|gb|EES

07623.11

hypothetical

protein

SORBIDRA

FT_04g0347

Sorghum 0.814545 60 [Sorghum XP 00245

1019 bicolor 455 bicolor] 255761094 4647

hypothetical

protein

SORBIDRA

FT 01g0042

90 [Sorghum

bicolor]

>gi|24191

7544|gb|EER

90688.11

hypothetical

protein

SORBIDRA

FT 01g0042

Sorghum 90 [Sorghum XP 00246

1020 bicolor 1 bicolor] 255761094 3690 946-966

1090 1021 Zea mays 0.836633 unknown 194696171 ACF8217 663 [Zea mays] 0

EXAMPLE 4

Verification of Expression of miRNAs Associated with Increased NUE Following identification of dsRNAs potentially involved in improvement of maize NUE using bioinformatics tools, as described in Examples 1-2 above, the actual mRNA levels were determined using reverse transcription assay followed by quantitative Real-Time PCR (qRT-PCR) analysis. RNA levels were compared between different tissues, developmental stages, growth conditions and/or genetic backgrounds incorporated. A correlation analysis between mRNA levels in different experimental conditions/genetic backgrounds was applied and used as evidence for the role of the gene in the plant.

Methods

Mobile nutrients such as N reach their targets and are then recycled, often executed in the form of simultaneous import and export of the nutrients from leaves. This dynamic nutrient cycling is termed remobilization or retranslocation, and thus leaf analyses are highly recommended. For that reason, root and leaf samples were freshly excised from maize plants grown as described above on Murashige-Skoog without Ammonium Nitrate (NH₄NO₃) (Duchefa). Experimental plants were grown either under optimal ammonium nitrate concentrations (100%) and used as a control group, or under stressful conditions of 10% or 1% ammonium nitrate used as stress-induced groups. Total RNA was extracted from the different tissues, using mirVana™ commercial kit (Ambion) following the protocol provided by the manufacturer. For measurement and verification of messenger RNA (mRNA) expression level of all genes, reverse transcription followed by quantitative real time PCR (qRT-PCR) was performed on total RNA extracted from each plant tissue (i.e., roots and leaves) from each experimental group as described above. To elaborate, reverse transcription was performed on 1 μg total RNA, using a miScript Reverse Transcriptase kit (Qiagen), following the protocol suggested by the manufacturer. Quantitative RT-PCR was performed on cDNA (0.1 ng/μΐ final concentration), using a miScript SYBR GREEN PCR (Qiagen) forward (based on the miR sequence itself) and reverse primers (supplied with the kit). All qRT-PCR reactions were performed in triplicates using an ABI7500 real-time PCR machine, following the recommended protocol for the machine. To normalize, the expression level of miRNAs associated with enhanced NUE between the different tissues and growth conditions of the maize plants, normalizer miRNAs were used for comparison. Normalizer miRNAs, which are miRNAs with unchanged expression level between tissues and growth conditions, were custom selected for each experiment. The normalization procedure consisted of second-degree polynomial fitting to a reference data (which is the median vector of all the data - excluding outliers) as described by Rosenfeld et al (2008, Nat Biotechnol, 26(4):462-469). A summary of primers for the differential miRNAs that was used in the qRT-PCR analysis is presented in Table 7a below. The results of the qRT-PCR analyses under different nitrogen concentrations (1% and 10% versus optimal 100%) are presented in Tables 7b-d below.

Table 7a: Primers of Small RNAs used for qRT-PCR Validation Analysis.

Table 7b: Results of qRT-PCR Validation Analysis on Differential Small RNAs - 1% Nitrogen vs. Control (100% Nitrogen).

Fold

p-value Change Direction Sequence/SEQ ID NO: miR Name

3.20E-03 1.68 up TTAGATGACCATC AGC AAAC A/ 10 zma-miR827

3.60E-03 1.96 up CCAAGTCGAGGGCAGACCAGGC/7 Predicted zma mir 48879

4.40E-02 1.55 up AGGATGCTGACGCAATGGGAT/9 Predicted zma mir 48486

1.30E-03 -3.16 down GTGAAGTGTTTGGGGGAACTC/4 zma-miR395b Table 7c: Results of qRT-PCR Validation Analysis on Differential Small RNAs -

1% Nitrogen vs. 10% Nitrogen.

Table 7d: Results of qRT-PCR Validation Analysis on Differential Small RNAs - 10% Nitrogen vs. Control (100% Nitrogen).

EXAMPLE 5

Gene Cloning and Creation of Binary Vectors for Plant Expression Cloning Strategy - the best validated miR As are cloned into pORE-El binary vectors for the generation of transgenic plants. The full-length open reading frame (ORF) comprising of the hairpin sequence of each selected miRNA, is synthesized by Genscript (Israel). The resulting clone is digested with appropriate restriction enzymes and inserted into the Multi Cloning Site (MCS) of a similarly digested binary vector through ligation using T4 DNA ligase enzyme (Promega, Madison, WI, USA).

EXAMPLE 6

Generation of Transgenic Model Plants Expressing the NUE small RNAs

Arabidoposis thaliana transformation is performed using the floral dip procedure following a slightly modified version of the published protocol (ref). Briefly, TO Plants are planted in small pots filled with soil. The pots are covered with aluminum foil and a plastic dome, kept at 4°C for 3-4 days, then uncovered and incubated in a growth chamber at 24°C under 16 hr light:8 hr dark cycles. A week prior to transformation all individual flowering stems are removed to allow for growth of multiple flowering stems instead. A single colony of Agrobacterium (GV3101) carrying the binary vectors (pORE-El), harboring the NUE miRNA hairpin sequences with additional flanking sequences both upstream and downstream of it, is cultured in LB medium supplemented with kanamycin (50 mg/L) and gentamycin (25 mg/L). Three days prior to transformation, each culture is incubated at 28°C for 48 hrs, shaking at 180 rpm. The starter culture is split the day before transformation into two cultures, which are allowed to grow further at 28°C for 24 hours at 180 rpm. Pellets containing the agrobacterium cells are obtained by centrifugation of the cultures at 5000 rpm for 15 minutes. The pellets are resuspended in an infiltration medium (10 mM MgCl₂, 5% sucrose, 0.044 μΜ BAP (Sigma) and 0.03% Tween 20) in double-distilled water.

Transformation of TO plants is performed by inverting each plant into the Agrobacterium suspension, keeping the flowering stem submerged for 5 minutes. Following inoculation, each plant is blotted dry for 5 minutes on both sides, and placed sideways on a fresh covered tray for 24 hours at 22°C. Transformed (transgenic) plants are then uncovered and transferred to a greenhouse for recovery and maturation. The transgenic TO plants are grown in the greenhouse for 3-5 weeks until the seeds are ready, which are then harvested from plants and kept at room temperature until sowing.

EXAMPLE 7

Selection of Transgenic Arabidopsis Plants Expressing the NUE Genes According to

Expression Level

Arabidopsis seeds are sown and sprayed with Basta (Bayer) on 1-2 weeks old seedlings, at least twice every few days. Only resistant plants, which are heterozygous for the transgene, survive. PCR on the genomic gene sequence is performed on the surviving seedlings using primers pORE-F2 (fwd, 5 '-TTTAGCGATGAACTTCACTC- 3', SEQ ID NO: 20) and a custom designed reverse primer based on each miR's sequence.

EXAMPLE 8

Evaluating Changes in Root Architecture in Transgenic Plants Many key traits in modern agriculture can be explained by changes in the root architecture of the plant. Root size and depth have been shown to logically correlate with drought tolerance, since deeper root systems can access water stored in deeper soil layers. Correspondingly, a highly branched root system provides better coverage of the soil and therefore can effectively absorb all micro and macronutrients available, resulting in enhanced NUE.

To test whether the transgenic plants produce a modified root structure, plants can be grown in agar plates placed vertically. A digital picture of the plates is taken every few days and the maximal length and total area covered by the plant roots are assessed. From every construct created, several independent transformation events are checked in replicates. To assess significant differences between root features, a statistical test, such as a Student's t-test, is employed in order to identify enhanced root features and to provide a statistical value to the findings.

EXAMPLE 9

Testing for increased Nitrogen Use Efficiency (NUE) To analyze whether the transgenic Arabidopsis plants are more responsive to nitrogen, plants are grown in two different nitrogen concentrations: (1) optimal nitrogen concentration (100% NH₄NO₃, which corresponds to 20.61 mM) or (2) nitrogen deficient conditions (1% or 10% NH₄NO₃, which corresponds to 0.2 and 2.06 mM, respectively). Plants are allowed to grow until seed production followed by an analysis of their overall size, time to flowering, yield, protein content of shoot and/or grain, and seed production. The parameters checked can be the overall size of the plant, wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Other parameters that may be tested are: the chlorophyll content of leaves (as nitrogen plant status and the degree of leaf greenness are highly correlated), amino acid and the total protein content of the seeds or other plant parts such as leaves or shoots and oil content. Transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher measured parameters levels than wild-type plants, are identified as nitrogen use efficient plants. EXAMPLE 10

Method for Generating Transgenic Maize Plants with Enhanced or Reduced microRNA Regulation of Target Genes

Target prediction enables two contrasting strategies; an enhancement (positive) or a reduction (negative) of microRNA regulation. Both these strategies have been used in plants and have resulted in significant phenotype alterations. For complete in-vivo assessment of the phenotypic effects of the differential miRNAs in this invention, the inventors implement both over-expression and down-regulation methods on all miRNAs found to associate with NUE as listed in Table 1. Reduction of miRNA regulation of target genes can be accomplished in one of two approaches:

Expressing a microRNA-Resistant Target

In this method, silent mutations are introduced in the microRNA binding site of the target gene so that the DNA and resulting RNA sequences are changed to prevent microRNA binding, but the amino acid sequence of the protein is unchanged.

Expressing a Target-mimic Sequence

Plant microRNAs usually lead to cleavage of their targeted gene, with this cleavage typically occurring between bases 10 and 11 of the microRNA. This position is therefore especially sensitive to mismatches between the microRNA and the target. It was found that expressing a DNA sequence that could potentially be targeted by a microRNA, but contains three extra nucleotides (ATC) between the two nucleotides that are predicted to hybridize with bases 10-11 of the microRNA (thus creating a bulge in that position), can inhibit the regulation of that microRNA on its native targets (Franco- Zorilla JM et al, Nat Genet 2007; 39(8): 1033-1037).

This type of sequence is referred to as a "target-mimic". Inhibition of the microRNA regulation is presumed to occur through physically capturing the microRNA by the target-mimic sequence and titering-out the microRNA, thereby reducing its abundance. This method was used to reduce the amount and, consequentially, the regulation of microRNA 399 in Arabidop sis. Table 8 - miRNA-Resistant Target Examples for Selected down-regulated miRNAs of the Invention.

1037

1017 - 1037 1123

1017 - 1037 1124

target:

TC422488

of Mir

Predicted

zma mir AGGATG

48486 is CTGACG Predicted located in CAATGG zma mir UTR GAT/9 48486

Table 9 - miRNA-Resistant Target Examples for Selected up-regulated miRNAs of

5 the Invention.

NCBI

Mir Mutated ORF Original

Bindi Nucleotide Nucleotide Nucleotide Protein Homolog

ng SEQ ID SEQ ID SEQ ID SEQ NCBI WMD3 MiR

Site NO: NO: NO: ID NO: Organism Accession Targets sequence MiR name

GTGAA

GRMZM GTGTTT

ACN3402 2G05127 GGGGG zma-

1127 1126 1125 Zea mays 3 0 T01 AACTC/4 miR395b

527 - 547 1128

527 - 547 1129

527 - 547 1130

527 - 547 1131

527 - 547 1132

527 - 547 1133

527 - 547 1134

AGAAG AGAGA

ACN3057 TC44193 GAGTAC zma-

1137 1136 1135 Zea mays 0 3 AGCCT/1 miR529

889 - 909 1138

889 - 909 1139 889 - 909 1140

889 - 909 1141

889 - 909 1142

889 - 909 1143

889 - 909 1144

889 - 909 1145

889 - 909 1146

889 - 909 1147

ACF8678 TC37411

1150 1149 1148 Zea mays 2 8

923 - 943 1151

923 - 943 1152

923 - 943 1153

923 - 943 1154

923 - 943 1155

923 - 943 1156

923 - 943 1157

923 - 943 1158

923 - 943 1159

923 - 943 1160

GRMZM

NP 00114 2G41480

1163 1162 1161 Zea mays 9534 5 T04

1396 - 1416 1164

1396 - 1416 1165

1396 - 1416 1166

1396 - 1416 1167

1396 - 1416 1168 1396 - 1416 1169

1396 - 1416 1170

1396 - 1416 1171

1396 - 1416 1172

1396 - 1416 1173

GRMZM

NP 00113 2G12601

1176 1175 1174 Zea mays 6945 8 T01

926- 946 1177

926- 946 1178

926- 946 1179

926- 946 1180

926- 946 1181

926- 946 1182

926- 946 1183

926- 946 1184

926- 946 1185

926- 946 1186

GRMZM

CAB5663 2G16091

1189 1188 1187 Zea mays 1 7 T01

589 - 609 1190

589 - 609 1191

589 - 609 1192

589 - 609 1193

589 - 609 1194

589 - 609 1195

589 - 609 1196

589 - 609 1197

589 - 1198 609

589 - 609 1199

target:

GRMZM

2G10151

1_T01 of

Mir zma- miR529 is located in

UTR

target:

TC37495

8 of Mir

zma- TAGCCA

miR1691 GGGAT

is located GATTTG zma- in UTR CCTG/2 miR1691 target:

TC39180

7 of Mir

zma- miR1691

is located

in UTR

Table 10 - Target Mimic Examples for Selected up-regulated miRNAs of the

Invention

Bulge

Bulge in Target Reverse

Binding Complement MiR

Sequence SEQ miR/SEQ ID sequence S MiR

Full Target Mimic Nucleotide Seq/SEQ ID NO: ID NO: NO: EQ ID NO: name

GAGTTCCC GTGAAGT

GAGTTCCTCC CCACTAAA GTTTGGG zma-

ACTAAGCAC CACTTCAC/ GGAACTC miR39

1208 TTCAT/1204 1200 /4 5b

ATGCAGCA GGAATCT

CTGCAGCAT TCACTATCA TGATGAT zma-

CACTATCAG AGATTCC/12 GCTGCAT/ miR17

11 GATTCT/1205 01 3 2e

AGGCTGTA AGAAGA

CGAGTGTGC CTCCTATCT GAGAGA zma-

TCCTATCTCT CTCTTCT/12 GTACAGC miR52

12 CTTCT/1206 02 CT/1 9

CAGGCAAA TAGCCAG

GTGGCAACT TCACTATCC GGATGAT zma-

CACTATCCTT CTGGCTA/12 TTGCCTG/ miR16

13 GGCTC/1207 03 2 91

Table 11 - Target Mimic Examples for Selected up-regulated miRNAs of the

Invention

Bulge in Bulge

Target Reverse

Binding Complement MiR MiR

Full Target Mimic Nucleotide Seq Sequence iniR sequence name

TGTTAG

CTGATC TTAGATG

TAGGTC TGTTTGCTG ACCATCA zma-

ATATAC/ ATCTAGGT GCAAACA/ miR82

18 16 CATCTAA/14 10 7 Pre die

TTCCCCC AGGATGC ted

TGCGCT ATCCCATTG TGACGCA zma

ATCAGC CGCTATCA ATGGGAT/ mir

TTCCT/17 GCATCCT/15 9 48486

Table 12 - Abbreviations of plant species

Common Name Organism Name Abbreviation

Peanut Arachis hypogaea ahy

Arabidopsis lyrata Arabidopsis lyrata aly

Rocky Mountain Columbine Aquilegia coerulea aqc

Tausch's goatgrass Aegilops taushii ata

Arabidopsis thaliana Arabidopsis thaliana ath

Grass Brachypodium distachyon bdi

Brassica napus canola ("liftit") Brassica napus bna

Brassica oleracea wild cabbage Brassica oleracea bol

Brassica rapa yellow mustard Brassica rapa bra

Clementine Citrus Clementine ecl

Orange Citrus sinensis csi

Trifoliate orange Citrus trifoliata ctr

Glycine max Glycine max gma

Wild soybean Glycine soja gso

Barley Hordeum vulgare hvu

Lotus japonicus Lotus japonicus lia

Medicago trancatula - Barrel Clover ("tiltan") Medicago truncatula mtr

Oryza sativa Oryza sativa os a

European spruce Picea abies pab

Physcomitrella patens (moss) Physcomitrella patens ppt

Pinus taeda - Loblolly Pine Pinus taeda pta

Populus trichocarpa - black cotton wood Populus trichocarpa ptc

Castor bean ("kikayon") Ricinus communis rco

Sorghum bicolor Dura Sorghum bicolor sbi tomato microtom Solanum lycopersicum sly

Selaginella moellendorffii Selaginella moellendorffii smo

Sugarcane Saccharum officinarum sof

Sugarcane Saccharum spp ssp

Triticum aestivum Triticum aestivum tae cacao tree and cocoa tree Theobroma cacao tec

Vitis vinifera Grapes Vitis vinifera wi corn Zea mays zma Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 10, 6-9, 21, 22, 23-37, 38-52, 1209, 1211, 1212, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

2. A transgenic plant exogenously expressing a polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 10, 6-9, 23-37, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant.

3. The method of claim 1 or transgenic plant of claim 2, wherein said exogenous polynucleotide encodes a precursor of said nucleic acid sequence.

4. The method or transgenic plant of claim 3, wherein said precursor of said nucleic acid sequence is at least 60 % identical to SEQ ID NO: 21, 22, 38-52, 1209, 1211, 1212.

5. The method of claim 1 or the transgenic plant of claim 2, wherein said exogenous polynucleotide encodes a miRNA or a precursor thereof.

6. The method of claim 1 or the transgenic plant of claim 2, wherein said exogenous polynucleotide encodes a siRNA or a precursor thereof.

7. The method of claim 1 or the transgenic plant of claim 2, wherein said exogenous polynucleotide is selected from the group consisting of SEQ ID NO: 10, 6-9, 21, 22, 23-37, 38-52, 1209, 1211, 1212.

8. An isolated polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NO: 6, 7 and 9, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of a plant.

9. The isolated polynucleotide of claim 8, wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NO: 6, 7 and 9.

10. The isolated polynucleotide of claim 8, wherein said polynucleotide encodes a precursor of said nucleic acid sequence.

11. The isolated polynucleotide of claim 8, wherein said polynucleotide encodes a miRNA or a precursor thereof.

12. The isolated polynucleotide of claim 8, wherein said polynucleotide encodes a siRNA or a precursor thereof.

13. A nucleic acid construct comprising the isolated polynucleotide of claim 8-12 under the regulation of a cis-acting regulatory element.

14. The nucleic acid construct of claim 13, wherein said cis-acting regulatory element comprises a promoter.

15. The nucleic acid construct of claim 14, wherein said promoter comprises a tissue-specific promoter.

16. The nucleic acid construct of claim 15, wherein said tissue-specific promoter comprises a root specific promoter.

17. A method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising 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 SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant.

18. A transgenic plant exogenous ly expressing a polynucleotide which downregulates an activity or expression of a gene encoding an RNAi molecule having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209.

19. An isolated polynucleotide which downregulates an activity or expression of a gene encoding an RNAi molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53- 56, 1209.

20. The method of claim 17, the transgenic plant of claim 18 or the isolated polynucleotide of claim 19, wherein said polynucleotide encodes a miRNA-Resistant Target as set forth in SEQ ID NO 1104-1124.

21. The method of claim 17, the transgenic plant of claim 18 or the isolated polynucleotide of claim 19, wherein said isolated polynucleotide encodes a target mimic as set forth in SEQ ID NO: 18 or 19.

22. A nucleic acid construct comprising the isolated polynucleotide of claim 19 under the regulation of a cis-acting regulatory element.

23. The nucleic acid construct of claim 22, wherein said cis-acting regulatory element comprises a promoter.

24. The nucleic acid construct of claim 23, wherein said promoter comprises a tissue-specific promoter.

25. The nucleic acid construct of claim 24, wherein said tissue-specific promoter comprises a root specific promoter.

26. The method of claim 1 or 17, further comprising growing the plant under limiting nitrogen conditions.

27. The method of claim 1 or 17, further comprising growing the plant under abiotic stress.

28. The method of claim 27, wherein said abiotic stress is selected from the group consisting of salinity, drought, water deprivation, flood, etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution and UV irradiation.

29. The method of claim 1 or 17, or the plant of claim 2 or 18, being a monocotyledon.

30. The method of claim 1 or 17, or the plant of claim 2 or 18, being a dicotyledon.

31. A method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

32. A transgenic plant exogenous ly expressing a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.

33. A nucleic acid construct comprising a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, and wherein said polynucleotide is under a transcriptional control of a cis- acting regulatory element.

34. The method of claim 31, the transgenic plant of claim 32 or the nucleic acid construct of claim 33, wherein said polynucleotide is selected from the group consisting of SEQ ID NO: 1022-1090.

35. The method of claim 31, the transgenic plant of claim 32 or the nucleic acid construct of claim 33, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 927-1021.

36. The nucleic acid construct of claim 33, wherein said cis-acting regulatory element comprises a promoter.

37. The nucleic acid construct of claim 36, wherein said promoter comprises a tissue-specific promoter.

38. The nucleic acid construct of claim 37, wherein said tissue-specific promoter comprises a root specific promoter.

39. The method of claim 31, further comprising growing the plant under limiting nitrogen conditions.

40. The method of claim 31, further comprising growing the plant under abiotic stress.

41. The method of claim 40, wherein said abiotic stress is selected from the group consisting of salinity, drought, water deprivation, flood, etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution and UV irradiation.

42. The method of claim 31, or the plant of claim 32, being a monocotyledon.

43. The method of claim 31 , or the plant of claim 32, being a dicotyledon.

44. A method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.

45. A transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.

46. A nucleic acid construct comprising a polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of a plant, said nucleic acid sequence being under the regulation of a cis-acting regulatory element.

47. The method of claim 44, the transgenic plant of claim 45 or the nucleic acid construct of claim 46, wherein said polynucleotide acts by a mechanism selected from the group consisting of sense suppression, antisense suppresion, ribozyme inhibition, gene disruption.

48. The nucleic acid construct of claim 46, wherein said cis-acting regulatory element comprises a promoter.

49. The nucleic acid construct of claim 48, wherein said promoter comprises a tissue-specific promoter.

50. The nucleic acid construct of claim 49, wherein said tissue-specific promoter comprises a root specific promoter.