US20150082487A1

US20150082487A1 - Polynucleotides, polypeptides encoded thereby, and methods of using same for increasing abiotic stress tolerance, biomass and/or yield in plants expressing same

Info

Publication number: US20150082487A1
Application number: US14/548,346
Authority: US
Inventors: Sharon Ayal; Eyal Emmanuel; Zur Granevitze; Alex Diber; Basia Judith Vinocur; Hagai Karchi; Yoav Herschkovitz
Original assignee: Evogene Ltd
Current assignee: Evogene Ltd
Priority date: 2008-12-29
Filing date: 2014-11-20
Publication date: 2015-03-19
Also published as: ZA201104002B; US20110265222A1; US20180312860A1; EP2373792A4; US8952218B2; AU2016203516B2; AU2009334312B2; AU2017251769B2; AU2009334312A1; AU2019253838A1; EP2373792A2; MX350550B; MX2011006998A; CA2744827C; CA3052515A1; CA2744827A1; US10975382B2; EP2373792B1; WO2010076756A3; AU2016203516A1

Abstract

Provided are isolated polynucleotides comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, or 671. Also provided are nucleic acid constructs comprising same, isolated polypeptides encoded thereby, transgenic cells and transgenic plants comprising same and methods of using same for increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.

Description

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No. 13/139,729 filed on Jun. 15, 2011, which is a National Phase of PCT Patent Application No. PCT/IB2009/055962 having International filing date of Dec. 28, 2009, which claims the benefit of priority of U.S. Provisional Patent Application Nos. 61/213,577 filed on Jun. 22, 2009; and 61/193,830 filed on Dec. 29, 2008. The contents of the above applications are all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to isolated polypeptides and polynucleotides, nucleic acid constructs comprising same, transgenic cells comprising same, transgenic plants exogenously expressing same and more particularly, but not exclusively, to methods of using same for increasing abiotic stress tolerance, growth rate, biomass, vigor, yield (e.g., seed yield, oil yield), oil content, fiber yield, fiber quality and/or fertilizer use efficiency (e.g., nitrogen use efficiency) of a plant.
Abiotic stress (ABS; also referred to as “environmental stress”) conditions such as salinity, drought, flood, suboptimal temperature and toxic chemical pollution, cause substantial damage to agricultural plants. Most plants have evolved strategies to protect themselves against these conditions. However, if the severity and duration of the stress conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Furthermore, most of the crop plants are highly susceptible to abiotic stress and thus necessitate optimal growth conditions for commercial crop yields. Continuous exposure to stress causes major alterations in the plant metabolism which ultimately leads to cell death and consequently yield losses.
The global shortage of water supply is one of the most severe agricultural problems affecting plant growth and crop yield and efforts are made to mitigate the harmful effects of desertification and salinization of the world's arable land. Water deficit is a common component of many plant stresses and occurs in plant cells when the whole plant transpiration rate exceeds the water uptake. In addition to drought, other stresses, such as salinity and low temperature, produce cellular dehydration.
Drought is a gradual phenomenon, which involves periods of abnormally dry weather that persists long enough to produce serious hydrologic imbalances such as crop damage and water supply shortage. In severe cases, drought can last many years and results in devastating effects on agriculture and water supplies. Furthermore, drought is associated with increase susceptibility to various diseases.
For most crop plants, the land regions of the world are too arid. In addition, overuse of available water results in increased loss of agriculturally-usable land (desertification), and increase of salt accumulation in soils adds to the loss of available water in soils.
Salinity, high salt levels, affects one in five hectares of irrigated land. This condition is only expected to worsen, further reducing the availability of arable land and crop production, since none of the top five food crops, i.e., wheat, corn, rice, potatoes, and soybean, can tolerate excessive salt. Detrimental effects of salt on plants result from both water deficit which leads to osmotic stress (similar to drought stress) and the effect of excess sodium ions on critical biochemical processes. As with freezing and drought, high salt causes water deficit; and the presence of high salt makes it difficult for plant roots to extract water from their environment. Soil salinity is thus one of the more important variables that determine whether a plant may thrive. In many parts of the world, sizable land areas are uncultivable due to naturally high soil salinity. Thus, salination of soils that are used for agricultural production is a significant and increasing problem in regions that rely heavily on agriculture, and is worsen by over-utilization, over-fertilization and water shortage, typically caused by climatic change and the demands of increasing population. Salt tolerance is of particular importance early in a plant's lifecycle, since evaporation from the soil surface causes upward water movement, and salt accumulates in the upper soil layer where the seeds are placed. On the other hand, germination normally takes place at a salt concentration which is higher than the mean salt level in the whole soil profile.
Germination of many crops is sensitive to temperature. A gene that would enhance germination in hot conditions would be useful for crops that are planted late in the season or in hot climates. In addition, seedlings and mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs, including leaves and particularly fruit, when transpiration is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and cyto skeleton, and impairs membrane function. Heat shock may produce a decrease in overall protein synthesis, accompanied by expression of heat shock proteins, e.g., chaperones, which are involved in refolding proteins denatured by heat.
Heat stress often accompanies conditions of low water availability. Heat itself is seen as an interacting stress and adds to the detrimental effects caused by water deficit conditions. Water evaporation increases along with the rise in daytime temperatures and can result in high transpiration rates and low plant water potentials. High-temperature damage to pollen almost always occurs in conjunction with drought stress, and rarely occurs under well-watered conditions. Combined stress can alter plant metabolism in various ways; therefore understanding the interaction between different stresses may be important for the development of strategies to enhance stress tolerance by genetic manipulation.
Excessive chilling conditions, e.g., low, but above freezing, temperatures affect crops of tropical origins, such as soybean, rice, maize, and cotton. Typical chilling damage includes wilting, necrosis, chlorosis or leakage of ions from cell membranes. The underlying mechanisms of chilling sensitivity are not completely understood yet, but probably involve the level of membrane saturation and other physiological deficiencies. For example, photoinhibition of photosynthesis (disruption of photosynthesis due to high light intensities) often occurs under clear atmospheric conditions subsequent to cold late summer/autumn nights. In addition, chilling may lead to yield losses and lower product quality through the delayed ripening of maize.
Salt and drought stress signal transduction consist of ionic and osmotic homeostasis signaling pathways. The ionic aspect of salt stress is signaled via the SOS pathway where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. The osmotic component of salt stress involves complex plant reactions that overlap with drought and/or cold stress responses.
Common aspects of drought, cold and salt stress response [Reviewed in Xiong and Zhu (2002) Plant Cell Environ. 25: 131-139] include: (a) transient changes in the cytoplasmic calcium levels early in the signaling event; (b) signal transduction via mitogen-activated and/or calcium dependent protein kinases (CDPKs) and protein phosphatases; (c) increases in abscisic acid levels in response to stress triggering a subset of responses; (d) inositol phosphates as signal molecules (at least for a subset of the stress responsive transcriptional changes; (e) activation of phospholipases which in turn generates a diverse array of second messenger molecules, some of which might regulate the activity of stress responsive kinases; (f) induction of late embryogenesis abundant (LEA) type genes including the CRT/DRE responsive COR/RD genes; (g) increased levels of antioxidants and compatible osmolytes such as proline and soluble sugars; and (h) accumulation of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals. Abscisic acid biosynthesis is regulated by osmotic stress at multiple steps. Both ABA-dependent and -independent osmotic stress signaling first modify constitutively expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream stress tolerance effector genes.
Several genes which increase tolerance to cold or salt stress can also improve drought stress protection, these include for example, the transcription factor AtCBF/DREB1, OsCDPK7 (Saijo et al. 2000, Plant J. 23: 319-327) or AVP1 (a vacuolar pyrophosphatase-proton pump, Gaxiola et al. 2001, Proc. Natl. Acad. Sci. USA 98: 11444-11449).
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies used to develop new lines of plants that exhibit tolerance to ABS are relatively inefficient since they are tedious, time consuming and of unpredictable outcome. Furthermore, limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to ABS tolerance are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways.
Genetic engineering efforts, aimed at conferring abiotic stress tolerance to transgenic crops, have been described in various publications [Apse and Blumwald (Curr Opin Biotechnol. 13:146-150, 2002), Quesada et al. (Plant Physiol. 130:951-963, 2002), Holmström et al. (Nature 379: 683-684, 1996), Xu et al. (Plant Physiol 110: 249-257, 1996), Pilon-Smits and Ebskamp (Plant Physiol 107: 125-130, 1995) and Tarczynski et al. (Science 259: 508-510, 1993)].
Various patents and patent applications disclose genes and proteins which can be used for increasing tolerance of plants to abiotic stresses. These include for example, U.S. Pat. Nos. 5,296,462 and 5,356,816 (for increasing tolerance to cold stress); U.S. Pat. No. 6,670,528 (for increasing ABST); U.S. Pat. No. 6,720,477 (for increasing ABST); U.S. application Ser. Nos. 09/938,842 and 10/342,224 (for increasing ABST); U.S. application Ser. No. 10/231,035 (for increasing ABST); WO2004/104162 (for increasing ABST and biomass); WO2007/020638 (for increasing ABST, biomass, vigor and/or yield); WO2007/049275 (for increasing ABST, biomass, vigor and/or yield).
Suboptimal nutrient (macro and micro nutrient) affect plant growth and development through the whole plant life cycle. One of the essential macronutrients for the plant is Nitrogen. Nitrogen is responsible for biosynthesis of amino acids and nucleic acids, prosthetic groups, plant hormones, plant chemical defenses, and the like. Nitrogen is often the rate-limiting element in plant growth and all field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Additional important macronutrients are Phosphorous (P) and Potassium (K), which have a direct correlation to yield and general plant tolerance.
Vegetable or seed oils are the major source of energy and nutrition in human and animal diet. They are also used for the production of industrial products, such as paints, inks and lubricants. In addition, plant oils represent renewable sources of long-chain hydrocarbons which can be used as fuel. Since the currently used fossil fuels are finite resources and are gradually being depleted, fast growing biomass crops may be used as alternative fuels or for energy feedstocks and may reduce the dependence on fossil energy supplies. However, the major bottleneck for increasing consumption of plant oils as bio-fuel is the oil price, which is still higher than fossil fuel. In addition, the production rate of plant oil is limited by the availability of agricultural land and water. Thus, increasing plant oil yields from the same growing area can effectively overcome the shortage in production space and can decrease vegetable oil prices at the same time.
Studies aiming at increasing plant oil yields focus on the identification of genes involved in oil metabolism as well as in genes capable of increasing plant and seed yields in transgenic plants. Genes known to be involved in increasing plant oil yields include those participating in fatty acid synthesis or sequestering such as desaturase [e.g., DELTA6, DELTA12 or acyl-ACP (Ssi2; Arabidopsis Information Resource (TAIR; Hypertext Transfer Protocol://World Wide Web (dot) arabidopsis (dot) org/), TAIR No. AT2G43710)], OleosinA (TAIR No. AT3G01570) or FAD3 (TAIR No. AT2G29980), and various transcription factors and activators such as Lec1 [TAIR No. AT1G21970, Lotan et al. 1998. Cell. 26; 93(7):1195-205], Lec2 [TAIR No. AT1G28300, Santos Mendoza et al. 2005, FEBS Lett. 579(20:4666-70], Fus3 (TAIR No. AT3G26790), ABI3 [TAIR No. AT3G24650, Lara et al. 2003. J Biol. Chem. 278(23): 21003-11] and Wri1 [TAIR No. AT3G54320, Cernac and Benning, 2004. Plant J. 40(4): 575-85].
Genetic engineering efforts aiming at increasing oil content in plants (e.g., in seeds) include upregulating endoplasmic reticulum (FAD3) and plastidal (FAD7) fatty acid desaturases in potato (Zabrouskov V., et al., 2002; Physiol Plant. 116:172-185); over-expressing the GmDof4 and GmDof11 transcription factors (Wang H W et al., 2007; Plant J. 52:716-29); over-expressing a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter (Vigeolas H, et al. 2007, Plant Biotechnol J. 5:431-41; U.S. Pat. Appl. No. 20060168684); using Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed (Katavic V, et al., 2000, Biochem Soc Trans. 28:935-7).
Various patent applications disclose genes and proteins which can increase oil content in plants. These include for example, U.S. Pat. Appl. No. 20080076179 (lipid metabolism protein); U.S. Pat. Appl. No. 20060206961 (the Ypr140w polypeptide); U.S. Pat. Appl. No. 20060174373 [triacylglycerols synthesis enhancing protein (TEP)]; U.S. Pat. Appl. Nos. 20070169219, 20070006345, 20070006346 and 20060195943 (disclose transgenic plants with improved nitrogen use efficiency which can be used for the conversion into fuel or chemical feedstocks); WO2008/122980 (polynucleotides for increasing oil content, growth rate, biomass, yield and/or vigor of a plant).
Cotton and cotton by-products provide raw materials that are used to produce a wealth of consumer-based products in addition to textiles including cotton foodstuffs, livestock feed, fertilizer and paper. The production, marketing, consumption and trade of cotton-based products generate an excess of $100 billion annually in the U.S. alone, making cotton the number one value-added crop.
Even though 90% of cotton's value as a crop resides in the fiber (lint), yield and fiber quality has declined due to general erosion in genetic diversity of cotton varieties, and an increased vulnerability of the crop to environmental conditions.
There are many varieties of cotton plant, from which cotton fibers with a range of characteristics can be obtained and used for various applications. Cotton fibers may be characterized according to a variety of properties, some of which are considered highly desirable within the textile industry for the production of increasingly high quality products and optimal exploitation of modem spinning technologies. Commercially desirable properties include length, length uniformity, fineness, maturity ratio, decreased fuzz fiber production, micronaire, bundle strength, and single fiber strength. Much effort has been put into the improvement of the characteristics of cotton fibers mainly focusing on fiber length and fiber fineness. In particular, there is a great demand for cotton fibers of specific lengths.
A cotton fiber is composed of a single cell that has differentiated from an epidermal cell of the seed coat, developing through four stages, i.e., initiation, elongation, secondary cell wall thickening and maturation stages. More specifically, the elongation of a cotton fiber commences in the epidermal cell of the ovule immediately following flowering, after which the cotton fiber rapidly elongates for approximately 21 days. Fiber elongation is then terminated, and a secondary cell wall is formed and grown through maturation to become a mature cotton fiber.
Several candidate genes which are associated with the elongation, formation, quality and yield of cotton fibers were disclosed in various patent applications such as U.S. Pat. No. 5,880,100 and U.S. patent application Ser. Nos. 08/580,545, 08/867,484 and Ser. No. 09/262,653 (describing genes involved in cotton fiber elongation stage); WO0245485 (improving fiber quality by modulating sucrose synthase); U.S. Pat. No. 6,472,588 and WO0117333 (increasing fiber quality by transformation with a DNA encoding sucrose phosphate synthase); WO9508914 (using a fiber-specific promoter and a coding sequence encoding cotton peroxidase); WO9626639 (using an ovary specific promoter sequence to express plant growth modifying hormones in cotton ovule tissue, for altering fiber quality characteristics such as fiber dimension and strength); U.S. Pat. No. 5,981,834, U.S. Pat. No. 5,597,718, U.S. Pat. No. 5,620,882, U.S. Pat. No. 5,521,708 and U.S. Pat. No. 5,495,070 (coding sequences to alter the fiber characteristics of transgenic fiber producing plants); U.S. patent applications U.S. 2002049999 and U.S. 2003074697 (expressing a gene coding for endoxyloglucan transferase, catalase or peroxidase for improving cotton fiber characteristics); WO 01/40250 (improving cotton fiber quality by modulating transcription factor gene expression); WO 96/40924 (a cotton fiber transcriptional initiation regulatory region associated which is expressed in cotton fiber); EP0834566 (a gene which controls the fiber formation mechanism in cotton plant); WO2005/121364 (improving cotton fiber quality by modulating gene expression); WO2008/075364 (improving fiber quality, yield/biomass/vigor and/or abiotic stress tolerance of plants).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, or 671, thereby increasing the abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant, comprising expressing within the plant an exogenous polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs:619, 617, 606, 615, 629, 1-49, 51-59, 113-343, 345-351, 353-358, 605, 607-614, 616, 618, 620-628, 630-638, 641, 642, 644, 644-646, 648-651, 670, and 671, thereby increasing the abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 352, 639, 640, or 643, thereby increasing the abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing nitrogen use efficiency, seed yield and/or oil content of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 50, 645, or 647, thereby increasing the nitrogen use efficiency, seed yield and/or oil content of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing seed yield, fiber yield and/or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:344, thereby increasing the seed yield, fiber yield and/or fiber quality of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80% identical to SEQ ID NO:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, or 672, thereby increasing the abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672, thereby increasing the abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80% identical to SEQ ID NO:99 or 598, thereby increasing the abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing nitrogen use efficiency, seed yield and/or oil content of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80% identical to SEQ ID NO:599 or 663, thereby increasing the nitrogen use efficiency, seed yield and/or oil content of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing nitrogen use efficiency, abiotic stress tolerance, seed yield and/or oil content of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80% identical to SEQ ID NO:110 or 665, thereby increasing the nitrogen use efficiency, abiotic stress tolerance, seed yield and/or oil content of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing seed yield, fiber yield and/or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80% identical to SEQ ID NO:590, thereby increasing the seed yield, fiber yield and/or fiber quality of the plant.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650-651, 670, or 671, wherein said nucleic acid sequence is capable of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs:619, 617, 606, 615, 629, 1-49, 51-59, 113-343, 345-351, 353-358, 605, 607-614, 616, 618, 620-628, 630-638, 641, 642, 644, 644-646, 648-651, 670, and 671.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide which comprises an amino acid sequence at least 80% homologous to the amino acid sequence set forth in SEQ ID NO: 75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, or 672, wherein said amino acid sequence is capable of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide which comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising the isolated polynucleotide of claim 12, 13, 14 or 15, and a promoter for directing transcription of said nucleic acid sequence in a host cell.
According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 80% homologous to SEQ ID NO:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, or 672, wherein said amino acid sequence is capable of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.
According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672
According to an aspect of some embodiments of the present invention there is provided a plant cell exogenously expressing the polynucleotide of claim 12, 13, 14 or 15, or the nucleic acid construct of claim 16.
According to an aspect of some embodiments of the present invention there is provided a plant cell exogenously expressing the polypeptide of claim 17 or 18.
According to some embodiments of the invention, the nucleic acid sequence is as set forth in SEQ ID NO:619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, or 671.
According to some embodiments of the invention, the polynucleotide consists of the nucleic acid sequence selected from the group consisting of SEQ ID NOs:619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, and 671.
According to some embodiments of the invention, the nucleic acid sequence encodes an amino acid sequence at least 80% homologous to SEQ ID NO:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, or 672.
According to some embodiments of the invention, the nucleic acid sequence encodes the amino acid sequence selected from the group consisting of SEQ ID NOs:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, and 672.
According to some embodiments of the invention, the plant cell forms part of a plant.
According to some embodiments of the invention, the method further comprising growing the plant expressing said exogenous polynucleotide under the abiotic stress.
According to some embodiments of the invention, the 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 yield comprises seed yield or oil yield.
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 schematic illustration of the pGI binary plasmid used for expressing the isolated polynucleotide sequences of some embodiments of the invention. RB—T-DNA right border; LB—T-DNA left border; H—HindIII restriction enzyme; X—XbaI restriction enzyme; B—BamHI restriction enzyme; S—SaiI restriction enzyme; Sm—SmaI restriction enzyme; R-I—EcoRI restriction enzyme; Sc—SacI/SstI/Ecl136II; (numbers)—Length in base-pairs; NOS pro=nopaline synthase promoter; NPT-II=neomycin phosphotransferase gene; NOS ter=nopaline synthase terminator; Poly-A signal (polyadenylation signal); GUSintron—the GUS reporter gene (coding sequence and intron) The isolated polynucleotide sequences of the invention were cloned into the vector while replacing the GUSintron reporter gene

FIG. 2 is a schematic illustration of the modified pGI binary plasmid used for expressing the isolated polynucleotide sequences of the invention. RB—T-DNA right border; LB—T-DNA left border; MCS—Multiple cloning site; RE—any restriction enzyme; (numbers)—Length in base-pairs; NOS pro=nopaline synthase promoter; NPT-II=neomycin phosphotransferase gene; NOS ter=nopaline synthase terminator; Poly-A signal (polyadenylation signal); GUSintron—the GUS reporter gene (coding sequence and intron) The isolated polynucleotide sequences of the invention were cloned into the vector while replacing the GUSintron reporter gene.

FIGS. 3A-F are images depicting visualization of root development of transgenic plants exogenously expressing the polynucleotide of some embodiments of the invention when grown in transparent agar plates under normal (FIGS. 3A-B), osmotic stress (15% PEG; FIGS. 3C-D) or nitrogen-limiting (FIGS. 3E-F) conditions. The different transgenes were grown in transparent agar plates for 17 days (7 days nursery and 10 days after transplanting). The plates were photographed every 3-4 days starting at day 1 after transplanting. FIG. 3A—An image of a photograph of plants taken following 10 after transplanting days on agar plates when grown under normal (standard) conditions. FIG. 3B—An image of root analysis of the plants shown in FIG. 3A in which the lengths of the roots measured are represented by arrows. FIG. 3C—An image of a photograph of plants taken following 10 days after transplanting on agar plates, grown under high osmotic (PEG 15%) conditions. FIG. 3D—An image of root analysis of the plants shown in FIG. 3C in which the lengths of the roots measured are represented by arrows. FIG. 3E—An image of a photograph of plants taken following 10 days after transplanting on agar plates, grown under low nitrogen conditions. FIG. 3F—An image of root analysis of the plants shown in FIG. 3E in which the lengths of the roots measured are represented by arrows.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention relates to polypeptides, polynucleotides, nucleic acid constructs and methods of increasing abiotic stress tolerance, fertilizer use efficiency (e.g., nitrogen use efficiency), growth, biomass, fiber development or quality, vigor and/or yield of a plant.
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 present inventors have identified novel polypeptides and polynucleotides which can be used to increase abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.
Thus, as shown in the Examples section which follows, the present inventors have utilized bioinformatics tools to identify polynucleotides which increase abiotic stress tolerance (ABST), fertilizer use efficiency [e.g., nitrogen use efficiency (NUE)], yield (e.g., seed yield, oil yield, oil content), growth rate, biomass, vigor and/or of a plant. Genes which affect the trait-of-interest were identified using digital expression profiles in specific tissues and conditions such as expression in roots; expression under stress conditions such as drought stress, ultraviolet (UV) irradiation, cold stress, heat stress, nutrient deficiency, stress hormones [for example as abscisic acid (ABA) and ethylene] etiolation conditions, salinity stress, waterlogging; and/or expression during plant development (Tables 1-5; Example 1 of the Examples section which follows; polynucleotide SEQ ID NOs:1-59 and 638; polypeptide SEQ ID NOs:60-112). Homologous polypeptides and polynucleotides having the same function were also identified (Table 6, Example 2 of the Examples section which follows; polynucleotide SEQ ID NOs:113-358; polypeptide SEQ ID NOs:359-604). The identified polynucleotides were cloned into binary vectors [Tables 7-10; Example 3; SEQ ID NOs:605-637, 639-651, 670, 671 (polynucleotides); SEQ ID NOs: 60, 63-73, 75, 77, 81-83, 86, 87, 90, 92, 93, 94, 95, 96, 99, 100, 101, 102, 652-669, 672 (polypeptides)], transformed into agrobacterium cells (Example 4), and further into Arabidopsis plants (Example 5). Transgenic plants over-expressing the identified polynucleotides were found to exhibit increased biomass [e.g., fresh and dry weight; leaf area and growth rate, rosette area, rosette diameter and growth rate of rosette area and diameter, plot coverage, leaf number], growth rate, yield (e.g., seed yield and weight), harvest index, roots growth (e.g., root length, root coverage, growth rate of root length and/or coverage), oil yield, oil percentage in seeds, weight of 1000 seeds (Tables 11-62; Examples 6, 7, 8, 9, and 11 of the Examples section which follows) under normal or limiting conditions (e.g., abiotic stress, nitrogen limiting conditions). Altogether, these results suggest the use of the novel polynucleotides and polypeptides of the invention for increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.
Thus, according to an aspect of some embodiments of the invention, there is provided method of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant, the method comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670 or 671, thereby increasing the abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of the plant.
As used herein the phrase “plant yield” refers to the amount (e.g., as determined by weight or size) or quantity (numbers) of tissues or organs produced per plant or per growing season. Hence increased yield could affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time.
It should be noted that a plant yield can be affected by various parameters including, but not limited to, plant biomass; plant vigor; 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 (florets) per panicle (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 (density); number of harvested organs in field; total leaf area; carbon assimilation and carbon partitioning (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 phrase “seed yield” refers to the number or weight of the seeds per plant, seeds per pod, or per growing area or to the weight of a single seed, or to the oil extracted per seed. Hence seed yield can be affected by seed dimensions (e.g., length, width, perimeter, area and/or volume), number of (filled) seeds and seed filling rate and by seed oil content. Hence increase seed yield per plant could affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time; and increase seed yield per growing area could be achieved by increasing seed yield per plant, and/or by increasing number of plants grown on the same given area.
The term “seed” (also referred to as “grain” or “kernel”) as used herein refers to a small embryonic plant enclosed in a covering called the seed coat (usually with some stored food), the product of the ripened ovule of gymnosperm and angiosperm plants which occurs after fertilization and some growth within the mother plant.
The phrase “oil content” as used herein refers to the amount of lipids in a given plant organ, either the seeds (seed oil content) or the vegetative portion of the plant (vegetative oil content) and is typically expressed as percentage of dry weight (10% humidity of seeds) or wet weight (for vegetative portion).
It should be noted that oil content is affected by intrinsic oil production of a tissue (e.g., seed, vegetative portion), as well as the mass or size of the oil-producing tissue per plant or per growth period.
In one embodiment, increase in oil content of the plant can be achieved by increasing the size/mass of a plant's tissue(s) which comprise oil per growth period. Thus, increased oil content of a plant can be achieved by increasing the yield, growth rate, biomass and vigor of the plant.
As used herein the phrase “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, which could also determine or affect the plant yield or the yield per growing area. An increase in plant biomass can be in the whole plant or in parts thereof such as aboveground (harvestable) parts, vegetative biomass, roots and seeds.
As used herein the phrase “growth rate” refers to the increase in plant organ/tissue size per time (can be measured in cm²per day).
As used herein the phrase “plant vigor” refers to the amount (measured by weight) of tissue produced by the plant in a given time. Hence increased vigor could determine or affect the plant yield or the yield per growing time or growing area. In addition, early vigor (seed and/or seedling) results in improved field stand.
It should be noted that a plant yield can be determined under stress (e.g., abiotic stress, nitrogen-limiting conditions) and/or non-stress (normal) conditions.
As used herein, the phrase “non-stress conditions” refers to the growth conditions (e.g., water, temperature, light-dark cycles, humidity, salt concentration, fertilizer concentration in soil, nutrient supply such as nitrogen, phosphorous and/or potassium), that do not significantly go beyond the everyday climatic and other abiotic conditions that plants may encounter, and which allow optimal growth, metabolism, reproduction and/or viability of a plant at any stage in its life cycle (e.g., in a crop plant from seed to a mature plant and back to seed again). Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given plant in a given geographic location. It should be noted that while the non-stress conditions may include some mild variations from the optimal conditions (which vary from one type/species of a plant to another), such variations do not cause the plant to cease growing without the capacity to resume growth.
The phrase “abiotic stress” as used herein refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant. Accordingly, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, water deprivation, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, atmospheric pollution or UV irradiation. The implications of abiotic stress are discussed in the Background section.
The phrase “abiotic stress tolerance” as used herein refers to the ability of a plant to endure an abiotic stress without suffering a substantial alteration in metabolism, growth, productivity and/or viability.
As used herein the phrase “water use efficiency (WUE)” refers to the level of organic matter produced per unit of water consumed by the plant, i.e., the dry weight of a plant in relation to the plant's water use, e.g., the biomass produced per unit transpiration.
As used herein the phrase “fertilizer use efficiency” refers to the metabolic process(es) which lead to an increase in the plant's yield, biomass, vigor, and growth rate per fertilizer unit applied. The metabolic process can be the uptake, spread, absorbent, accumulation, relocation (within the plant) and use of one or more of the minerals and organic moieties absorbed by the plant, such as nitrogen, phosphates and/or potassium.
As used herein the phrase “fertilizer-limiting conditions” refers to growth conditions which include a level (e.g., concentration) of a fertilizer applied which is below the level needed for normal plant metabolism, growth, reproduction and/or viability.
As used herein the phrase “nitrogen use efficiency (NUE)” refers to the metabolic process(es) which lead to an increase in the plant's yield, biomass, vigor, and growth rate per nitrogen unit applied. The metabolic process can be the uptake, spread, absorbent, accumulation, relocation (within the plant) and use of nitrogen absorbed by the plant.
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 normal plant metabolism, growth, reproduction and/or viability.
Improved plant NUE and FUE 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. Thus, the polynucleotides and polypeptides of some embodiments of the invention positively affect plant yield, seed yield, and plant biomass. In addition, the benefit of improved plant NUE will certainly improve crop quality and biochemical constituents of the seed such as protein yield and oil yield.
It should be noted that improved ABST will confer plants with improved vigor also under non-stress conditions, resulting in crops having improved biomass and/or yield e.g., elongated fibers for the cotton industry, higher oil content.
As used herein the term “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 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, increase in abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant as compared to a native plant [i.e., a plant not modified with the biomolecules (polynucleotide or polypeptides) of the invention, e.g., a non-transformed plant of the same species which is grown under the same growth conditions).
The phrase “expressing within the plant an exogenous polynucleotide” as used herein refers to upregulating the expression level of an exogenous polynucleotide within the plant by introducing the exogenous polynucleotide into a plant cell or plant and expressing by recombinant means, as further described herein below.
As used herein “expressing” refers to expression at the mRNA and optionally polypeptide level.
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 and/or a polypeptide 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.
The term “endogenous” as used herein refers to any polynucleotide or polypeptide which is present and/or naturally expressed within a plant or a cell thereof.
According to some embodiments of the invention the exogenous polynucleotide comprises a nucleic acid sequence which is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, and 671.
Identity (e.g., percent homology) 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.
According to some embodiments of the invention the exogenous polynucleotide is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the polynucleotide selected from the group consisting of SEQ ID NOs: 619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, and 671.
According to some embodiments of the invention the exogenous polynucleotide is set forth by SEQ ID NO:619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, or 671.
According to an aspect of some embodiments of the invention, there is provided a method of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant, comprising expressing within the plant an exogenous polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs:619, 617, 606, 615, 629, 1-49, 51-59, 113-343, 345-351, 353-358, 605, 607-614, 616, 618, 620-628, 630-638, 641, 642, 644, 644-646, 648-651, 670, and 671, thereby increasing the abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of the plant.
According to some embodiments of the invention the exogenous polynucleotide is set forth by the nucleic acid sequence selected from the group consisting of SEQ ID NOs:619, 617, 606, 615, 629, 1-49, 51-59, 113-343, 345-351, 353-358, 605, 607-614, 616, 618, 620-628, 630-638, 641, 642, 644, 644-646, 648-651, 670, and 671.
According to an aspect of some embodiments of the invention, there is provided a method of increasing abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the polynucleotide selected from the group consisting of SEQ ID NOs:352, 639, 640, and 643, thereby increasing the abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of the plant.
According to an aspect of some embodiments of the invention, there is provided a method of increasing abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 352, 639, 640, and 643, thereby increasing the abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of the plant.
According to some embodiments of the invention the exogenous polynucleotide is set forth by the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 352, 639, 640, and 643.
According to an aspect of some embodiments of the invention, there is provided a method of increasing nitrogen use efficiency, seed yield and/or oil content of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the polynucleotide selected from the group consisting of SEQ ID NOs:50, 645, and 647, thereby increasing the nitrogen use efficiency, seed yield and/or oil content of the plant.
According to an aspect of some embodiments of the invention, there is provided a method of increasing nitrogen use efficiency, seed yield and/or oil content of a plant, comprising expressing within the plant an exogenous polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs:50, 645 and 647, thereby increasing the nitrogen use efficiency, seed yield and/or oil content of the plant.
According to some embodiments of the invention the exogenous polynucleotide is set forth by the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 50, 645 and 647.
According to an aspect of some embodiments of the invention, there is provided a method of increasing seed yield, fiber yield and/or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the polynucleotide set forth by SEQ ID NO:344, thereby increasing the seed yield, fiber yield and/or fiber quality of the plant.
According to an aspect of some embodiments of the invention, there is provided a method of increasing seed yield, fiber yield and/or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:344, thereby increasing the seed yield, fiber yield and/or fiber quality of the plant.
According to some embodiments of the invention the exogenous polynucleotide is set forth by the nucleic acid sequence set forth in SEQ ID NO:344.
As used herein the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
The term “isolated” refers to at least partially separated from the natural environment e.g., from a plant cell.
As used herein the phrase “complementary polynucleotide sequence” refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
As used herein the phrase “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
As used herein the phrase “composite polynucleotide sequence” refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
According to some embodiments of the invention, the exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, and 672.
Homology (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastP or 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.
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. If orthologues in rice were sought, the sequence-of-interest would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI. 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.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence set forth by SEQ ID NO: 75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, or 672.
According to an aspect of some embodiments of the invention, the method of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant, is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672, thereby increasing the abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of the plant.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence set forth by SEQ ID NO: 75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, or 672.
According to an aspect of some embodiments of the invention, the method of increasing abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of a plant, is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:99 and 598, thereby increasing the abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of the plant.
According to an aspect of some embodiments of the invention, the method of increasing abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of a plant is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 99 and 598, thereby increasing the abiotic stress tolerance, nitrogen use efficiency, fiber yield and/or fiber quality of the plant.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence set forth by SEQ ID NO: 99 or 598.
According to an aspect of some embodiments of the invention, the method of increasing nitrogen use efficiency, seed yield and/or oil content of a plant is effected expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:599 and 663, thereby increasing the nitrogen use efficiency, seed yield and/or oil content of the plant.
According to an aspect of some embodiments of the invention, the method of increasing nitrogen use efficiency, seed yield and/or oil content of a plant is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 599 and 663, thereby increasing the nitrogen use efficiency, seed yield and/or oil content of the plant.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence set forth by SEQ ID NO: 599 or 663.
According to an aspect of some embodiments of the invention, the method of increasing nitrogen use efficiency, abiotic stress tolerance, seed yield and/or oil content of a plant is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:110 and 665, thereby increasing the nitrogen use efficiency, abiotic stress tolerance, seed yield and/or oil content of the plant.
According to an aspect of some embodiments of the invention, the method of increasing nitrogen use efficiency, abiotic stress tolerance, seed yield and/or oil content of a plant is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs:110 and 665, thereby increasing the nitrogen use efficiency, abiotic stress tolerance, seed yield and/or oil content of the plant.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence set forth by SEQ ID NO: 110 or 665.
According to an aspect of some embodiments of the invention, the method of increasing seed yield, fiber yield and/or fiber quality of a plant is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to the amino acid sequence set forth by SEQ ID NO:590, thereby increasing the seed yield, fiber yield and/or fiber quality of the plant.
According to an aspect of some embodiments of the invention, the method of increasing seed yield, fiber yield and/or fiber quality of a plant is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence set forth by SEQ ID NO:590, thereby increasing the seed yield, fiber yield and/or fiber quality of the plant.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence set forth by SEQ ID NO:590.
Nucleic acid sequences encoding the polypeptides of the present invention may be optimized for expression. Non-limiting examples of optimized nucleic acid sequences are provided in SEQ ID NOs: 670 (BDL103_long), 639 (BDL11) and 643 (BDL17) which encode optimized polypeptide comprising the amino acid sequences set forth by SEQ ID NOs: 96, 661 and 101, respectively. 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 (Hypertext Transfer Protocol://World Wide Web (dot) kazusa (dot) or (dot) 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.
Thus, the invention encompasses nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
The invention provides an isolated polynucleotide comprising a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the polynucleotide selected from the group consisting of SEQ ID NOs: 619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650-651, 670, and 671.
According to some embodiments of the invention the nucleic acid sequence is capable of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.
According to some embodiments of the invention the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650-651, 670, and 671.
According to some embodiments of the invention the isolated polynucleotide consists of the nucleic acid sequence selected from the group consisting of SEQ ID NOs:619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, and 671.
According to some embodiments of the invention the isolated polynucleotide is set forth by SEQ ID NO: 619, 617, 606, 615, 629, 1-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650-651, 670, and 671.
According to an aspect of some embodiments of the invention, there is provided an isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs:619, 617, 606, 615, 629, 1-49, 51-59, 113-343, 345-351, 353-358, 605, 607-614, 616, 618, 620-628, 630-638, 641, 642, 644, 644-646, 648-651, 670, and 671.
The invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide which comprises an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, and 672.
According to some embodiments of the invention the amino acid sequence is capable of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.
The invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide which comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672.
The invention provides an isolated polypeptide comprising an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, and 672.
According to some embodiments of the invention the isolated polypeptide is capable of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.
According to some embodiments of the invention, the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672.
According to some embodiments of the invention, the polypeptide is set forth by SEQ ID NO: 75, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, or 672.
The invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or man induced, either randomly or in a targeted fashion.
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 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. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily 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, Cyclonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cyclonia 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., Eucalypfus 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 fleckii, 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 such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, poplar and cotton.
According to some embodiments of the invention, there is provided a plant cell exogenously expressing the polynucleotide of some embodiments of the invention, the nucleic acid construct of some embodiments of the invention and/or the polypeptide of some embodiments of the invention.
According to some embodiments of the invention, expressing the exogenous polynucleotide of the invention within the plant is effected by transforming one or more cells of the plant with the exogenous polynucleotide, followed by generating a mature plant from the transformed cells and cultivating the mature plant under conditions suitable for expressing the exogenous polynucleotide within the mature plant.
According to some embodiments of the invention, the transformation is effected by introducing to the plant cell a nucleic acid construct which includes the exogenous polynucleotide of some embodiments of the invention and at least one promoter for directing transcription of the exogenous polynucleotide in a host cell (a plant cell). Further details of suitable transformation approaches are provided hereinbelow.
According to some embodiments of the invention, there is provided a nucleic acid construct comprising the isolated polynucleotide of the invention, and a promoter for directing transcription of the nucleic acid sequence of the isolated polynucleotide in a host cell.
According to some embodiments of the invention, the isolated polynucleotide is operably linked to the promoter sequence.
A coding nucleic acid sequence is “operably 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.
As used herein, the term “promoter” refers to a region of DNA which lies upstream of the transcriptional initiation site of a gene to which RNA polymerase binds to initiate transcription of RNA. The promoter controls where (e.g., which portion of a plant) and/or when (e.g., at which stage or condition in the lifetime of an organism) the gene is expressed.
Any suitable promoter sequence can be used by the nucleic acid construct of the present invention. Preferably the promoter is a constitutive promoter, a tissue-specific, or an abiotic stress-inducible promoter.
Suitable constitutive promoters include, for example, CaMV 35S promoter (SEQ ID NO:675; Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter (SEQ ID NO:674; see PCT Publication No. WO04081173A2); 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. 81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al, Plant J November;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 U.S. Pat. No. 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, et al., 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 (EMBO3:1409-15, 1984), Barley ltrl promoter, barley B1, 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 Glb-1 (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), sorgum gamma-kafirin (PMB 32:1029-35, 1996)], embryo specific promoters [e.g., rice OSH1 (Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma of 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].
Suitable abiotic stress-inducible promoters include, but not limited to, salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al., Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such as maize rab 17 gene promoter (Pla et. al., Plant Mol. Biol. 21:259-266, 1993), maize rab28 gene promoter (Busk et. al., Plant J. 11:1285-1295, 1997) and maize Ivr2 gene promoter (Pelleschi et. al., Plant Mol. Biol. 39:373-380, 1999); heat-inducible promoters such as heat tomato hsp80-promoter from tomato (U.S. Pat. No. 5,187,267).
The nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication. According to some embodiments of the invention, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible with propagation in cells. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
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.
There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., 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: 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.
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. Therefore, 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. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. 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.
According to some embodiments of the invention, the transgenic plants are generated by transient transformation of leaf cells, meristematic cells or the whole plant.
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), Gal-on 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 protein 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 polynucleotide is provided in which the native coat protein coding sequence has been deleted from a viral polynucleotide, 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 polynucleotide, and ensuring a systemic infection of the host by the recombinant plant viral polynucleotide, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native polynucleotide sequence within it, such that a protein is produced. The recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign) polynucleotide 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 polynucleotide sequence is included. The non-native polynucleotide 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 polynucleotide 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 polynucleotide 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 polynucleotide. 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 polynucleotide 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 polynucleotide 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 polynucleotide to produce a recombinant plant virus. The recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral polynucleotide is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.
Techniques for inoculation of viruses to plants may be found in Foster and Taylor, eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods in Virology” 7 vols, Academic Press, New York 1967-1984; Hill, S. A. “Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D. G. A. “Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa, eds. “Principles and Techniques in Plant Virology”, Van Nostrand-Reinhold, New York.
In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.
A technique for introducing exogenous polynucleotide 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 polynucleotide is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts. The exogenous polynucleotides 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 polynucleotide includes, in addition to a gene of interest, at least one polynucleotide stretch which is derived from the chloroplast's genome. In addition, the exogenous polynucleotide 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 polynucleotide. Further details relating to this technique are found in U.S. Pat. No. 4,945,050; and U.S. Pat. No. 5,693,507 which are incorporated herein by reference. A polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
Since processes which increase oil content, yield, growth rate, biomass, vigor and/or abiotic stress tolerance of a plant can involve multiple genes acting additively or in synergy (see, for example, in Quesda et al., Plant Physiol. 130:951-063, 2002), the present invention also envisages expressing a plurality of exogenous polynucleotides in a single host plant to thereby achieve superior effect on oil content, yield, growth rate, biomass, vigor and/or abiotic stress tolerance.
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 than 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. To enable co-translation of the different polypeptides encoded by the polycistronic messenger RNA, the polynucleotide sequences can be inter-linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule encoding the different polypeptides described above will be translated from both the capped 5′ end and the two internal IRES sequences of the polycistronic RNA molecule to thereby produce in the cell all different polypeptides. 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 in a single host plant 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 abiotic stress tolerance, water use efficiency, fertilizer use efficiency, growth, biomass, yield and/or vigor traits, using conventional plant breeding techniques.
According to some embodiments of the invention, the method further comprising growing the plant expressing the exogenous polynucleotide under the abiotic stress.
Non-limiting examples of abiotic stress conditions include, salinity, drought, water deprivation, excess of water (e.g., flood, waterlogging), etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution and UV irradiation.
Thus, the invention encompasses plants exogenously expressing the polynucleotide(s), the nucleic acid constructs and/or polypeptide(s) of the invention. Once expressed within the plant cell or the entire plant, the level of the polypeptide encoded by the exogenous polynucleotide can be determined by methods well known in the art such as, activity assays, Western blots using antibodies capable of specifically binding the polypeptide, Enzyme-Linked Immuno Sorbent Assay (ELISA), radio-immunoassays (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence and the like.
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-in 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., abiotic stress tolerance, increased yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant). 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), micro-satellites and single nucleotide polymorphism (SNP), DNA fingerprinting (DFP), amplified fragment length polymorphism (AFLP), expression level polymorphism, polymorphism of the encoded polypeptide 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 and polypeptides 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 or the polypeptide of the invention are screened to identify those that show the greatest increase of the desired plant trait.
The effect of the transgene (the exogenous polynucleotide encoding the polypeptide) on abiotic stress tolerance can be determined using known methods such as detailed below and in the Examples section which follows.
Abiotic Stress Tolerance—
Transformed (i.e., expressing the transgene) and non-transformed (wild type) plants are exposed to an abiotic stress condition, such as water deprivation, suboptimal temperature (low temperature, high temperature), nutrient deficiency, nutrient excess, a salt stress condition, osmotic stress, heavy metal toxicity, anaerobiosis, atmospheric pollution and UV irradiation.
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), or by culturing the plants in a hyperosmotic growth medium [e.g., 50% Murashige-Skoog medium (MS medium)]. 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, 100 mM, 200 mM, 400 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 wilting 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 mannitol 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 germination experiments, the medium is supplemented for example with 50 mM, 100 mM, 200 mM NaCl or 100 mM, 200 mM NaCl, 400 mM mannitol.
Drought Tolerance Assay/Osmoticum Assay—
Tolerance to drought is performed to identify the genes conferring better plant survival after acute water deprivation. To analyze whether the transgenic plants are more tolerant to drought, an osmotic stress produced by the non-ionic osmolyte sorbitol in the medium can be performed. Control and transgenic plants are germinated and grown in plant-agar plates for 4 days, after which they are transferred to plates containing 500 mM sorbitol. The treatment causes growth retardation, then both control and transgenic plants are compared, by measuring plant weight (wet and dry), yield, and by growth rates measured as time to flowering.
Conversely, soil-based drought screens are performed with plants overexpressing the polynucleotides detailed above. Seeds from control Arabidopsis plants, or other transgenic plants overexpressing the polypeptide of the invention are germinated and transferred to pots. Drought stress is obtained after irrigation is ceased accompanied by placing the pots on absorbent paper to enhance the soil-drying rate. 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.
Cold Stress Tolerance—
To analyze cold stress, 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 both 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—
Heat stress tolerance is achieved 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.
Water Use Efficiency—
can be determined as the biomass produced per unit transpiration. To analyze WUE, leaf relative water content can be measured in control and transgenic plants. Fresh weight (FW) is immediately recorded; then leaves are soaked for 8 hours in distilled water at room temperature in the dark, and the turgid weight (TW) is recorded. Total dry weight (DW) is recorded after drying the leaves at 60° C. to a constant weight. Relative water content (RWC) is calculated according to the following Formula I:
RWC=[(FW−DW)/(TW−DW)]×100 Formula I
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 Example 6, hereinbelow and in Yanagisawa et al (Proc Natl Acad Sci USA. 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 Arabidopsis plants are more responsive to nitrogen, plant are grown in 0.75-1.5 mM (nitrogen deficient conditions) or 6-10 mM (optimal nitrogen concentration). Plants are allowed to grow for additional 20 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 Dof1 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×MS [Murashige-Skoog] supplemented with a selection agent are transferred to two nitrogen-limiting conditions: MS media in which the combined nitrogen concentration (NH₄NO₃and KNO₃) was 0.2 mM or 0.05 mM. 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 T1 seeds are available are sown on selective media and at least 25 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, 25 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 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 NO₃ ⁻ to NO₂ ⁻ (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 NaNO₂. The procedure is described in details in Samonte et al. 2006 Agron. J. 98:168-176.
Germination Tests—
Germination tests compare the percentage of seeds from transgenic plants that could complete the germination process to the percentage of seeds from control plants that are treated in the same manner. Normal conditions are considered for example, incubations at 22° C. under 22-hour light 2-hour dark daily cycles. Evaluation of germination and seedling vigor is conducted between 4 and 14 days after planting. The basal media is 50% MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497).
Germination is checked also at unfavorable conditions such as cold (incubating at temperatures lower than 10° C. instead of 22° C.) or using seed inhibition solutions that contain high concentrations of an osmolyte such as sorbitol (at concentrations of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM, and up to 1000 mM) or applying increasing concentrations of salt (of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl).
The effect of the transgene on plant's vigor, growth rate, biomass, yield and/or oil content can be determined using known methods.
Plant Vigor—
The plant vigor can be calculated by the increase in growth parameters such as leaf area, fiber length, rosette diameter, plant fresh weight and the like per time.
Growth Rate—
The growth rate can be measured using digital analysis of growing plants. For example, images of plants growing in greenhouse on plot basis can be captured every 3 days and the rosette area can be calculated by digital analysis. Rosette area growth is calculated using the difference of rosette area between days of sampling divided by the difference in days between samples.
Evaluation of growth rate can be done by measuring plant biomass produced, rosette area, leaf size or root length per time (can be measured in cm²per day of leaf area).
Relative growth area can be calculated using Formula II.
Relative growth rate area=Regression coefficient of area along time course Formula II:
Thus, the relative growth area rate is in units of 1/day and length growth rate is in units of 1/day.
Seed Yield—
Evaluation of the seed yield per plant can be done by measuring the amount (weight or size) or quantity (i.e., number) of dry seeds produced and harvested from 8-16 plants and divided by the number of plants.
For example, the total seeds from 8-16 plants can be collected, weighted using e.g., an analytical balance and the total weight can be divided by the number of plants. Seed yield per growing area can be calculated in the same manner while taking into account the growing area given to a single plant. Increase seed yield per growing area could be achieved by increasing seed yield per plant, and/or by increasing number of plants capable of growing in a given area.
In addition, seed yield can be determined via the weight of 1000 seeds. The weight of 1000 seeds can be determined as follows: seeds are scattered on a glass tray and a picture is taken. Each sample is weighted and then using the digital analysis, the number of seeds in each sample is calculated.
The 1000 seeds weight can be calculated using formula III:
1000 Seed Weight=number of seed in sample/sample weight X 1000 Formula III:
The Harvest Index can be calculated using Formula IV
Harvest Index=Average seed yield per plant/Average dry weight Formula IV:
Grain Protein Concentration—
Grain protein content (g grain protein m⁻²) is estimated as the product of the mass of grain N (g grain N m⁻²) multiplied by the N/protein conversion ratio of k-5.13 (Mosse 1990, supra). The grain protein concentration is estimated as the ratio of grain protein content per unit mass of the grain (g grain protein kg⁻¹grain).
Fiber Length—
Fiber length can be measured using fibrograph. The fibrograph system was used to compute length in terms of “Upper Half Mean” length. The upper half mean (UHM) is the average length of longer half of the fiber distribution. The fibrograph measures length in span lengths at a given percentage point (Hypertext Transfer Protocol://WorldWide Web (dot) cottoninc (dot) com/ClassificationofCotton/?Pg=4#Length).
According to some embodiments of the invention, increased yield of corn may be manifested as one or more of the following: increase in the number of plants per growing area, increase in the number of ears per plant, increase in the number of rows per ear, number of kernels per ear row, kernel weight, thousand kernel weight (1000-weight), ear length/diameter, increase oil content per kernel and increase starch content per kernel.
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.
Increased yield of canola 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, among others. An increase in yield may also result in modified architecture, or may occur because of modified architecture.
Increased yield of cotton may be manifested by an increase in one or more of the following: number of plants per growing area, number of bolls per plant, number of seeds per boll, increase in the seed filling rate, increase in thousand seed weight (1000-weight), increase oil content per seed, improve fiber length, fiber strength, among others. An increase in yield may also result in modified architecture, or may occur because of modified architecture.
Oil Content—
The oil content of a plant can be determined by extraction of the oil from the seed or the vegetative portion of the plant. Briefly, lipids (oil) can be removed from the plant (e.g., seed) by grinding the plant tissue in the presence of specific solvents (e.g., hexane or petroleum ether) and extracting the oil in a continuous extractor. Indirect oil content analysis can be carried out using various known methods such as Nuclear Magnetic Resonance (NMR) Spectroscopy, which measures the resonance energy absorbed by hydrogen atoms in the liquid state of the sample [See for example, Conway T F. and Earle F R., 1963, Journal of the American Oil Chemists' Society; Springer Berlin/Heidelberg, ISSN: 0003-021X (Print) 1558-9331 (Online)]; the Near Infrared (NI) Spectroscopy, which utilizes the absorption of near infrared energy (1100-2500 nm) by the sample; and a method described in WO/2001/023884, which is based on extracting oil a solvent, evaporating the solvent in a gas stream which forms oil particles, and directing a light into the gas stream and oil particles which forms a detectable reflected light.
Thus, the present invention is of high agricultural value for promoting the yield of commercially desired crops (e.g., biomass of vegetative organ such as poplar wood, or reproductive organ such as number of seeds or seed biomass).
Any of the transgenic plants described hereinabove or parts thereof may be processed to produce a feed, meal, protein or oil preparation, such as for ruminant animals.
The transgenic plants described hereinabove, which exhibit an increased oil content can be used to produce plant oil (by extracting the oil from the plant).
The plant oil (including the seed oil and/or the vegetative portion oil) produced according to the method of the invention may be combined with a variety of other ingredients. The specific ingredients included in a product are determined according to the intended use. Exemplary products include animal feed, raw material for chemical modification, biodegradable plastic, blended food product, edible oil, biofuel, cooking oil, lubricant, biodiesel, snack food, cosmetics, and fermentation process raw material. Exemplary products to be incorporated to the plant oil include animal feeds, human food products such as extruded snack foods, breads, as a food binding agent, aquaculture feeds, fermentable mixtures, food supplements, sport drinks, nutritional food bars, multi-vitamin supplements, diet drinks, and cereal foods.
According to some embodiments of the invention, the oil comprises a seed oil.
According to some embodiments of the invention, the oil comprises a vegetative portion oil.
According to some embodiments of the invention, the plant cell forms a part of a plant.
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, Md. (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, Conn. (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, Calif. (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

Identifying Putative Genes which Increase Abiotic Stress Tolerance, Yield, Biomass, Growth Rate and/or Fiber Development and Quality

The present inventors have identified genes which increase abiotic stress-tolerance (ABST), growth rate, biomass, fiber development or quality, vigor, yield (e.g., seed yield, oil yield), oil content, and nitrogen use efficiency. All nucleotide sequence datasets used here were originated from publicly available databases. Sequence data from 80 different plant species was introduced into a single, comprehensive database. Other information on gene expression, protein annotation, enzymes and pathways were also incorporated. Major databases used include:
Genomes
Arabidopsis genome [TAIR genome version 6 (Hypertext Transfer Protocol://World Wide Web (dot) arabidopsis (dot) org/)]
Rice genome [IRGSP build 4.0 (Hypertext Transfer Protocol://rgp (dot) dna (dot) affrc (dot) go (dot) jp/IRGSP/)].
Poplar [Populus trichocarpa release 1.1 from JGI (assembly release v1.0) (Hypertext Transfer Protocol://World Wide Web (dot) genome (dot) jgi-psf (dot) org/)]
Brachypodium [JGI 4× assembly Hypertext Transfer Protocol://World Wide Web (dot) brachpodium (dot) org)]
Soybean [DOE-JGI SCP, version Glyma0 (Hypertext Transfer Protocol://World Wide Web (dot) phytozome (dot) net/)]
Grape International Grape Genome Program Genome Assembly (Hypertext Transfer Protocol://World Wide Web (dot) genoscope (dot) cns (dot) fr/externe/Download/Projets/Projet_mL/data/assembly/
Castobean [TIGR/J Craig Venter Institute 4× assembly (Hypertext Transfer Protocol://msc (dot) jcv (dot) org/)]
Sorghum [DOE-JGI SCP, version Sbi1 Hypertext Transfer Protocol://World Wide Web (dot) phytozome (dot) net/)].
Partially assembled genome of Maize [Hypertext Transfer Protocol://maizesequence (dot) org/]
Expressed EST and mRNA sequences were extracted from
GeneBank versions 154, 157, 160, 161, 164, 165, 166 (Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/dbEST/)
RefSeq (Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/RefSeq/).
TAIR (Hypertext Transfer Protocol://World Wide Web (dot) arabidopsis (dot) org/).
Protein and pathway databases
Uniprot (Hypertext Transfer Protocol://World Wide Web.expasy.uniprot.org/).
AraCyc (Hypertext Transfer Protocol://World Wide Web (dot) arabidopsis (dot) org/biocyc/index (dot) jsp).
ENZYME (Hypertext Transfer Protocol://expasy.org/enzyme/).
Microarray datasets were downloaded from
GEO (Hypertext Transfer Protocol://World Wide Web.ncbi.nlm.nih.gov/geo/)
TAIR (Hypertext Transfer Protocol://World Wide Web.arabidopsis.org/).
Proprietary cotton fiber microarray data
QTL information
Gramene (Hypertext Transfer Protocol://World Wide Web (dot) gramene (dot) org/qtl/).
Database Assembly was performed to build a wide, rich, reliable annotated and easy to analyze database comprised of publicly available genomic mRNA, ESTs DNA sequences, data from various crops as well as gene expression, protein annotation and pathway data QTLs, and other relevant information.
Database assembly is comprised of a toolbox of gene refining, structuring, annotation and analysis tools enabling to construct a tailored database for each gene discovery project. Gene refining and structuring tools enable to reliably detect splice variants and antisense transcripts, generating understanding of various potential phenotypic outcomes of a single gene. The capabilities of the “LEADS” platform of Compugen LTD for analyzing human genome have been confirmed and accepted by the scientific community (“Widespread Antisense Transcription”, Yelin, et al. (2003) Nature Biotechnology 21, 379-85; “Splicing of Alu Sequences”, Lev-Maor, et al. (2003) Science 300 (5623), 1288-91; “Computational analysis of alternative splicing using EST tissue information”, Xie H et al. Genomics 2002), and have been proven most efficient in plant genomics as well.
EST Clustering and Gene Assembly—
For gene clustering and assembly of organisms with available genome sequence data (arabidopsis, rice, castorbean, grape, brachypodium, poplar, soybean, sorghum) the genomic LEADS version (GANG) was employed. This tool allows most accurate clustering of ESTs and mRNA sequences on genome, and predicts gene structure as well as alternative splicing events and anti-sense transcription.
For organisms with no available full genome sequence data, “expressed LEADS” clustering software was applied.
Gene Annotation—
Predicted genes and proteins were annotated as follows:
Blast search (Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov (dot) library (dot) vu (dot) edu (dot) au/BLAST/) against all plant UniProt (Hypertext Transfer Protocol://World Wide Web (dot) expasy (dot) uniprot (dot) org/) sequences was performed.
Open reading frames of each putative transcript were analyzed and longest ORF with higher number of homologs was selected as predicted protein of the transcript.
The predicted proteins were analyzed by InterPro (Hypertext Transfer Protocol://World Wide Web (dot) ebi (dot) ac (dot) uk/interpro/).
Blast against proteins from AraCyc and ENZYME databases was used to map the predicted transcripts to AraCyc pathways.
Predicted proteins from different species were compared using blast algorithm (Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov (dot) library (dot) vu (dot) edu (dot) au/BLAST/) to validate the accuracy of the predicted protein sequence, and for efficient detection of orthologs.
Gene Expression Profiling—
Few data sources were exploited for gene expression profiling, namely microarray data and digital expression profile (see below). According to gene expression profile, a correlation analysis was performed to identify genes which are co-regulated under different development stages and environmental conditions.
Publicly available microarray datasets were downloaded from TAIR and NCBI GEO sites, renormalized, and integrated into the database. Expression profiling is one of the most important resource data for identifying genes important for ABST. Moreover, when homolog genes from different crops were responsive to ABST, the genes are marked as “highly predictive to improve ABST”.
A digital expression profile summary was compiled for each cluster according to all keywords included in the sequence records comprising the cluster. Digital expression, also known as electronic Northern Blot, is a tool that displays virtual expression profile based on the EST sequences forming the gene cluster. The tool can provide the expression profile of a cluster in terms of plant anatomy (in what tissues/organs is the gene expressed), developmental stage (the developmental stages at which a gene can be found) and profile of treatment (provides the physiological conditions under which a gene is expressed such as drought, cold, pathogen infection, etc). Given a random distribution of ESTs in the different clusters, the digital expression provides a probability value that describes the probability of a cluster having a total of N ESTs to contain X ESTs from a certain collection of libraries. For the probability calculations are taken into consideration: a) the number of ESTs in the cluster, b) the number of ESTs of the implicated and related libraries, c) the overall number of ESTs available representing the species. Thereby clusters with low probability values are highly enriched with ESTs from the group of libraries of interest indicating a specialized expression.
The results of the digital and microarray gene expression data are provided in Tables 1-4, hereinbelow.
Below are summarized the key criteria used to select the genes which expression thereof in a plant can be used to increase ABST, WUE, NUE, FUE, biomass, yield and oil content. The overexpression Fold (“Fold”) is calculated as the ratio between the number of ESTs found in a gene or an orthologue group for a certain category (“Keyword”) and the number of expected ESTs according to a normal distribution. A probabilistic value (P-value) was estimated for the calculated overexpression folds. Genes were selected based on the results presented in Tables 1-4 below and other computational filtering combined with manual curation as detailed below.
LAB25, LAB31, LAB33, LAB34, LAB45 and LAB51 were selected since they are highly expressed in roots and under drought stress conditions (as shown in Table 1 hereinbelow).

TABLE 1

Digital expression of LAB25, LAB31, LAB33, LAB34,
LAB45 and LAB51 in roots and under drought stress

	Anatomy		Treatment
	Root		Drought stress

	Genes	fold	p-value	fold	p-value

LAB25	5.39	1.125E−52	1.984434	0.0404051
LAB31	10.00	6.034E−09	7.00	8.6157E−06
LAB33	2.66	7.272E−05	3.25	0.00090165
LAB34	3.38	1.474E−05	9.55	6.8734E−08
LAB45	2.22	1.7E−07	14.11	4.2333E−14
LAB51	2.10	0.0046312	4.00	0.0131351

Table 1. Digital expression of the indicated genes in root and under drought stress. Provided are the fold increase and the calculated p-values of expression of the gene in the indicated tissue or condition as compared to the randomly expected expression. Results were considered statistically significant if the p-value was lower than 0.05.

LAB4, LAB7, LAB14 and LAB49 were selected since they are highly expressed in roots and under UV radiation, cold stress or heat stress (as shown in Table 2 hereinbelow).

TABLE 2

Digital expression of LAB4, LAB7, LAB14 and LAB49 in roots, under UV
irradiation, cold stress or heat stress

Anatomy

Treatment

Root

UV irradiation

Cold stress

Heat stress

Genes	fold	p-value	fold	p-value	fold	p-value	fold	p-value

LAB4	4.45	2.005E−10
LAB7	2.48	6.421E−08			2.37	0.0303
LAB14	2.15	0.0319954	3.64	0.00019			2	0.0570
LAB49	4.17	8.6877E−11

Table 2. Digital expression of the indicated genes in roots, under UV irradiation, cold stress or heat stress. Provided are the fold increase and the calculated p-values of expression of the gene in the indicated tissue or condition as compared to the randomly expected expression. Results were considered statistically significant if the p-value was lower than 0.05. Blank cells indicate that either the gene is not expressed or data is not available.

LAB5, LAB13, LAB16, LAB18, LAB20, LAB22, LAB3, LAB24, LAB35, LAB38, LAB39, LAB40, LAB50 and LAB51 were selected since they are highly expressed under drought stress and possibly nutrient deficiencies, cold stress or plant development or stress hormones (as shown in Table 3 hereinbelow).

TABLE 3

Digital expression of LAB5, LAB13, LAB16, LAB18, LAB20, LAB22, LAB3,
LAB24, LAB35, LAB38, LAB39, LAB40, LAB50 and LAB51 under drought stress
and possibly nutrient deficiencies, cold stress or plant development or stress hormones

Nutrient deficiencies

			Plant development
Drought stress		Cold	or stress
fold	p-value	stress	hormones

	3.46	0.00188373	fold	p-value	fold	p-value	fold	p-value

LAB5	3.13	0.0400183
LAB13	3.00	0.00017491
LAB16	4.00	0.00458478
LAB18	4.95	4.2144E−05
LAB20	8.88	3.4638E−22
LAB22	3.00	0.00978408			3.17	0.0379553
LAB3	2.37	5.7818E−08
LAB24	14.11	4.2333E−14
LAB35	4.00	0.00207373			3.00	0.0072537
LAB38	2.35	0.00067594
LAB39	8.93	2.6849E−08	3.06	0.0144515
LAB40	7.00	5.6733E−05
LAB50	3.44	1.1207E−06			3.15	0.012142

Table 3. Digital expression of the indicated genes under drought stress, possibly nutrient deficiencies, cold stress or plant development or stress hormones. Provided are the fold increase and the calculated p-values of expression of the gene in the indicated tissue or condition as compared to the randomly expected expression. Results were considered statistically significant if the p-value was lower than 0.05. Blank cells indicate that either the gene is not expressed or data is not available.

LAB9, LAB21, LAB32, LAB15, LAB17, LAB30, LAB36, and LAB39 were selected since they are highly expressed under etiolatlion condition, plant development or stress hormones, salinity stress or waterlogging (as shown in Table 4 hereinbelow).

TABLE 4

Digital expression of LAB9, LAB21, LAB32, LAB15, LAB17, LAB30, LAB36,
and LAB39 under etiolatlion condition, plant development or stress hormones, salinity
stress or waterlogging

Etiolated

Plant development or stress hormones

fold

p-value

Salinity stress

Waterlogging

	2.91	0.0160756	fold	p-value	fold	p-value	fold	p-value

LAB9	2.23	0.00043618
LAB21	4.65	5.5967E−17
LAB32
LAB15					1.0	0.0705542
LAB17					2.0	0.0420927
LAB30			6.00	7.4196E−05
LAB36			3.66	3.338E−06	4.7	9.3682E−06
LAB39

Table 4. Digital expression of the indicated genes under etiolatlion condition, plant development or stress hormones, salinity stress or waterlogging. Provided are the fold increase and the calculated p-values of expression of the gene in the indicated tissue or condition as compared to the randomly expected expression. Results were considered statistically significant if the p-value was lower than 0.05. Blank cells indicate that either the gene is not expressed or data is not available.

Overall, 51 genes were identified to have a major impact on ABST, nitrogen use efficiency, yield (e.g., seed yield), oil content, growth rate and/or vigor when overexpressed in plants. The identified genes, their curated polynucleotide and polypeptide sequences, as well as their updated sequences according to Genebank database are summarized in Table 5, hereinbelow.

TABLE 5

Identified genes which can be used to increase ABST, fiber development (quality
and yield), yield, biomass, growth rate, nitrogen use efficiency, fertilizer
use efficiency, water use efficiency, and/or oil content of a plant

			SEQ ID NO:	SEQ ID NO:
Gene Name	Cluster Name	Organism	Polynuc.	Polypep.

LAB4	rice\|gb157.2\|AA751809	rice	1	60
LAB5	sorghum\|gb161.xeno\|AW922806	sorghum	2	61
LAB7	rice\|gb157.2\|AA754242	rice	3	62
LAB8	rice\|gb157.2\|AA754407	rice	4	63
LAB9	rice\|gb157.2\|AB004799	rice	5	64
LAB11	rice\|gb157.2\|AK070868	rice	6	65
LAB13	rice\|gb157.2\|AT003625	rice	7	66
LAB14	rice\|gb157.2\|AU056017	rice	8	67
LAB15	barley\|gb157.3\|BF623077	barley	9	68
LAB2	barley\|gb157.3\|BE195266	barley	10	69
LAB16	cotton\|gb164\|BE052656	cotton	11	70
LAB17	sorghum\|gb161.xeno\|AI724026	sorghum	12	71
LAB18	sorghum\|gb161.xeno\|BE359151	sorghum	13	72
LAB20	rice\|gb157.2\|AW070136	rice	14	73
LAB21	barley\|gb157.3\|BE421259	barley	15	74
LAB22	sorghum\|gb161.xeno\|AW678130	sorghum	16	75
LAB3	canola\|gb161\|CD831005	canola	17	76
LAB23	barley\|gb157.3\|BI947386	barley	18	77
LAB24	sorghum\|gb161.xeno\|AW433371	sorghum	19	78
LAB25	barley\|gb157.3\|X84056	barley	20	79
LAB30	sorghum\|gb161.xeno\|BE362140	sorghum	21	80
LAB31	canola\|gb161\|H74460	canola	22	81
LAB32	barley\|gb157.3\|AL499903	barley	23	82
LAB33	sorghum\|gb161.xeno\|AW676682	sorghum	24	83
LAB34	soybean\|gb166\|CF921741	soybean	25	84
LAB35	wheat\|gb164\|BE497867	wheat	26	85
LAB36	sorghum\|gb161.xeno\|H55004	sorghum	27	86
LAB38	wheat\|gb164\|BE412185	wheat	28	87
LAB39	sorghum\|gb161.xeno\|BG048297	sorghum	29	88
LAB40	wheat\|gb164\|BE488436	wheat	30	89
LAB41	wheat\|gb164\|X52472	wheat	31	90
LAB43	barley\|gb157.3\|BF624177	barley	32	91
LAB45	sorghum\|gb161.crp\|AI855293	sorghum	33	92
LAB49	rice\|gb157.2\|BE040470	rice	34	93
LAB50	rice\|gb157.2\|BI305323	rice	35	94
LAB51	wheat\|gb164\|BI751966	wheat	36	95
BDL103_P1	rice\|gb157.2\|BE228840	rice	37	96
BDL11	arabidopsis\|gb165\|AT5G12460	arabidopsis	38	97
BDL12	arabidopsis\|gb165\|AT4G08530	arabidopsis	39	98
BDL14	arabidopsis\|gb165\|AT1G53690	arabidopsis	40	99
BDL166	arabidopsis\|gb165\|AT1G71691	arabidopsis	41	100
BDL17	arabidopsis\|gb165\|AT5G36680	arabidopsis	42	101
BDL210	arabidopsis\|gb165\|AT5G22810	arabidopsis	43	102
CTF113	cotton\|gb164\|AI727515	cotton	44	103
CTF163	cotton\|gb164\|CO123733	cotton	45	104
CTF175	cotton\|gb164\|AW187393	cotton	46	105
CTF180	cotton\|gb164\|BG440663	cotton	47	106
CTF205	cotton\|gb164\|AI725800	cotton	48	107
CTF215	cotton\|gb164\|AI729467	cotton	49	108
CTF225	cotton\|gb164\|AW187127	cotton	50	109
CTF226	cotton\|gb164\|AI730124	cotton	51	110
LAB2	barley\|gb157.3\|BE195266	barley	52	69
LAB3	canola\|gb161\|CD831005	canola	53	76
LAB32	barley\|gb157.3\|AL499903	barley	54	82
LAB38	wheat\|gb164\|BE412185	wheat	55	87
LAB51	wheat\|gb164\|BI751966	wheat	56	95
BDL17	arabidopsis\|gb165\|AT5G36680	arabidopsis	57	111
CTF163	cotton\|gb164\|CO123733	cotton	58	104
CTF205	cotton\|gb164\|AI725800	cotton	59	112
BDL103_P2	rice\|gb157.2\|BE228840	rice	638	96

Table 5. Provided are the identified genes, their annotation, organism and polynucleotide and polypeptide sequence identifiers. SEQ ID NOs: 52-59 are polynucleotide sequences which were uncovered after cloning the gene. SEQ ID NO: 638 is a computational curated sequence.

Example 2

Identification of Homologues which Affect ABST, WUE, NUE, FUE, Yield, Growth Rate, Vigor, Biomass and Oil Content

The concepts of orthology and paralogy have been applied to functional characterizations and classifications on the scale of whole-genome comparisons. Orthologs and paralogs constitute two major types of homologs: The first evolved from a common ancestor by specialization, and the latter are related by duplication events. It is assumed that paralogs arising from ancient duplication events are likely to have diverged in function while true orthologs are more likely to retain identical function over evolutionary time.
To further investigate and identify putative ortholog genes of genes affecting abiotic stress tolerance, nitrogen use efficiency, fertilizer use efficiency, yield (e.g., seed yield, oil yield, biomass, grain quantity and/or quality), growth rate, vigor, biomass, oil content, and/or water use efficiency (presented in Table 5, above) all sequences were aligned using the BLAST (/Basic Local Alignment Search Tool/). Sequences sufficiently similar were tentatively grouped. These putative orthologs were further organized under a Phylogram—a branching diagram (tree) assumed to be a representation of the evolutionary relationships among the biological taxa. Putative ortholog groups were analyzed as to their agreement with the phylogram and in cases of disagreements these ortholog groups were broken accordingly. Expression data was analyzed and the EST libraries were classified using a fixed vocabulary of custom terms such as developmental stages (e.g., genes showing similar expression profile through development with up regulation at specific stage, such as at the seed filling stage) and/or plant organ (e.g., genes showing similar expression profile across their organs with up regulation at specific organs such as root). The annotations from all the ESTs clustered to a gene were analyzed statistically by comparing their frequency in the cluster versus their abundance in the database, allowing the construction of a numeric and graphic expression profile of that gene, which is termed “digital expression”. The rationale of using these two complementary methods with methods of phenotypic association studies of QTLs, and phenotype expression correlation is based on the assumption that true orthologs are likely to retain identical function over evolutionary time. These methods provide different sets of indications on function similarities between two homologous genes, similarities in the sequence level—identical amino acids in the protein domains and similarity in expression profiles.
The search and identification of homologous genes involves the screening of sequence information available, for example, in public databases, which include but are not limited to the DNA Database of Japan (DDBJ), Genbank, and the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL) or versions thereof or the MIPS database. A number of different search algorithms have been developed, including but not limited to the suite of programs referred to as BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequence queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology: 76-80, 1994; Birren et al., Genome Analysis, I: 543, 1997). Such methods involve alignment and comparison of sequences. The BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Other such software or algorithms are GAP, BESTFIT, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
The homologous genes may belong to the same gene family. The analysis of a gene family may be carried out using sequence similarity analysis. To perform this analysis one may use standard programs for multiple alignments e.g. Clustal W. A neighbor-joining tree of the proteins homologous to the genes of some embodiments of the invention may be used to provide an overview of structural and ancestral relationships. Sequence identity may be calculated using an alignment program as described above. It is expected that other plants will carry a similar functional gene (orthologue) or a family of similar genes and those genes will provide the same preferred phenotype as the genes presented here. Advantageously, these family members may be useful in the methods of some embodiments of the invention. Example of other plants include, but not limited to, barley (Hordeum vulgare), Arabidopsis (Arabidopsis thaliana), maize (Zea mays), cotton (Gossypium), Oilseed rape (Brassica napus), Rice (Oryza sativa), Sugar cane (Saccharum officinarum), Sorghum (Sorghum bicolor), Soybean (Glycine max), Sunflower (Helianthus annuus), Tomato (Lycopersicon esculentum) and Wheat (Triticum aestivum).
The above-mentioned analyses for sequence homology is preferably carried out on a full-length sequence, but may also be based on a comparison of certain regions such as conserved domains. The identification of such domains would also be well within the realm of the person skilled in the art and would involve, for example, a computer readable format of the nucleic acids of some embodiments of the invention, the use of alignment software programs and the use of publicly available information on protein domains, conserved motifs and boxes. This information is available in the PRODOM (Hypertext Transfer Protocol://World Wide Web (dot) biochem (dot) ucl (dot) ac (dot) uk/bsm/dbbrowser/protocol/prodomqry (dot) html), PIR (Hypertext Transfer Protocol://pir (dot) Georgetown (dot) edu/) or Pfam (Hypertext Transfer Protocol://World Wide Web (dot) sanger (dot) ac (dot) uk/Software/Pfam/) database. Sequence analysis programs designed for motif searching may be used for identification of fragments, regions and conserved domains as mentioned above. Preferred computer programs include, but are not limited to, MEME, SIGNALSCAN, and GENESCAN.
A person skilled in the art may use the homologous sequences provided herein to find similar sequences in other species and other organisms. Homologues of a protein encompass, peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. To produce such homologues, amino acids of the protein may be replaced by other amino acids having similar properties (conservative changes, such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or 3-sheet structures). Conservative substitution Tables are well known in the art [see for example Creighton (1984) Proteins. W.H. Freeman and Company]. Homologues of a nucleic acid encompass nucleic acids having nucleotide substitutions, deletions and/or insertions relative to the unmodified nucleic acid in question and having similar biological and functional activity as the unmodified nucleic acid from which they are derived.
Table 6, hereinbelow, lists a summary of orthologous and homologous sequences of the polynucleotide sequences (SEQ ID NOs:1-59 and 638) and polypeptide sequences (SEQ ID NOs:60-112) presented in Table 5, which were identified using NCBI BLAST (BlastP) and needle (EMBOSS package) having at least 80% identity to the selected polypeptides and which are expected to posses the same role in abiotic stress tolerance (ABST), water use efficiency (WUE), nitrogen use efficiency (NUE), fertilizer use efficiency (FUE), biomass increment, growth rate increment, yield, vigor, fiber quality and/or yield and/or oil content of plants.

TABLE 6

Homologues of the identified genes of the invention which can increase ABST, fiber
development (quality and yield), biomass, growth rate, nitrogen use efficiency,
fertilizer use efficiency, water use efficiency, yield and/or oil content of a plant

Polynucl.			Polypep.	Homology
SEQ ID	Gene		SEQ ID	to SEQ	% Global
NO:	Name	Organism/Cluster name	NO:	ID NO:	identity	Algor.

113	LAB4_H0	sorghum\|gb161.crp\|AW747731	359	60	82.6	blastp
114	LAB4_H1	switchgrass\|gb167\|DN143443	360	60	81.6	blastp
115	LAB5_H0	barley\|gb157.3\|BE412466	361	61	83.1	blastp
116	LAB5_H1	barley\|gb157.3\|BF623020	362	61	82.3	blastp
117	LAB5_H2	barley\|gb157.3\|BI953964	363	61	82.3	blastp
118	LAB5_H3	brachypodium\|gb169\|AF181661	364	61	85.61	tblastn
119	LAB5_H4	cenchrus\|gb166\|BM084156	365	61	85.1	blastp
120	LAB5_H5	fescue\|gb161\|DT683694	366	61	87.7	blastp
121	LAB5_H6	maize\|gb170\|BI325281	367	61	94.7	blastp
122	LAB5_H7	rice\|gb170\|OS06G46950	368	61	82.3	blastp
123	LAB5_H8	rye\|gb164\|BE637379	369	61	80	tblastn
124	LAB5_H9	spruce\|gb162\|CO219921	370	61	83.1	blastp
125	LAB5_H10	sugarcane\|gb157.3\|BQ529602	371	61	93.3	blastp
126	LAB5_H11	sugarcane\|gb157.3\|BQ535202	372	61	89.23	tblastn
127	LAB5_H12	sugarcane\|gb157.3\|CA072503	373	61	94.8	blastp
128	LAB5_H13	sugarcane\|gb157.3\|CA082920	374	61	94.1	blastp
129	LAB5_H14	sugarcane\|gb157.3\|CA085102	375	61	89.1	blastp
130	LAB5_H15	sugarcane\|gb157.3\|CA090891	376	61	94.1	blastp
131	LAB5_H16	sugarcane\|gb157.3\|CA122790	377	61	90.5	blastp
132	LAB5_H17	switchgrass\|gb167\|DN145030	378	61	80.6	blastp
133	LAB5_H18	switchgrass\|gb167\|FE635988	379	61	89.3	blastp
134	LAB5_H19	switchgrass\|gb167\|FL774816	380	61	85.5	tblastn
135	LAB5_H20	wheat\|gb164\|AF181661	381	61	84.6	blastp
136	LAB5_H21	wheat\|gb164\|BE417364	382	61	83.1	blastp
137	LAB5_H22	wheat\|gb164\|BF484215	383	61	80.6	blastp
138	LAB7_H0	barley\|gb157.3\|AL501769	384	62	83	blastp
139	LAB7_H1	brachypodium\|gb169\|BE471170	385	62	82.8	blastp
140	LAB7_H2	maize\|gb170\|AW042403	386	62	80.4	blastp
141	LAB7_H3	maize\|gb170\|T69041	387	62	81.8	blastp
142	LAB7_H4	sorghum\|gb161.crp\|BE356561	388	62	82.3	blastp
143	LAB7_H5	sugarcane\|gb157.3\|CA091573	389	62	83.1	blastp
144	LAB7_H6	switchgrass\|gb167\|DN142661	390	62	81.4	blastp
145	LAB7_H7	switchgrass\|gb167\|FE615102	391	62	81.2	blastp
146	LAB7_H8	wheat\|gb164\|BE443254	392	62	84	blastp
147	LAB7_H9	wheat\|gb164\|BE471170	393	62	83.1	blastp
148	LAB7_H10	wheat\|gb164\|BF293813	394	62	82.6	blastp
149	LAB8_H0	rice\|gb170\|OS03G22790	395	63	98.85	tblastn
150	LAB15_H0	wheat\|gb164\|BM137033	396	68	87.63	tblastn
151	LAB15_H1	wheat\|gb164\|BM138703	397	68	83.51	tblastn
152	LAB15_H2	wheat\|gb164\|CD882022	398	68	81.5	blastp
153	LAB2_H0	brachypodium\|gb169\|DV485170	399	69	81.8	blastp
154	LAB2_H1	fescue\|gb161\|DT694419	400	69	83.2	blastp
155	LAB16_H0	antirrhinum\|gb166\|AJ787590	401	70	81.2	blastp
156	LAB16_H1	apple\|gb171\|CN580957	402	70	86.4	blastp
157	LAB16_H2	apricot\|gb157.2\|CB824020	403	70	85.1	blastp
158	LAB16_H3	arabidopsis\|gb165\|AT4G38580	404	70	83.7	blastp
159	LAB16_H4	b_juncea\|gb164\|EVGN00544315151807	405	70	83.7	blastp
160	LAB16_H5	b_oleracea\|gb161\|AM058105	406	70	82.4	blastp
161	LAB16_H6	b_oleracea\|gb161\|ES942384	407	70	81.7	tblastn
162	LAB16_H7	b_rapa\|gb162\|EX025293	408	70	83	blastp
163	LAB16_H8	barley\|gb157.3\|BF258224	409	70	84.97	tblastn
164	LAB16_H9	bean\|gb167\|CA910356	410	70	85.1	blastp
165	LAB16_H10	cacao\|gb167\|CU476614	411	70	95.5	blastp
166	LAB16_H11	canola\|gb161\|CD817401	412	70	83	blastp
167	LAB16_H12	canola\|gb161\|CN736951	413	70	81.7	blastp
168	LAB16_H13	cassava\|gb164\|BI325222	414	70	84.42	tblastn
169	LAB16_H14	castorbean\|09v1\|EG691829	415	70	87	blastp
170	LAB16_H15	catharanthus\|gb166\|FD421293	416	70	84.4	blastp
171	LAB16_H16	chestnut\|gb170\|SRR006295S0010879	417	70	81.3	blastp
172	LAB16_H17	chickpea\|09v1\|FE673275	418	70	81.4	blastp
173	LAB16_H18	cichorium\|gb171\|EH697988	419	70	80	blastp
174	LAB16_H19	citrus\|gb166\|CN184469	420	70	80.5	blastp
175	LAB16_H20	coffea\|gb157.2\|DV666808	421	70	80.6	blastp
176	LAB16_H21	cowpea\|gb166\|FC458156	422	70	83.1	blastp
177	LAB16_H22	cowpea\|gb166\|FF538669	423	70	80.8	blastp
178	LAB16_H23	grape\|gb160\|BM436505	424	70	86.5	blastp
179	LAB16_H24	ipomoea\|gb157.2\|BJ555808	425	70	82.6	blastp
180	LAB16_H25	kiwi\|gb166\|FG420453	426	70	81.8	blastp
181	LAB16_H26	liquorice\|gb171\|FS257949	427	70	80.5	blastp
182	LAB16_H27	lotus\|09v1\|GO007127	428	70	84.6	blastp
183	LAB16_H28	medicago\|09v1\|BE320877	429	70	81.4	blastp
184	LAB16_H29	melon\|gb165\|AM726967	430	70	83.1	blastp
185	LAB16_H30	papaya\|gb165\|EX255354	431	70	86.5	blastp
186	LAB16_H31	peach\|gb157.2\|BU039481	432	70	86.4	blastp
187	LAB16_H32	peanut\|gb171\|CX018165	433	70	82.2	blastp
188	LAB16_H33	peanut\|gb171\|ES491048	434	70	82.8	blastp
189	LAB16_H34	pepper\|gb171\|BM060814	435	70	81.8	blastp
190	LAB16_H35	periwinkle\|gb164\|FD421293	436	70	84.4	blastp
191	LAB16_H36	poplar\|gb170\|AJ534494	437	70	85.1	blastp
192	LAB16_H37	poplar\|gb170\|BI129301	438	70	83.8	blastp
193	LAB16_H38	potato\|gb157.2\|BG098018	439	70	82.5	blastp
194	LAB16_H39	potato\|gb157.2\|BG098308	440	70	82.5	blastp
195	LAB16_H40	prunus\|gb167\|BU039481	441	70	86.4	blastp
196	LAB16_H41	radish\|gb164\|EV544328	442	70	83.7	blastp
197	LAB16_H42	soybean\|gb168\|BE315834	443	70	85.1	blastp
198	LAB16_H43	spurge\|gb161\|DV122649	444	70	85.71	tblastn
199	LAB16_H44	strawberry\|gb164\|CO817272	445	70	83.8	blastp
200	LAB16_H45	tomato\|gb164\|AA824901	446	70	81.8	blastp
201	LAB16_H46	triphysaria\|gb164\|EX989778	447	70	81.8	blastp
202	LAB16_H47	walnuts\|gb166\|CB303653	448	70	80.5	blastp
203	LAB17_H0	barley\|gb157.3\|BE231003	449	71	91	blastp
204	LAB17_H1	brachypodium\|gb169\|BE498333	450	71	91	blastp
205	LAB17_H2	cenchrus\|gb166\|EB657534	451	71	84.5	blastp
206	LAB17_H3	fescue\|gb161\|DT685866	452	71	91.6	blastp
207	LAB17_H4	leymus\|gb166\|EG394438	453	71	90.3	blastp
208	LAB17_H5	maize\|gb170\|AW498181	454	71	94.2	blastp
209	LAB17_H6	pseudoroegneria\|gb167\|FF340520	455	71	90.3	blastp
210	LAB17_H7	rice\|gb170\|OS04G17100	456	71	93.5	blastp
211	LAB17_H8	sugarcane\|gb157.3\|CA073067	457	71	85.16	tblastn
212	LAB17_H9	sugarcane\|gb157.3\|CA075729	458	71	96.8	blastp
213	LAB17_H10	sugarcane\|gb157.3\|CA078804	459	71	96.8	blastp
214	LAB17_H11	sugarcane\|gb157.3\|CA116673	460	71	96.1	blastp
215	LAB17_H12	sugarcane\|gb157.3\|CA118688	461	71	97.4	blastp
216	LAB17_H13	sugarcane\|gb157.3\|CA119291	462	71	96.1	blastp
217	LAB17_H14	sugarcane\|gb157.3\|CA222723	463	71	94.2	blastp
218	LAB17_H15	switchgrass\|gb167\|DN143094	464	71	85.9	blastp
219	LAB17_H16	switchgrass\|gb167\|FL792168	465	71	80.8	blastp
220	LAB17_H17	wheat\|gb164\|BE498333	466	71	90.3	blastp
221	LAB17_H18	wheat\|gb164\|BF474623	467	71	89.7	blastp
222	LAB17_H19	wheat\|gb164\|CV760043	468	71	89.7	blastp
223	LAB18_H0	switchgrass\|gb167\|DN140747	469	72	80.6	blastp
224	LAB20_H0	sugarcane\|gb157.3\|CA130714	470	73	82.6	blastp
225	LAB21_H0	aquilegia\|gb157.3\|DR914842	471	74	82.9	blastp
226	LAB21_H1	arabidopsis\|gb165\|AT3G47340	472	74	80.6	blastp
227	LAB21_H2	b_oleracea\|gb161\|X84448	473	74	80.4	blastp
228	LAB21_H3	b_rapa\|gb162\|CV545962	474	74	80.3	blastp
229	LAB21_H4	barley\|gb157.3\|BI948886	475	74	87.5	blastp
230	LAB21_H5	bean\|gb167\|AJ133522	476	74	81.9	blastp
231	LAB21_H6	bean\|gb167\|CB542570	477	74	83.1	blastp
232	LAB21_H7	cacao\|gb167\|CA797951	478	74	83.5	blastp
233	LAB21_H8	castorbean\|09v1\|EE256522	479	74	82.1	blastp
234	LAB21_H9	centaurea\|gb166\|EL931554	480	74	81.2	blastp
235	LAB21_H10	citrus\|gb166\|BQ623162	481	74	83	blastp
236	LAB21_H11	cotton\|gb164\|AI054642	482	74	83.1	blastp
237	LAB21_H12	cotton\|gb164\|BF277939	483	74	81.8	blastp
238	LAB21_H13	cotton\|gb164\|CD486005	484	74	84	blastp
239	LAB21_H14	cowpea\|gb166\|FC458174	485	74	83.3	blastp
240	LAB21_H15	cowpea\|gb166\|FC461749	486	74	82.8	blastp
241	LAB21_H16	kiwi\|gb166\|FG404880	487	74	81.8	blastp
242	LAB21_H17	lettuce\|gb157.2\|DW062326	488	74	83.1	blastp
243	LAB21_H18	maize\|gb170\|AW076472	489	74	85.1	blastp
244	LAB21_H19	medicago\|09v1\|AW126175	490	74	80.7	blastp
245	LAB21_H20	monkeyflower\|09v1\|GO982561	491	74	81.4	blastp
246	LAB21_H21	oak\|gb170\|CU656355	492	74	82.8	blastp
247	LAB21_H22	oil_palm\|gb166\|EL681380	493	74	87	blastp
248	LAB21_H23	peach\|gb157.2\|BU043116	494	74	82	blastp
249	LAB21_H24	poplar\|gb170\|BI138803	495	74	82.8	blastp
250	LAB21_H25	poplar\|gb170\|BU814657	496	74	83.6	blastp
251	LAB21_H26	potato\|gb157.2\|CK258159	497	74	82.1	blastp
252	LAB21_H27	prunus\|gb167\|BU043116	498	74	82.7	blastp
253	LAB21_H28	pseudoroegneria\|gb167\|FF342746	499	74	98.3	blastp
254	LAB21_H29	radish\|gb164\|AB050900	500	74	80.6	blastp
255	LAB21_H30	sorghum\|gb161.crp\|AW286475	501	74	84.96	tblastn
256	LAB21_H31	soybean\|gb168\|AW126284	502	74	83.1	blastp
257	LAB21_H32	soybean\|gb168\|AW720554	503	74	82.3	blastp
258	LAB21_H33	soybean\|gb168\|GMU55874	504	74	81.48	tblastn
259	LAB21_H34	soybean\|gb168\|GMU77678	505	74	83.5	blastp
260	LAB21_H35	soybean\|gb168\|GMU77679	506	74	83	blastp
261	LAB21_H36	sugarcane\|gb157.3\|BQ535363	507	74	85.5	blastp
262	LAB21_H37	sugarcane\|gb157.3\|BQ535939	508	74	85.5	blastp
263	LAB21_H38	sunflower\|gb162\|AF037363	509	74	81.3	blastp
264	LAB21_H39	sunflower\|gb162\|AF190728	510	74	80.9	blastp
265	LAB21_H40	sunflower\|gb162\|DY931765	511	74	80.7	blastp
266	LAB21_H41	tomato\|gb164\|BG127495	512	74	82.5	blastp
267	LAB21_H42	triphysaria\|gb164\|AF014055	513	74	81.9	blastp
268	LAB21_H43	wheat\|gb164\|BE403264	514	74	98.6	blastp
269	LAB21_H44	wheat\|gb164\|BE403866	515	74	98.6	blastp
270	LAB21_H45	wheat\|gb164\|BE430398	516	74	87.9	blastp
271	LAB22_H0	maize\|gb170\|BG833173	517	75	81.7	blastp
272	LAB22_H1	maize\|gb170\|BI423707	518	75	86.4	blastp
273	LAB22_H2	sugarcane\|gb157.3\|BQ536240	519	75	90.9	blastp
274	LAB22_H3	sugarcane\|gb157.3\|BQ536340	520	75	91	blastp
275	LAB22_H4	sugarcane\|gb157.3\|BU103170	521	75	88.2	blastp
276	LAB22_H5	sugarcane\|gb157.3\|CA116439	522	75	89.4	blastp
277	LAB22_H6	switchgrass\|gb167\|FL733549	523	75	85.9	blastp
278	LAB22_H7	wheat\|gb164\|CA484841	524	75	100	blastp
279	LAB3_H0	arabidopsis\|gb165\|AT1G15380	525	76	89.1	blastp
280	LAB3_H1	b_rapa\|gb162\|EX016736	526	76	98.9	blastp
281	LAB3_H2	canola\|gb161\|CD830331	527	76	81.6	blastp
282	LAB3_H3	canola\|gb161\|CN731229	528	76	81.6	blastp
283	LAB3_H4	radish\|gb164\|EV527368	529	76	94.3	blastp
284	LAB3_H5	radish\|gb164\|EV547102	530	76	81	blastp
285	LAB23_H0	pseudoroegneria\|gb167\|FF341473	531	77	89.9	blastp
286	LAB23_H1	wheat\|gb164\|BE516915	532	77	87.6	blastp
287	LAB23_H2	wheat\|gb164\|BE517204	533	77	88.48	tblastn
288	LAB24_H0	maize\|gb170\|BE552559	534	78	80.4	blastp
289	LAB24_H1	sugarcane\|gb157.3\|CA087195	535	78	86.1	blastp
290	LAB24_H2	switchgrass\|gb167\|FL736257	536	78	80	blastp
291	LAB25_H0	leymus\|gb166\|EG374989	537	79	85.6	blastp
292	LAB25_H1	leymus\|gb166\|EG375019	538	79	91.3	blastp
293	LAB25_H2	pseudoroegneria\|gb167\|FF339936	539	79	90.3	blastp
294	LAB25_H3	rye\|gb164\|BE493752	540	79	84.2	blastp
295	LAB25_H4	wheat\|gb164\|TAU73210	541	79	89.2	blastp
296	LAB25_H5	wheat\|gb164\|TAU73211	542	79	91.5	blastp
297	LAB25_H6	wheat\|gb164\|WHTWCOR	543	79	90.1	blastp
298	LAB31_H0	b_oleracea\|gb161\|AM387244	544	81	94.6	blastp
299	LAB31_H1	b_rapa\|gb162\|AT000569	545	81	97.9	blastp
300	LAB31_H2	canola\|gb161\|DY012596	546	81	95.2	blastp
301	LAB31_H3	radish\|gb164\|EV537620	547	81	87.1	blastp
302	LAB32_H0	pseudoroegneria\|gb167\|FF342820	548	82	89.1	blastp
303	LAB33_H0	sugarcane\|gb157.3\|BQ530200	549	83	88.1	blastp
304	LAB34_H0	bean\|gb167\|BQ481761	550	84	86.1	blastp
305	LAB34_H1	soybean\|gb168\|AI755294	551	84	90.9	blastp
306	LAB35_H0	barley\|gb157.3\|Y07823	552	85	92.4	blastp
307	LAB35_H1	brachypodium\|gb169\|Y07823	553	85	83.96	tblastn
308	LAB36_H0	maize\|gb170\|DW878104	554	86	88.3	blastp
309	LAB38_H0	barley\|gb157.3\|AL450676	555	87	91	blastp
310	LAB38_H1	barley\|gb157.3\|BE438884	556	87	91.4	blastp
311	LAB38_H2	barley\|gb157.3\|BF625343	557	87	84.9	blastp
312	LAB38_H3	barley\|gb157.3\|BG299345	558	87	86.12	tblastn
313	LAB38_H4	leymus\|gb166\|CD808961	559	87	94.3	blastp
314	LAB38_H5	pseudoroegneria\|gb167\|FF340156	560	87	95.9	blastp
315	LAB38_H6	wheat\|gb164\|AF495872	561	87	97.1	blastp
316	LAB38_H7	wheat\|gb164\|BE591570	562	87	98.4	blastp
317	LAB39_H0	switchgrass\|gb167\|FE640133	563	88	84.2	blastp
318	LAB39_H1	switchgrass\|gb167\|FL746283	564	88	84.6	blastp
319	LAB39_H2	wheat\|gb164\|CA484141	565	88	99.2	blastp
320	LAB40_H0	barley\|gb157.3\|BF258976	566	89	96.9	blastp
321	LAB40_H1	brachypodium\|gb169\|BE488436	567	89	84.4	blastp
322	LAB41_H0	barley\|gb157.3\|BE437787	568	90	90.5	blastp
323	LAB43_H0	leymus\|gb166\|EG377283	569	91	92.5	blastp
324	LAB43_H1	wheat\|gb164\|AL822945	570	91	91.7	blastp
325	LAB43_H2	wheat\|gb164\|BE413988	571	91	93.2	blastp
326	LAB43_H3	wheat\|gb164\|CA610190	572	91	92.8	blastp
327	LAB45_H0	apple\|gb171\|CN488819	573	92	80	blastp
328	LAB45_H1	barley\|gb157.3\|AL502429	574	92	88.8	blastp
329	LAB45_H2	basilicum\|gb157.3\|DY328093	575	92	80.7	blastp
330	LAB45_H3	brachypodium\|gb169\|BE403542	576	92	90.3	blastp
331	LAB45_H4	cotton\|gb164\|AI727046	577	92	80.42	tblastn
332	LAB45_H5	fescue\|gb161\|DT697400	578	92	83.1	blastp
333	LAB45_H6	leymus\|gb166\|EG380210	579	92	87.9	blastp
334	LAB45_H7	maize\|gb170\|LLAI855293	580	92	96.4	blastp
335	LAB45_H8	medicago\|09v1\|AW690268	581	92	80.76	tblastn
336	LAB45_H9	poplar\|gb170\|BI124748	582	92	80.5	blastp
337	LAB45_H10	potato\|gb157.2\|BF053337	583	92	80	tblastn
338	LAB45_H11	rice\|gb170\|OS02G50350	584	92	92.4	blastp
339	LAB45_H12	switchgrass\|gb167\|FL865538	585	92	96.2	blastp
340	LAB45_H13	tomato\|gb164\|BG126074	586	92	80.19	tblastn
341	LAB45_H14	wheat\|gb164\|BE403542	587	92	88.8	blastp
342	LAB51_H0	barley\|gb157.3\|BE421767	588	95	85.2	blastp
343	LAB51_H1	wheat\|gb164\|CA615952	589	95	93.1	blastp
344	BDL103_H0	barley\|gb157.3\|BI954496	590	96	82.75	tblastn
345	BDL103_H1	barley\|gb157.3\|BI956043	591	96	83.53	tblastn
346	BDL103_H2	brachypodium\|gb169\|BE497565	592	96	85.1	blastp
347	BDL103_H3	leymus\|gb166\|EG378510	593	96	84.6	blastp
348	BDL103_H4	pseudoroegneria\|gb167\|FF346555	594	96	81.89	tblastn
349	BDL103_H5	wheat\|gb164\|BE497565	595	96	82.68	tblastn
350	BDL103_H6	wheat\|gb164\|BF428885	596	96	83.14	tblastn
351	BDL166_H0	b_rapa\|gb162\|CX267860	597	100	81.77	tblastn
352	BDL166_H1	canola\|gb161\|CD820129	598	100	90.4	blastp
353	CTF113_H0	castorbean\|09v1\|XM002524611	599	103	81	blastp
354	CTF113_H1	poplar\|gb170\|BI124993	600	103	80.6	blastp
355	CTF113_H2	poplar\|gb170\|CV228068	601	103	81.1	blastp
356	CTF180_H0	castorbean\|09v1\|EG657203	602	106	80.7	blastp
357	CTF215_H0	castorbean\|09v1\|XM002514996	603	108	81.2	blastp
358	CTF215_H1	poplar\|gb170\|AI162434	604	108	82.3	blastp

Table 6: Provided are polynucleotides and polypeptides which are homologous to the identified polynucleotides or polypeptides of Table 5. Homol. = homologue; Algor. = Algorithm; Polynucl. = polynucleotide; Polypep. = polypeptide. Homology was calculated as % of identity over the aligned sequences. The query sequences were polynucleotide sequences SEQ ID NOs: 1-59 and 638) or polypeptides sequences SEQ ID NOs: 60-112, and the subject sequences are protein sequences identified in the database based on greater than 80% identity to the predicted translated sequences of the query nucleotide sequences.

Example 3

Gene Cloning and Generation of Binary Vectors for Plant Expression

To validate their role in improving ABST, yield, growth rate, vigor, biomass, nitrogen use efficiency and/or oil content selected genes were over-expressed in plants, as follows.
Cloning Strategy
Genes listed in Examples 1 and 2 hereinabove were cloned into binary vectors for the generation of transgenic plants. For cloning, the full-length open reading frames (ORFs) were identified. EST clusters and in some cases mRNA sequences were analyzed to identify the entire open reading frame by comparing the results of several translation algorithms to known proteins from other plant species.
In order to clone the full-length cDNAs, reverse transcription (RT) followed by polymerase chain reaction (PCR; RT-PCR) was performed on total RNA extracted from leaves, roots or other plant tissues, growing under normal conditions. Total RNA extraction, production of cDNA and PCR amplification was performed using standard protocols described elsewhere (Sambrook J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, New York.) which are well known to those skilled in the art. PCR products were purified using PCR purification kit (Qiagen)
Usually, 2 sets of primers were prepared for the amplification of each gene, via nested PCR (meaning first amplifying the gene using external primers and then using the produced PCR product as a template for a second PCR reaction, where the internal set of primers are used). Alternatively, one or two of the internal primers were used for gene amplification, both in the first and the second PCR reactions (meaning only 2-3 primers were designed for a gene). To facilitate further cloning of the cDNAs, an 8-12 bp extension was added to the 5′ of each internal primer. The primer extension includes an endonuclease restriction site. The restriction sites were selected using two parameters: (a) the restriction site does not exist in the cDNA sequence; and (b) the restriction sites in the forward and reverse primers are designed such that the digested cDNA is inserted in the sense direction into the binary vector utilized for transformation. In Table 7 below, primers used for cloning selected genes are provided.

TABLE 7

PCR primers for cloning
selected genes of the invention

	Restriction
Gene	Enzymes used	Primers used for
Name	for cloning	amplification (SEQ ID NOs:)

BDL103S	SalI, XbaI	BDL103_Short_F_SalI SEQ ID NO: 677
		AATGTCGACTCTGGGCTCAGGGATAGG
		BDL103_NR_XbaI SEQ ID NO: 678
		TATCTAGACTACTAAAAGGAATTATCTAGCAGAGG

BDL12	SalI, SacI	BDL12_gDNA_NF_SalI SEQ ID NO: 679
		AATGTCGACGTTCTATCCCCAACTCTAAATG
		BDL12_gDNA_NR_SacI SEQ ID NO: 680
		AGAGCTCCTTAAAGTTCTATCGAGATAGTGC

BDL14	SalI, XbaI	BDL14_ORF_F1_SalI SEQ ID NO: 681
		AATGTCGACAACAATGGATCTACAACAGTCCGAAAC
		BDL14_ORF_F1_SalI SEQ ID NO: 681
		AATGTCGACAACAATGGATCTACAACAGTCCGAAAC
		BDL14_ORF_NR_XbaI SEQ ID NO: 682
		AATCTAGACACTCAGACAGCTGGGTATTAAAC
		BDL14_ORF_ER_SacI SEQ ID NO: 683
		AGAGCTCGTTGTGGCACTCAGACAGCTG

BDL166	XbaI, SacI	BDL166_NF_XbaI SEQ ID NO: 684
		AATCTAGAAAAGTTACACCTTACTAAACACAAAC
		BDL166_NR_SacI SEQ ID NO: 685
		TGAGCTCTCTTGTTGATAGTCTTCATAATCG

BDL210	SalI, XbaI	BDL210_NF_SalI SEQ ID NO: 686
		AAAGTCGACAACAAAGTTATGGGTTTCTCG
		BDL210_EF_SalI SEQ ID NO: 687
		AAAGTCGACGAGCAACAAAGTTATGGGTTTC
		BDL210_NR_XbaI SEQ ID NO: 688
		ATTCTAGATTAGGATGATCAGGAGATGAGAGAG
		BDL210_ER_XbaI SEQ ID NO: 689
		ATTCTAGACTAAAGTAGAGAGATGGATGATCAGG

CTF113		CTF113_ORF_F_Sm SEQ ID NO: 690
		GACCCGGGAAACGATGGAGGATCTTGCC
		CTF113_ORF_R_Sc SEQ ID NO: 691
		CAGAGCTCTTGGAATTGAAATGTCATTACAGAG

CTF163	SalI, XbaI	CTF163_NF_SalI SEQ ID NO: 692
		AAAGTCGACGAACTGGTTGTTCTTGGCTATG
		CTF163_NR_XbaI SEQ ID NO: 693
		ATTCTAGACCAGATGAACTTGGCTTTATC

CTF175	EcoRV, SacI	CTF175_ORF_NF_EcRV SEQ ID NO: 694
		AGGATATCTTTCGATCACCGTGATGGC
		CTF175_ORF_EF_EcRV SEQ ID NO: 695
		AAGATATCAGAGCATTTCGATCACCGTG
		CTF175_ORF_NR_Sc SEQ ID NO: 696
		GCGAGCTCGTAGTGACGTCACCGGTTC
		CTF175_ORF_ER_Sc SEQ ID NO: 697
		TCGAGCTCCTCACCTTTCACTATCACCC

CTF180	SalI, SacI	CTF180_NF_SalI SEQ ID NO: 698
		AAAGTCGACTTCGATGTGGGATAACTGAATC
		CTF180_ER_SacI SEQ ID NO: 699
		AACGAGCTCATTCAACAACCTAACCATCTTTG
		CTF180_NR_SacI SEQ ID NO: 700
		AATGAGCTCTTTTCTTTACAGTGGAATCTGC
		CTF180_ER_SacI SEQ ID NO: 699
		AACGAGCTCATTCAACAACCTAACCATCTTTG

CTF205		CTF205_EF_SalI SEQ ID NO: 701
		AAAGTCGACGAAAACACAGATGGAAGATATTAAAC
		CTF205_ER_XbaI SEQ ID NO: 702
		ATTCTAGATGGACTTACAGGTCAAGAAGGTAG

CTF215	SalI, XbaI	CTF215_NF_SalI SEQ ID NO: 703
		AAAGTCGACAAGTTTGGAAAGAGATGAATCC
		CTF215_NR_XbaI SEQ ID NO: 704
		ATTCTAGACTAAGCAAGCAGAAACAAAATATAGC

CTF226	SalI, XbaI	CTF226_NF_SalI SEQ ID NO: 705
		AAAGTCGACGCCAAGGTCAAACGAAGG
		CTF226_EF_SalI SEQ ID NO: 706
		AAAGTCGACCAAAAGCCAAGGTCAAACG
		CTF226_NR_XbaI SEQ ID NO: 707
		ATTCTAGACTAAACTTATGCAACATGAGCTGG
		CTF226_ER_XbaI SEQ ID NO: 708
		ACTCTAGAAAGTCATTATCCTAGTTCAGTTTGC

LAB11	SalI, XbaI	LAB11_NF_SalI SEQ ID NO: 709
		AAAGTCGACATCTACTGCCTTTGACCGATG
		LAB11_NR_XbaI SEQ ID NO: 710
		AATTCTAGATTACAGTTAAGTGAGGACATTCTTGG

LAB13	SalI, XbaI	LAB13_NF_SalI SEQ ID NO: 711
		AAAGTCGACCCCAAGATCGATATAAATTTCC
		LAB13_NR_XbaI SEQ ID NO: 712
		AACTCTAGAAACCACCATGCTTGCTCATC

LAB14	EcoRV, EcoRV	LAB14_NF_EcoRV SEQ ID NO: 713
		AATGATATCTTCCATTGTTACACGCGTTC
		LAB14_NR_EcoRV SEQ ID NO: 714
		AATGATATCTTAGGTGATTTAAAGCCAGAGGG

LAB16	SalI, XbaI	LAB16_NF_SalI SEQ ID NO: 715
		AAAGTCGACAACCAGACAAGAGAGAAAACAAG
		LAB16_NR_XbaI SEQ ID NO: 716
		AATTCTAGATTACAATCACATAACAGAACAAGCAG

LAB17	EcoRV, PstI	LAB17_NF_EcoRV SEQ ID NO: 717
		AATGATATCTTGTTTCGTTTTCCCTTAGC
		LAB17_NR_PstI SEQ ID NO: 718
		AATCTGCAGTCACCAGTTCACCACCATCTAC

LAB2	EcoRV, PstI	LAB2_NF_EcoRV SEQ ID NO: 719
		AATGATATCTTGCCGGTCGATCTTGAG
		LAB2_EF_EcoRV SEQ ID NO: 720
		AATGATATCCCTATATCTCCCTCCTCCTCC

LAB2		LAB2_NR_PstI SEQ ID NO: 721
		AATCTGCAGTCAGCCACGGACTACCTACATGAC
		LAB2_ER_PstI SEQ ID NO: 722
		AACCTGCAGACAATTTCATTCTGTGGGTTC

LAB20	SmaI	LAB2O_NF SEQ ID NO: 723
		CCTCAGAAAATCACCGTACG
		LAB2O_NR_SmaI SEQ ID NO: 724
		TAACCCGGGCCTATGAACAGATATCTGACATGATC

LAB21	SalI, XbaI	LAB21_NF_SalI SEQ ID NO: 725
		TTAGTCGACGGAGAGAGATCTTCTAGCTACATAC
		LAB21_NR_XbaI SEQ ID NO: 726
		TAATCTAGATCACAGGACAGGACACCATCAAC

LAB22	SalI, XbaI	LAB22_NF_SalI SEQ ID NO: 727
		TTAGTCGACGGAGACAAAGATGGAGAACAAC
		LAB22_NR_XbaI SEQ ID NO: 728
		TATTCTAGACCGAAATTAAACAACAAGTACAC

LAB23	EcoRV, EcoRV	LAB23_NF_EcoRV SEQ ID NO: 729
		AAAGATATCGGAGGTACATATAGCTAGCGAAG
		LAB23_NR_EcoRV SEQ ID NO: 730
		AATGATATCCTAACAAAATCCACGACTCCACTG

LAB24	SalI, XbaI	LAB24_NF_SalI SEQ ID NO: 731
		AAAGTCGACGAGAGAGGATGGTGAGCAGC
		LAB24_NR_XbaI SEQ ID NO: 732
		AATTCTAGATTACGTGTAGTCATCAAATCACGC

LAB25	SalI, XbaI	LAB25_NF_SalI SEQ ID NO: 733
		AATGTCGACTCTAGCTCCCACGAGTCTTTAG
		LAB25_NR_XbaI SEQ ID NO: 734
		AATTCTAGATTACAACAATTTAATGGAGGTCCG

LAB3	SalI, XbaI	LAB3_NF_SalI SEQ ID NO: 735
		TTAGTCGACGAGCAAAAAATGAAGGAGAAC
		LAB3_NR_XbaI SEQ ID NO: 736
		TATTCTAGATTACAGAGATTGTTAAGGTTGGACC

LAB31	EcoRV, PstI	LAB31 NF EcoRV SEQ ID NO: 737
		AAAGATATCTCACAATTTCATTCACAAGTCG
		LAB31 NR PstI SEQ ID NO: 738
		AATCTGCAGTTTTCAAATCCAAACCCAAC

LAB32	SalI, XbaI	LAB32_NF_SalI SEQ ID NO: 739
		AAAGTCGACCTTTCCTTTCCTTTCCATCC
		LAB32_EF_SalI SEQ ID NO: 740
		AATTCTAGAAGCCATCACCACGCATTAC
		LAB32_NR_XbaI SEQ ID NO: 741
		AATTCTAGAAGCACTGAGCAGCCTTCATC
		LAB32_ER_XbaI SEQ ID NO: 740
		AATTCTAGAAGCCATCACCACGCATTAC

LAB33	EcoRV, EcoRV	LAB33 NF EcoRV SEQ ID NO: 742
		TCAGATATCCATCGCATCGCATCCATC
		LAB33 NR EcoRV SEQ ID NO: 743
		ATAGATATCGCTGCCTGCTTCTGATCTG

LAB34	SalI, XbaI	LAB34_NF_SalI SEQ ID NO: 744
		AAAGTCGACGCTAGTGAGATACCATGGACAAC
		LAB34_NR_XbaI SEQ ID NO: 745
		AAATCTAGATTACTTCTATGCTGGAATGACTTTG

LAB35		LAB35_NF_SalI SEQ ID NO: 746
		AAAGTCGACCAGATCGCGATGAAGTCTTG
		LAB35_EF_SalI SEQ ID NO: 747
		AAAGTCGACAGGGGAGAAGAGAGAGAGACAG
		LAB35_NR_XbaI SEQ ID NO: 748
		AAATCTAGATTAGCTCGTTCATTTAGCCTCAG
		LAB35 ER Xba SEQ ID NO: 749
		TCCTCTAGAGAGTTTATTCCTCGACGATGC

LAB36		LAB36_NF_SalI SEQ ID NO: 750
		AAAGTCGACCAGTGTAGAGCAAGAGGTGTGG
		LAB36_EF_SalI SEQ ID NO: 751
		AAAGTCGACTCGTCTCGATCAGTGTAGAGC
		LAB36_NR_XbaI SEQ ID NO: 752
		AAATCTAGATTACGTCGTTCATTTAGCCTTTG
		LAB36_ER_XbaI SEQ ID NO: 753
		AATTCTAGACAATTATTCCACAGGACATCAC

LAB38	EcoRV	LAB38_NF_EcoRV SEQ ID NO: 754
		AAAGATATCAGGAGATATGGCCCAGAGG
		LAB38_EF_EcoRV SEQ ID NO: 755
		TTAGATATCCTGCTTGCAATACTTAGTAGAGG
		LAB38_NR_EcoRV SEQ ID NO: 756
		AAAGATATCTTAACGTACTCTCAGGTGAGGCG
		LAB38_ER_EcoRV SEQ ID NO: 757
		TAAGATATCTTTATTTATTCACCGGAGCAAC

LAB39	SalI, XbaI	LAB39_NF_SalI SEQ ID NO: 758
		AAAGTCGACCAAAATAGCAGAGATGGGAGG
		LAB39_NR_XbaI SEQ ID NO: 759
		AAATCTAGATCACGGTAATCAGTTCAGCATGG

LAB40	SalI, XbaI	LAB40_NF_SalI SEQ ID NO: 760
		AAAGTCGACACACTACCAACATGGAAACATAC
		LAB40_EF_SalI SEQ ID NO: 761
		AAAGTCGACGCTGAATCGGCACACACTAC
		LAB40_NR_XbaI SEQ ID NO: 762
		AATTCTAGATGACCATCATCAGTTCATTGC
		LAB40_ER_XbaI SEQ ID NO: 763
		AATTCTAGAGGAGTGAGGACTTTACAAAATG

LAB41	SalI, XbaI	LAB41_NF_SalI SEQ ID NO: 764
		AAAGTCGACAAGAGCTGCGAGAGGAAGG
		LAB41_NR_XbaI SEQ ID NO: 765
		AAATCTAGATTAACATCAATTGTCAGTCATCGG

LAB45	SalI, StuI	LAB45_NF_SalI SEQ ID NO: 766
		AAAGTCGACATTCTTATCAAAACAGAGGAACC
		LAB45_EF_SalI SEQ ID NO: 767
		AAAGTCGACCTCCCTCAGATTCTTATCAAAAC
		LAB45_NR_XbaI SEQ ID NO: 768
		AAATCTAGATTAGCATCAGTTGGATACCATG
		LAB45_ER_XbaI SEQ ID NO: 769
		AAATCTAGATTAAGTCACAAGTTGAAGCATGGTG

LAB49	EcoRV, EcoRV	LAB49_NF_EcoRV SEQ ID NO: 770
		AAAGATATCACGATCAGCCATGAAGAGC
		LAB49_NR_EcoRV SEQ ID NO: 771
		AAAGATATCTTATTAAGCTGGCTGGTTGTGAC

LABS		LAB5_EF_EcoRV SEQ ID NO: 772
		AAAGATATCCTCTTCCACAATCCACATTCC
		LAB5_ER_PstI SEQ ID NO: 773
		AATCTGCAGTGACGATCCATCTATGAACAAC

LAB50		LAB5O_NF_SalI SEQ ID NO: 774
		AAAGTCGACCACGGAGAAAAGAAAGATCG
		LAB5O_NR_XbaI SEQ ID NO: 775
		AAATCTAGATTAAAACTCCGGCTGCTAGACC

LAB51	SalI, XbaI	LAB51_NF_SalI SEQ ID NO: 776
		AAAGTCGACAGTACTTCGGTTGATGGCTTC
		LAB51_EF_SalI SEQ ID NO: 777
		AAAGTCGACCTCTGCTCGTCTCTGCATTTAG

LAB51		LAB51_NR_XbaI SEQ ID NO: 778
		AAATCTAGATTAAACACTTATGTATGCACGCTTAG
		LAB51_ER_XbaI SEQ ID NO: 779
		AAATCTAGATTATCCACACCAAGACCAAGACAG

Provided are primers and the restriction sites and enzymes used for cloning selected genes (polynucleotides, provided by gene name) identified herein.

TABLE 8

Restriction enzymes and cloning vectors used to clone selected genes of the invention

		Restriction enzymes	Restriction enzymes
		used for cloning into	used for cloning into	Restriction enzymes
Gene		binary vector-	binary vector-	used for digesting
name	Binary vector	FORWARD	REVERSE	the binary vector

BDL103	pBXYN (pGI_35S)	XbaI	Sac I	XbaI, Sac I
BDL103	pBXYN (pGI_35S)	SalI	EcoR I	SalI, EcoR I
BDL11	pM (pMBLArt)	NotI	Not I	NotI, Not I
BDL12	pBXYN (pGI_35S)	HindIII	EcoR I	HindIII, EcoR I
BDL14	pBXYN (pGI_35S)	HindIII	EcoRI	HindIII, EcoRI
BDL166	pQXYN	XbaI	EcoRI	XbaI, EcoRI
BDL17	pM (pMBLArt)	NotI	Not I	NotI, Not I
BDL17S	pM (pMBLArt)	NotI	Not I	NotI, Not I
BDL210	pQXYN	SalI	EcoRI	SalI, EcoRI
CTF113	pBXYN (pGI_35S)	SmaI	Sac I	SmaI, SacI
CTF163	pQXYN	SalI	SacI	SalI, SacI
CTF175	pBXYN (pGI_35S)	EcoRV	Sac I	SmaI, SacI
CTF180	pQXYN	SalI	EcoRI	SalI, EcoRI
CTF205	pQXYN	SalI	SacI	SalI, SacI
CTF215	pQXYN	SalI	SacI	SalI, SacI
CTF226	pQXYN	SalI	SacI	SalI, SacI
LAB11	pQFN	SalI	EcoRI	SalI, EcoRI
LAB13	pQFN	SalI	EcoRI	SalI, EcoRI
LAB14	pQYN	BamHI	SmaI	BamHI, Ecl136II
LAB15	pQFN	SalI	Ecl136II	SalI, StuI
LAB16	pQFN	SalI	EcoRI	SalI, EcoRI
LAB17	pQFN	EcoRV	SmaI	StuI, StuI
LAB18	pQFN	SalI	Ecl136II	SalI, StuI
LAB2	pQFN	EcoRV	SmaI	StuI, StuI
LAB20	pQYN	HindIII	SmaI	HindIII, Ecl136II
LAB21	pQFN	SalI	EcoRI	SalI, EcoRI
LAB22	pQFN	SalI	XbaI	SalI, XbaI
LAB23	pQFN	EcoRV	EcoRV	StuI, StuI
LAB24	pQYN_6669	SalI	EcoRI	SalI, EcoRI
LAB25	pQFN	SalI	EcoRI	SalI, EcoRI
LAB3	pQFN	SalI	EcoRI	SalI, EcoRI
LAB31	pQYN	BamHI	SmaI	BamHI, Ecl136II
LAB32	pQFN	SalI	EcoRI	SalI, EcoRI
LAB33	pQFN	EcoRV	EcoRV	StuI, StuI
LAB34	pQFN	SalI	EcoRI	SalI, EcoRI
LAB35	pQFN	SalI	Ecl136II	SalI, StuI
LAB36	pQFN	SalI	EcoRV	SalI, StuI
LAB38	pQYN	BamHI	SmaI	BamHI, Ecl136II
LAB39	pQFN	SalI	EcoRI	SalI, EcoRI
LAB4	pQFN	EcoRV	EcoRV	SmaI, SmaI
LAB40	pQFN	SalI	EcoRI	SalI, EcoRI
LAB41	pQYN_6669	SalI	EcoRI	SalI, EcoRI
LAB45	pQFN	SalI	BamHI	SalI, BamHI
LAB49	pQFN	EcoRV	Ecl136II	StuI, StuI
LAB5	pQFN	EcoRV	KpnI	StuI, KpnI
LAB50	pQFN	SalI	BamHI	SalI, BamHI
LAB51	pQYN_6669	SalI	EcoRI	SalI, EcoRI
LAB8	pQFN	BamHI	XhoI	BamHI, XhoI
LAB9	pQFN	BamHI	KpnI	BamHI, KpnI

Table 8: Provided are the restriction enzymes and cloning vectors used for cloning selected genes of the invention.

TABLE 9

Primers used for colony screening of the binary plasmid

	Colony Screening FP	FP SEQ	Colony Screening RP	RP SEQ
Gene Name	Name	ID NO:	Name	ID NO:

BDL103_Long	35S_1F	780	NOS R	784
BDL103_Short	BDL103_Short_F_SalI	677	101_ER	785
BDL11_GA	35S_1F	780	101_R	786
BDL12	101 EF	781	BDL12_gDNA_NR_SacI	680
BDL14	BDL14_ORF_F1_SalI	681	101_R	786
BDL166	35S_1F	780	BDL166_NR_SacI	685
BDL17	35S_1F	780	101_R	786
BDL17	101-F	782	BDL17_GA_R	787
BDL210	35S_1F	780	BDL210_NR_XbaI	688
CTF113	35S_1F	780	NOS R	784
CTF163	35S_1F	780	CTF163_NR_XbaI	693
CTF175	35S_1F	780	NOS R	784
CTF180	35S_1F	780	CTF180_NR_SacI	700
CTF205	35S_1F	780	CTF205_ER_XbaI	702
CTF215	35S_1F	780	CTF215_NR_XbaI	704
CTF226	35S_1F	780	CTF226_NR_XbaI	707
LAB11	6669 F	783	LAB11_NR_XbaI	710
LAB13	6669 F	783	101_R	786
LAB14	p6669-F	783	101_ER	785
LAB15	p6669-F	783	101_ER	785
LAB16	6669 F	783	LAB16_NR_XbaI	716
LAB17	p6669-F	783	LAB17_NR_PstI	718
LAB18	p6669-F	783	101_ER	785
LAB2	p6669-F	783	LAB2_NR_PstI	721
LAB20	101-F	782	LAB20_NR_SmaI	724
LAB21	6669 F	783	LAB21_NR_XbaI	726
LAB22	6669 F	783	LAB22_NR_XbaI	728
LAB23	p6669-F	783	LAB14_NR_EcoRV	714
LAB24	6669 F	783	LAB24_NR_XbaI	732
LAB25	6669 F	783	LAB25_NR_XbaI	734
LAB3	6669 F	783	LAB3_NR_XbaI	736
LAB31	LAB31 NF EcoRV	737	101 EF	781
LAB32	6669 F	783	LAB32_NR_XbaI	741
LAB33	p6669-F	783	LAB33_R1_seq	788
LAB34	6669 F	783	LAB34_NR_XbaI	745
LAB35	6669 F	783	LAB35_NR_XbaI	748
LAB36	6669 F	783	LAB36_NR_XbaI	752
LAB38	LAB38_NF_EcoRV	754	101 EF	781
LAB39	6669 F	783	LAB39_NR_XbaI	759
LAB4	6669 F	783	LAB4_R_GA	789
LAB40	6669 F	783	LAB40_NR_XbaI	762
LAB41	6669 F	783	LAB41_NR_XbaI	765
LAB45	6669 F	783	LAB45_NR_XbaI	790
LAB49	6669 F	783	LAB49_NR_EcoRV	771
LAB5	p6669-F	783	101_ER	785
LAB50	6669 F	783	LAB50_NR_XbaI	775
LAB51	6669 F	783	LAB51_NR_XbaI	778
LAB8	6669 F	783	LAB8_GA_rev	791
LAB9	6669 F	783	LAB9_GA_rev	792

Table 9. Provided are the forward primers (FP) and reverse primers (RP) along with their sequence identifiers used for screening of colonies harboring the cloned genes of some embodiments of the invention.

TABLE 10

Cloned genes from cDNA libraries or genomic DNA and the polypeptides encoded thereby

Polynuc.

Polypep.

Gene

High copy

Amplified from

SEQ ID

Name	plasmid	Organism	Origin	NO:	NO:

BDL103_Long			GeneArt	670	96
BDL103_Short	pGXN	RICE Oryza sativa	cDNA-	671	672
	(pKG + Nos + 35S)	L. Japonica ND	RICE
BDL11	pGXN		GeneArt	639	661
	(pKG + Nos + 35S)
BDL12	pGXN	ARABIDOPSIS	gDNA	640	662
	(pKG + Nos + 35S)	Arabidopsis thaliana ND
BDL14	pGXN	ARABIDOPSIS	cDNA	641	99
	(pKG + Nos + 35S)	Arabidopsis thaliana ND
BDL166	pGXN	ARABIDOPSIS	cDNA	642	100
	(pKG + Nos + 35S)	Arabidopsis thaliana ND
BDL17	pGXN		GeneArt	643	101
	(pKG + Nos + 35S)
BDL17	pGN_Napin		GeneArt	643	101
BDL210	pGXN	ARABIDOPSIS	cDNA	644	102
	(pKG + Nos + 35S)	Arabidopsis thaliana ND
CTF113	pKS(Pks_J)	Cotton	cDNA	645	663
CTF163	pGXN	COTTON	cDNA	646	664
	(pKG + Nos + 35S)	Gossypium barbadense ND
CTF175	pKS(Pks_J)	Cotton	cDNA	647	665
CTF180	pGXN	COTTON	cDNA	648	666
	(pKG + Nos + 35S)	Gossypium barbadense ND
CTF205	pGXN	COTTON	cDNA	649	667
	(pKG + Nos + 35S)	Gossypium barbadense ND
CTF215	pGXN	COTTON	cDNA	650	668
	(pKG + Nos + 35S)	Gossypium barbadense ND
CTF226	pGXN	COTTON	cDNA	651	669
	(pKG + Nos + 35S)	Gossypium barbadense ND
LAB11	pGXN	RICE Oryza sativa	cDNA	609	65
	(pKG + Nos + 35S)	L. Japonica ND
LAB13	pGXN	RICE Oryza sativa	cDNA	610	66
	(pKG + Nos + 35S)	L. Japonica ND
LAB14	pKSJ_6669a	RICE Oryza sativa	cDNA	611	67
		L. Japonica ND
LAB15			Gene Art	612	68
LAB16	pGXN	COTTON	cDNA	614	70
	(pKG + Nos + 35S)	Gossypium barbadense ND
LAB17	pKSJ_6669a	SORGHUM	cDNA	615	71
		Sorghum bicolor
		Monsanto S5
LAB18			GeneArt	616	72
LAB2	pKS(Pks_J)	BARLEY	cDNA	613	69
		Hordeum vulgare L. ND
LAB20	pUC19_pr6669	RICE Oryza sativa	cDNA	617	73
		L. Japonica ND
LAB21	pGXN	BARLEY	cDNA	618	653
	(pKG + Nos + 35S)	Hordeum vulgare L. ND
LAB22	pGXN	SORGHUM	cDNA	619	75
	(pKG + Nos + 35S)	Sorghum bicolor
		Monsanto S5
LAB23	pKSJ_6669a	BARLEY	cDNA	621	77
		Hordeum vulgare L. ND
LAB24	pGXN	SORGHUM	cDNA	622	655
	(pKG + Nos + 35S)	Sorghum bicolor
		Monsanto S5
LAB25	pGXN	BARLEY	cDNA	623	656
	(pKG + Nos + 35S)	Hordeum vulgare L. ND
LAB3	pGXN	COTTON	cDNA	620	654
	(pKG + Nos + 35S)	Gossypium hirsutum Akala
LAB31	pKSJ_6669a	COTTON	cDNA	624	81
		Gossypium hirsutum Akala
LAB32	pGXN	BARLEY	cDNA	625	82
	(pKG + Nos + 35S)	Hordeum vulgare L. ND
LAB33	pKS(Pks_J)	SORGHUM	cDNA	626	83
		Sorghum bicolor
		Monsanto S5
LAB34	pGXN	SOYBEAN	cDNA	627	657
	(pKG + Nos + 35S)	Glycine max ND
LAB35	Topo B	WHEAT	cDNA	628	658
		Triticum aestivum L. ND
LAB36	Topo B	SORGHUM	cDNA	629	86
		Sorghum bicolor
		Monsanto S5
LAB38	pKSJ_6669a	WHEAT	cDNA	630	87
		Triticum aestivum L. ND
LAB39	pGXN	SORGHUM	cDNA	631	659
	(pKG + Nos + 35S)	Sorghum bicolor
		Monsanto S5
LAB4			GeneArt	605	60
LAB40	pGXN	SORGHUM	cDNA	632	660
	(pKG + Nos + 35S)	Sorghum bicolor
		Monsanto S5
LAB41	pGXN	WHEAT	cDNA	633	90
	(pKG + Nos + 35S)	Triticum aestivum L. ND
LAB45	TopoB_LAB45	SORGHUM	cDNA + part	634	92
		Sorghum bicolor	from GA
		Monsanto S5
LAB49	pKSJ_6669a	RICE Oryza sativa	cDNA	635	93
		L. Japonica ND
LAB5	Topo B	SORGHUM	cDNA	606	652
		Sorghum bicolor
		Monsanto S5
LAB50	Topo B	RICE Oryza sativa	cDNA	636	94
		L. Japonica ND
LAB51	pGXN	WHEAT	cDNA	637	95
	(pKG + Nos + 35S)	Triticum aestivum L. ND
LAB8			GeneArt	607	63
LAB9			GeneArt	608	64

Table 10. Provided are the cloned and synthetic genes, the polypeptides encoded thereby along with their sequence identifiers. Also provided are the source of DNA used for cloning (cDNA or genomic DNA) and the organism from which the gene was cloned. Polynuc. = polynucleotide; Polypep. = polypeptide. BDL103 short was amplified from pGXN_BDL103. pGXN_BDL103 was amplified from cDNA- RICE Oryza sativa L. Japonica ND. LAB45 was composed of a part cloned from cDNA with the primers indicated and a part ordered from GA

PCR products were digested with the restriction endonucleases (Roche, Switzerland) according to the sites design in the primers (Table 7). Each digested PCR product was inserted into a high copy vector originated from pBlue-script KS plasmid vector (pBlue-script KS plasmid vector, Hypertext Transfer Protocol://World Wide Web (dot) stratagene (dot) com/manuals/212205 (dot) pdf) or pUC19 (New England BioLabs Inc). In case of the high copy vector originated from pBlue-script KS plasmid vector (pGXN) the PCR product was inserted in the high copy plasmid upstream to the NOS terminator (SEQ ID NO:673) originated from pBI 101.3 binary vector (GenBank Accession No. U12640, nucleotides 4417 to 4693) and down stream to the 35S promoter (SEQ ID NO:675). In other cases (pKSJ_—6669a or pUC19_pr6669) the At6669 promoter (SEQ ID NO:674) was already cloned into the pBlue-script KS or pUC19 respectively, so the gene was introduced downstream of the promoter.
Sequencing of the inserted genes was performed, using the ABI 377 sequencer (Applied Biosystems). In all the cases, after confirming the sequences of the cloned genes, the cloned cDNA accompanied with the NOS terminator was introduced into a modified pGI binary vector containing the At6669 promoter via digestion with appropriate restriction endonucleases. In other cases the cloned cDNA accompanied with the At6669 promoter was introduced into a pGI vector (that hasn't already contained the At6669 promoter). In any case the insert was followed by single copy of the NOS terminator (SEQ ID NO: 673). Part of the genes were introduced into a binary vector pGI containing the 35S promoter. The digested products and the linearized plasmid vector were ligated using T4 DNA ligase enzyme (Roche, Switzerland).
Several DNA sequences of the selected genes were synthesized by GeneArt (Hypertext Transfer Protocol://World Wide Web (dot) geneart (dot) com/). Synthetic DNA is designed in silico. Suitable restriction enzymes sites are added to the cloned sequences at the 5′ end and at the 3′ end to enable later cloning into the desired binary vector.
The pPI plasmid vector is constructed by inserting a synthetic poly-(A) signal sequence, originating from pGL3 basic plasmid vector (Promega, GenBank Accession No. U47295; nucleotides 4658-4811) into the HindIII restriction site of the binary vector pBI101.3 (Clontech, GenBank Accession No. U12640). pGI (FIG. 1) is similar to pPI, but the original gene in the back bone is GUS-Intron, rather than GUS.
The modified pGI vector (FIG. 2) is a modified version of the pGI vector in which the cassette is inverted between the left and right borders so the gene and its corresponding promoter are close to the right border and the NPTII gene is close to the left border.
At6669, the Arabidopsis thaliana promoter sequence (set forth in SEQ ID NO: 674) was inserted in the pGI binary vector, upstream to the cloned genes, followed by DNA ligation and binary plasmid extraction from positive E. coli colonies, as described above. Colonies were analyzed by PCR using the primers covering the insert which were designed to span the introduced promoter and gene. Positive plasmids were identified, isolated and sequenced as described above.
Some genes were cloned downstream of the Napin promoter (SEQ ID NO:676) and upstream to the NOS terminator in the pMBLArt vector. The vector displays resistance to Basta.
Promoters used: Arabidopsis At6669 promoter (SEQ ID NO: 674; which is SEQ ID NO: 61 of WO04081173), Napin (SEQ ID NO: 676) and 35S (SEQ ID NO: 675).

Example 4

Transforming Agrobacterium Tumefaciens Cells with Binary Vectors Harboring Putative Genes

Each of the binary vectors described in Example 3 above were used to transform Agrobacterium cells. Two additional binary constructs, having a GUS/Luciferase reporter gene replacing the selected gene (positioned downstream of the At6669 promoter), were used as negative controls.
The binary vectors were introduced to Agrobacterium tumefaciens GV301, or LB4404 competent cells (about 10⁹cells/mL) by electroporation. The electroporation was performed using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and EC-2 electroporation program (Biorad). The treated cells were cultured in LB liquid medium at 28° C. for 3 hours, then plated over LB agar supplemented with gentamycin (50 mg/L; for Agrobacterium strains GV301) or streptomycin (300 mg/L; for Agrobacterium strain LB4404) and kanamycin (50 mg/L) at 28° C. for 48 hours. Agrobacterium colonies developed on the selective media were analyzed by PCR using the primers which are designed to span the inserted sequence in the pPI plasmid. The resulting PCR products were isolated and sequenced as described in Example 3 above, to verify that the correct polynucleotide sequences were properly introduced to the Agrobacterium cells.

Example 5

Transformation of Arabidopsis Thaliana Plants with the Identified Polynucleotides of the Invention

Arabidopsis thaliana Columbia plants (T0 plants) were transformed according to the Floral Dip procedure [Clough S J, Bent A F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6): 735-43] and Desfeux C, Clough S J, Bent A F. (2000) [Female reproductive tissues are the primary targets of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol. 123(3): 895-904] with minor modifications. Briefly, T₀Plants were sown in 250 ml pots filled with wet peat-based growth mix. The pots were 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 18-24° C. under 16/8 hour light/dark cycles. The T₀plants were ready for transformation six days before anthesis.
Single colonies of Agrobacterium carrying the binary constructs were generated as described in Example 4 above. Colonies were cultured in LB medium supplemented with kanamycin (50 mg/L) and gentamycin (50 mg/L). The cultures were incubated at 28° C. for 48 hours under vigorous shaking and then centrifuged at 4000 rpm for 5 minutes. The pellets comprising the Agrobacterium cells were re-suspended in a transformation medium containing half-strength (2.15 g/L) Murashige-Skoog (Duchefa); 0.044 μM benzylamino purine (Sigma); 112 μg/L B5 Gambourg vitamins (Sigma); 5% sucrose; and 0.2 ml/L Silwet L-77 (OSI Specialists, CT) in double-distilled water, at pH of 5.7.
Transformation of T₀plants was performed by inverting each plant into an Agrobacterium suspension, such that the above ground plant tissue was submerged for 3-5 seconds. Each inoculated T₀plant was immediately placed in a plastic tray, then covered with clear plastic dome to maintain humidity and was kept in the dark at room temperature for 18 hours, to facilitate infection and transformation. Transformed (transgenic) plants were then uncovered and transferred to a greenhouse for recovery and maturation. The transgenic T₀plants were grown in the greenhouse for 3-5 weeks until siliques were brown and dry. Seeds were harvested from plants and kept at room temperature until sowing.
For generating T₁and T₂transgenic plants harboring the genes, seeds collected from transgenic T₀plants were surface-sterilized by soaking in 70% ethanol for 1 minute, followed by soaking in 5% sodium hypochloride and 0.05% triton for 5 minutes. The surface-sterilized seeds were thoroughly washed in sterile distilled water then placed on culture plates containing half-strength Murashige-Skoog (Duchefa); 2% sucrose; 0.8% plant agar; 50 mM kanamycin; and 200 mM carbenicylin (Duchefa). The culture plates were incubated at 4° C. for 48 hours then transferred to a growth room at 25° C. for an additional week of incubation. Vital T₁ Arabidopsis plants were transferred to a fresh culture plates for another week of incubation. Following incubation the T₁plants were removed from culture plates and planted in growth mix contained in 250 ml pots. The transgenic plants were allowed to grow in a greenhouse to maturity. Seeds harvested from T₁plants were cultured and grown to maturity as T₂plants under the same conditions as used for culturing and growing the T₁plants.

Example 6

Transgenic Plants Overexpressing the Polynucleotides of Some Embodiments of the Invention Exhibit Increased ABST, NUE, Biomass and/or Growth Rate

Plants transgenic to the polynucleotides of some embodiments of the invention were assayed for fertilizer use efficiency in a tissue culture assay.
Assay 1: Plant Growth at Nitrogen Deficiency Under Tissue Culture Conditions—
The present inventors have found the nitrogen use efficiency (NUE) assay to be relevant for the evaluation of the ABST candidate genes, since nitrogen limiting conditions encourage root elongation, increases root coverage and allows detecting the potential of the plant to generate a better root system under drought conditions. In addition, there are indications in the literature that biological mechanisms of NUE and drought tolerance are linked (Wesley et al., 2002 Journal of Experiment Botany Vol 53, No. 366, pp. 13-25).
Surface sterilized seeds were sown in basal media [50% Murashige-Skoog medium (MS) supplemented with 0.8% plant agar as solidifying agent] in the presence of Kanamycin (for selecting only transgenic plants). After sowing, plates were transferred for 2-3 days for stratification at 4° C. and then grown at 25° C. under 12-hour light 12-hour dark daily cycles for 7 to 10 days. At this time point, seedlings randomly chosen were carefully transferred to plates with nitrogen-limiting conditions: 0.5 MS media in which the combined nitrogen concentration (NH₄NO₃and KNO₃) is 0.75 mM (nitrogen deficient conditions) or 15 mM [Normal (optimal) nitrogen concentration]. Each plate contains 5 seedlings of same event, and 3-4 different plates (replicates) for each event. For each polynucleotide of the invention at least four independent transformation events were analyzed from each construct. Plants expressing the polynucleotides of the invention were compared to the average measurement of the control plants (generated by transformation of plant with an empty vector under the same promoter or a vector comprising the GUS reporter gene under the same promoter) used in the same experiment.
Digital Imaging—
A laboratory image acquisition system, which consists of a digital reflex camera (Canon EOS 300D) attached with a 55 mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which included 4 light units (4×150 Watts light bulb) and located in a darkroom, is used for capturing images of plantlets sawn in agar plates.
The image capturing process was repeated every 3-4 days starting at day 1 till day 10 (see for example the images in FIGS. 3A-F).
An image analysis system was used, which consists of a personal desktop computer (Intel P4 3.0 GHz processor) and a public domain program—ImageJ 1.39 (Java based image processing program which was developed at the U.S. National Institutes of Health and freely available on the internet at Hypertext Transfer Protocol://rsbweb (dot) nih (dot) gov/). Images were captured in resolution of 10 Mega Pixels (3888×2592 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group standard) format. Next, analyzed data was saved to text files and processed using the JMP statistical analysis software (SAS institute).
Seedling Analysis—
Using the digital analysis seedling data was calculated, including leaf area, root coverage and root length.
The relative growth rate for the various seedling parameters was calculated according to the following formulas V, VI and VII.
Relative growth rate of leaf area=Regression coefficient of leaf area along time course. Formula V:
Relative growth rate of root coverage=Regression coefficient of root coverage along time course. Formula VI:
Relative growth rate of root length=Regression coefficient of root length along time course. Formula VII:
At the end of the experiment, plantlets were removed from the media and weighed for the determination of plant fresh weight. Plantlets were then dried for 24 hours at 60° C., and weighed again to measure plant dry weight for later statistical analysis. Growth rate was determined by comparing the leaf area coverage, root coverage and root length, between each couple of sequential photographs, and results were used to resolve the effect of the gene introduced on plant vigor, under osmotic stress, as well as under normal or optimal conditions. Similarly, the effect of the gene introduced on biomass accumulation, under osmotic stress as well as under optimal conditions, was determined by comparing the plants' fresh and dry weight to that of control plants (containing an empty vector or the GUS reporter gene under the same promoter). From every construct created, 3-5 independent transformation events were examined in replicates.
Statistical Analyses—
To identify genes conferring significantly improved tolerance to abiotic stresses or enlarged root architecture, the results obtained from the transgenic plants were compared to those obtained from control plants. To identify outperforming genes and constructs, results from the independent transformation events tested were analyzed separately. To evaluate the effect of a gene event over a control the data was analyzed by Student's t-test and the p value was calculated. Results were considered significant if p≦0.1. The JMP statistics software package was used (Version 5.2.1, SAS Institute Inc., Cary, N.C., USA).
Experimental Results
The genes presented in Tables 11-18, hereinbelow, were found to increase ABST by improving root performance, plant growth characteristic and plant biomass when grown under limiting nitrogen growth conditions as compared to control plants.
Tables 11-18 depict analyses of root growth (root length and root coverage; Tables 11 and 12); plant biomass (plant fresh, dry weight and leaf area; Tables 13 and 14); root growth rate (relative growth rate of root length and root coverage; Tables 15 and 16); and leaf area and leaf area growth rate (relative growth rate of leaf area; Tables 17 and 18) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mM N)] in plants overexpressing the polynucleotides of some embodiments of the invention under the regulation of a constitutive promoter [35S (SEQ ID NO:675) or At6669 (SEQ ID NO:674)]. Evaluation of each gene was performed by testing the performance of several events. Some of the genes were evaluated in more than one tissue culture assay and the second experiment confirmed the significant increment in plant biomass. Event with p-value <0.05 was considered statistically significant.

TABLE 11

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the 35S promoter exhibit
improved plant roots under nitrogen deficient conditions

Gene

Plant Root length [cm]

Gene

Plant Root Coverage [cm²]

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

BDL103	8033.1	6.07	2.24E−04	22.7	CTF113	5871.2	15.17	3.7E−02	19
BDL103	8033.4	5.55	1.28E−02	12.3	Control		12.79		0.00
Control		4.95			BDL103	8033.1	8.63	1.3E−03	34
CTF163	11012.2	5.46	4.92E−02	16	Control		6.45		0.00
CTF163	11012.7	5.97	3.81E−03	27	CTF163	11011.2	7.03	2.1E−02	42
Control		4.71		0.00	CTF163	11012.2	7.59	2.8E−02	53
CTF163	11011.2	5.86	7.62E−03	36	CTF163	11012.7	8.03	5.5E−04	62
CTF163	11012.2	6.16	1.70E−03	43	Control		4.96		0.00
CTF163	11012.4	5.64	3.36E−02	31	CTF226	10982.1	8.81	8.8E−03	63
CTF163	11012.7	6.52	6.74E−03	52	CTF226	10982.3	10.72	9.1E−03	98
Control		4.30			Control		5.42		0.00
CTF226	10982.3	7.02	9.00E−06	38	CTF205	11972.3	6.81	7.3E−04	44
Control		5.08		0.00	Control		4.74		0.00
CTF205	11972.3	5.49	4.87E−02	14
Control		4.83		0.00

Table 11: Analyses of plant roots (root length and root coverage) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mM N)] as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 12

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the At6669 promoter exhibit
improved plant roots under nitrogen deficient conditions

Gene

Plant Root length [cm]

Gene

Plant Root Coverage [cm²]

Name	Event #	Ave.	p-value	incr.	Name	Event #	Ave.	p-value	% incr.

LAB31	11423.4	6.72	5.38E−03	17	LAB31	11423.4	9.25	3.9E−03	47
LAB13	11482.2	7.12	1.50E−02	24	LAB13	11482.2	8.91	4.7E−02	42
LAB41	11554.3	6.74	1.68E−02	17	LAB41	11551.2	8.47	3.3E−02	35
LAB23	11572.6	7.38	2.39E−04	28	LAB41	11554.3	9.09	1.6E−02	45
Control		5.77			LAB23	11572.6	12.36	5.5E−05	96
LAB11	11024.4	6.90	1.01E−02	22	Control		6.29		0.00
LAB22	11064.6	6.46	1.27E−02	14	LAB11	11024.4	9.65	4.3E−02	54
Control		5.66			Control		6.28		0.00
LAB32	11162.2	7.16	5.30E−03	16	LAB38	11434.4	8.50	4.5E−02	30
LAB34	11171.4	6.80	4.04E−02	11	Control		6.55		0.00
LAB38	11434.4	6.82	3.42E−02	11	LAB4	11962.1	6.17	2.4E−02	20
Control		6.14			LAB4	11964.2	6.63	2.3E−02	29
LAB20	11131.1	6.50	2.83E−02	17	Control		5.13		0.00
LAB20	11132.7	6.63	3.94E−03	19	LAB20	11131.1	7.66	3.0E−02	38
LAB9	12284.1	6.31	1.42E−02	13	LAB9	12284.1	9.08	6.5E−03	64
LAB45	12361.1	6.60	1.08E−02	19	LAB9	12286.1	8.19	8.4E−03	48
LAB45	12363.2	6.81	5.63E−03	23	LAB45	12361.1	9.06	1.3E−02	63
LAB45	12364.2	6.56	1.99E−02	18	LAB45	12361.2	8.20	3.5E−02	48
LAB45	12365.1	7.02	5.96E−03	26	LAB45	12363.2	7.28	1.0E−02	31
LAB8	12423.3	6.28	3.68E−02	13	LAB45	12365.1	11.21	1.5E−03	102
LAB8	12425.4	6.88	5.67E−04	24	LAB8	12423.3	7.43	2.9E−03	34
Control		5.56			LAB8	12425.4	10.37	1.3E−02	87
LAB32	11162.2	6.17	4.14E−03	25	Control		5.55		0.00
LAB31	11421.5	5.74	2.02E−02	16	LAB20	11131.1	7.32	9.7E−03	42
LAB31	11423.4	5.66	4.38E−02	14	LAB20	11131.2	7.28	7.5E−03	42
LAB13	11482.2	6.19	3.14E−03	25	LAB24	11191.5	6.51	4.7E−02	27
LAB8	12423.4	5.74	3.33E−02	16	LAB24	11193.5	6.62	4.5E−02	29
Control		4.95		0.00	LAB49	11281.2	6.48	3.6E−02	26
LAB20	11131.1	6.09	1.16E−02	20	LAB49	11283.5	8.10	1.4E−02	58
LAB20	11131.2	6.81	9.93E−03	34	LAB3	11331.1	6.71	1.7E−02	30
LAB20	11132.7	5.97	7.15E−04	18	Control		5.14		0.00
LAB20	11134.4	5.81	1.05E−02	14
LAB24	11191.5	6.43	7.00E−05	27
LAB24	11192.1	6.15	3.99E−03	21
LAB24	11193.5	6.55	2.20E−04	29
LAB24	11193.6	6.00	3.51E−02	18
LAB49	11281.2	6.09	5.70E−05	20
LAB49	11281.4	6.29	9.66E−03	24
LAB49	11283.1	6.01	2.98E−03	18
LAB49	11283.5	6.69	4.50E−03	32
LAB3	11331.1	6.50	4.00E−06	28
LAB3	11333.1	6.55	2.33E−04	29
LAB3	11333.5	6.05	1.00E−04	19
LAB3	11334.1	5.83	2.89E−02	15
LAB5	11443.3	6.07	1.28E−04	20
LAB5	11444.1	5.99	7.11E−04	18
LAB36	11583.1	6.02	1.37E−02	19
LAB36	11584.5	6.23	4.30E−05	23
LAB36	11585.5	6.74	3.65E−03	33
Control		5.08
LAB2	11234.2	5.79	0.025	15.69%
LAB2	11231.1	5.93	0.007	18.61%
Control		5
LAB2	11231.1	5.8	0.023	13.73%
Control		5.1

Table 12: Analyses of plant roots (root length and root coverage) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mMN)] as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 13

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the 35S promoter exhibit
improved plant biomass under nitrogen deficient conditions

Gene

Plant Fresh Weight [g]

Gene

Plant Dry Weight [g]

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

CTF163	11011.2	0.10		0.00	CTF215	11072.1	0.0089	1.5E−03	53
Control		0.14	4.1E−02	31	Control		0.0058
CTF215	11072.1	0.16	2.1E−02	27
Control		0.13		0.00
CTF226	10982.1	0.20	1.0E−03	49
CTF226	10982.3	0.18	4.7E−02	40
Control		0.13		0.00

Table 13: Analyses of plant Biomass (fresh weight and dry weight) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mMN)] as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 14

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the At6669 promoter exhibit
improved plant biomass under nitrogen deficient conditions

Gene

Plant Fresh Weight [g]

Gene

Plant Dry Weight [g]

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB31	11421.5	0.130	1.9E−03	62	LAB31	11423.4	0.0057	3.0E−05	39
LAB31	11423.4	0.125	2.6E−04	55	LAB41	11554.3	0.0062	6.8E−03	52
LAB13	11482.2	0.119	8.8E−03	48	LAB23	11572.6	0.0079	8.3E−04	93
LAB41	11554.3	0.119	7.0E−03	48	LAB23	11573.4	0.0051	3.8E−03	24
LAB23	11572.6	0.164	3.7E−03	105	LAB23	11574.2	0.0051	3.7E−02	26
Control		0.080		0.00	Control		0.0041		0.00
LAB11	11022.3	0.142	4.0E−02	83	LAB11	11024.4	0.0069	2.3E−04	81
LAB11	11024.4	0.155	4.6E−04	99	LAB16	11032.2	0.0048	2.9E−02	25
Control		0.078		0.00	Control		0.0038		0.00
LAB15	11642.2	0.107	4.0E−02	39	LAB32	11163.2	0.0061	2.0E−02	67
Control		0.077		0.00	LAB25	11341.2	0.0047	3.8E−02	28
LAB18	11653.4	0.087	3.5E−02	27	LAB38	11434.4	0.0045	2.8E−02	24
Control		0.068		0.00	LAB15	11642.2	0.0053	2.4E−04	45
LAB20	11131.2	0.120	6.5E−04	85	Control		0.0037		0.00
LAB9	12281.2	0.100	1.1E−02	55	LAB18	11653.4	0.0042	3.1E−02	19
LAB9	12286.1	0.108	4.8E−02	67	Control		0.0035		0.00
LAB45	12361.2	0.088	2.6E−03	35	LAB20	11131.2	0.0056	9.5E−04	91
Control		0.065		0.00	Control		0.0029		0.00
LAB32	11162.2	0.107	8.4E−03	44	LAB51	11561.5	0.0070	2.9E−02	92
LAB31	11423.4	0.119	3.0E−02	60	LAB8	12423.1	0.0049	5.0E−02	36
LAB13	11484.2	0.110	8.7E−03	47	Control		0.0036		0.00
LAB51	11561.5	0.139	3.6E−03	86	LAB49	11283.5	0.0054	8.3E−03	52
LAB51	11563.2	0.105	3.8E−02	41	LAB5	11444.5	0.0059	2.5E−03	65
Control		0.075		0.00	LAB36	11585.5	0.0052	8.7E−05	45
LAB20	11131.1	0.095	4.8E−02	29	Control		0.0036		0.00
LAB20	11134.4	0.098	2.2E−02	33
LAB49	11283.5	0.109	6.3E−04	48
LAB3	11333.5	0.094	1.5E−02	28
LAB3	11334.1	0.096	2.5E−02	30
LAB36	11583.1	0.101	4.0E−03	37
LAB36	11584.5	0.090	3.5E−02	23
LAB36	11585.5	0.117	2.1E−03	59
Control		0.073		0.00

Table 14: Analyses of plant Biomass (fresh weight and dry weight) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mMN)] as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 15

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the 35S promoter exhibit
improved plant biomass and growth rate under nitrogen deficient conditions

	Relative growth rate of		Relative growth rate of root
	root length (regression		coverage (regression
Gene	coefficient)	Gene	coefficient)

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

BDL103	8033.1	0.571	2.2E−03	28.0%	BDL103	8033.1	0.96	2.2E−03	38
BDL103	8033.4	0.543	8.5E−03	21.9%	Control		0.70		0.0
Control		0.446		0.0%	CTF163	11011.2	0.74	2.0E−02	33
CTF163	11012.2	0.525	5.0E−04	28.2%	Control		0.55		0.0
CTF163	11012.7	0.517	5.3E−03	26.2%	CTF163	11011.2	0.84	1.8E−03	48
CTF215	11073.4	0.507	1.9E−02	23.7%	CTF163	11012.2	0.91	1.4E−03	61
Control		0.410		0.0%	CTF163	11012.7	0.95	2.9E−05	67
CTF163	11011.2	0.542	5.7E−04	43.0%	Control		0.57		0.0
CTF163	11012.2	0.573	2.2E−04	51.2%	CTF226	10982.1	1.06	6.7E−05	68
CTF163	11012.4	0.503	1.7E−02	32.8%	CTF226	10982.3	1.25	1.0E−06	99
CTF163	11012.7	0.572	1.2E−04	50.9%	CTF226	10985.1	0.80	4.9E−02	28
Control		0.379		0.0%	Control		0.63		0.0
CTF226	10982.3	0.646	1.5E−04	34.8%	CTF205	11972.3	0.76	1.2E−03	37
Control		0.480		0.0%	Control		0.56		0.0

Table 15: Analyses of root growth rate (relative growth rate of root length and root coverage) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mM N)] as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 16

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the At6669 promoter exhibit
improved plant biomass and growth rate under nitrogen deficient conditions

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB31	11422.1	0.58	2.8E−02	24	LAB31	11421.5	1.01	3.0E−02	42
LAB31	11423.4	0.62	1.3E−03	31	LAB31	11423.4	1.09	2.8E−03	54
LAB13	11482.2	0.58	2.5E−02	24	LAB13	11482.2	1.05	1.2E−02	49
LAB41	11551.2	0.59	4.0E−02	26	LAB41	11551.2	1.00	2.4E−02	41
LAB23	11572.6	0.62	4.0E−03	31	LAB41	11554.3	1.07	5.6E−03	51
Control		0.47		0.0	LAB23	11572.6	1.45	1.0E−06	105
LAB32	11162.2	0.63	6.1E−03	22	Control		0.71		0.0
LAB34	11171.4	0.62	6.5E−03	22	LAB11	11024.4	1.13	6.0E−03	59
LAB38	11434.4	0.62	9.8E−03	20	Control		0.71		0.0
Control		0.51		0.0	LAB32	11162.2	1.00	5.1E−03	36
LAB4	11964.2	0.57	2.6E−02	28	LAB34	11171.4	0.96	7.1E−03	30
Control		0.44		0.0	LAB38	11434.4	1.01	3.4E−03	36
LAB20	11131.1	0.58	4.8E−02	27	LAB15	11642.2	1.05	5.2E−03	42
LAB45	12365.1	0.60	4.0E−02	30	LAB15	11644.1	0.96	8.0E−03	30
LAB8	12425.4	0.59	3.7E−02	28	Control		0.74		0.0
Control		0.46		0.0	LAB4	11964.2	0.85	4.3E−02	41
LAB20	11131.2	0.56	1.4E−04	42	Control		0.60		0.0
LAB20	11132.1	0.46	3.5E−02	17	LAB4	11964.2	0.75	2.9E−02	32
LAB24	11191.5	0.51	1.7E−04	31	Control		0.56		0.0
LAB24	11193.5	0.48	3.3E−03	22	LAB20	11131.1	0.88	3.5E−03	39
LAB24	11193.6	0.49	4.4E−03	24	LAB20	11132.7	0.81	3.7E−02	27
LAB49	11281.2	0.50	7.1E−04	26	LAB9	12284.1	1.06	1.7E−05	68
LAB49	11283.5	0.57	1.7E−05	46	LAB9	12286.1	0.96	2.4E−04	51
LAB3	11331.1	0.52	1.0E−05	33	LAB45	12361.1	1.05	4.6E−05	66
LAB3	11333.1	0.49	9.0E−04	25	LAB45	12361.2	0.95	1.3E−03	50
LAB3	11334.1	0.45	4.4E−02	15	LAB45	12363.2	0.83	1.2E−02	30
LAB5	11443.3	0.45	5.0E−02	13	LAB45	12364.2	1.16	2.7E−04	83
LAB5	11444.1	0.53	1.4E−05	34	LAB45	12365.1	1.30	0.0E+00	105
LAB5	11444.5	0.48	6.6E−03	22	LAB8	12423.3	0.88	2.0E−03	39
LAB36	11584.5	0.50	2.7E−04	27	LAB8	12425.4	1.22	2.0E−06	92
LAB36	11585.5	0.53	1.2E−04	34	Control		0.63		0.0
Control		0.39		0.0	LAB20	11131.1	0.80	1.5E−02	41
					LAB20	11131.2	0.85	3.4E−03	49
					LAB24	11191.5	0.74	4.9E−02	30
					LAB24	11193.6	0.76	4.7E−02	34
					LAB49	11281.2	0.74	4.0E−02	31
					LAB49	11283.5	0.97	4.0E−04	72
					LAB3	11331.1	0.76	3.1E−02	34
					LAB36	11584.5	0.74	4.3E−02	31
					LAB36	11585.5	0.85	1.2E−02	51
					Control		0.57		0.0

Table 16: Analyses of root growth rate (relative growth rate of root length and root coverage) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mM N)] as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 17

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the 35S promoter exhibit
improved plant biomass and growth rate under nitrogen deficient conditions

			Relative growth rate of
Gene	Leaf area (cm²)	Gene	Leaf area

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

CTF113	5871.2	0.70	3.6E−02	21.6%	CTF163	11011.2	0.07	1.7E−02	24
Control		0.58		0.0%	Control		0.06		0.00
CTF163	11011.2	0.77	3.0E−02	28.4%	CTF215	11072.1	0.08	4.6E−02	30
Control		0.60		0.0%	Control		0.06		0.00
CTF215	11072.1	0.83	1.8E−02	27.9%	CTF226	10982.1	0.11	1.1E−03	47
Control		0.65		0.0%	CTF226	10982.3	0.11	7.9E−03	45
CTF226	10982.1	1.16	8.3E−04	44.1%	Control		0.08		0.00
CTF226	10982.3	1.10	1.6E−02	36.4%
Control		0.80		0.0%
CTF180	11371.1	0.59	4.2E−02	16.2%
Control		0.51		0.0%

Table 17: Analyses of leaf area and leaf area growth rate (relative growth rate of leaf area) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mM N)] as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 18

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the At6669 promoter exhibit
improved plant biomass and growth rate under nitrogen deficient conditions

			Relative growth rate of leaf
			area (regression
Gene	Leaf area (cm²)	Gene	coefficient)

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB31	11421.5	0.79	4.9E−04	51.0%	LAB31	11421.5	0.08	1.0E−05	53.6%
LAB31	11423.4	0.69	2.4E−03	32.1%	LAB31	11423.4	0.07	2.7E−05	45.4%
LAB23	11572.6	0.93	7.0E−04	76.7%	LAB13	11482.2	0.06	2.1E−02	23.5%
Control		0.52		0.0%	LAB13	11484.2	0.06	1.7E−02	25.0%
LAB11	11024.4	0.80	3.3E−05	48.2%	LAB41	11551.2	0.07	2.0E−03	34.5%
Control		0.54		0.0%	LAB41	11554.3	0.07	9.9E−04	49.2%
LAB32	11163.2	0.67	5.2E−03	17.9%	LAB23	11572.6	0.09	0.0E+00	86.1%
LAB17	11534.1	0.84	8.6E−03	48.4%	Control		0.05		0.0%
LAB15	11642.2	0.68	5.3E−03	20.5%	LAB11	11024.4	0.08	4.4E−03	39.2%
Control		0.57		0.0%	Control		0.06		0.0%
LAB4	11964.2	0.52	4.0E−02	30.3%	LAB25	11341.1	0.07	4.9E−02	24.6%
Control		0.40		0.0%	LAB17	11534.1	0.08	1.8E−03	41.4%
LAB20	11131.2	0.51	3.1E−04	43.7%	Control		0.06		0.0%
LAB9	12281.2	0.45	2.2E−02	28.1%	LAB20	11131.2	0.05	1.3E−03	37.3%
Control		0.35		0.0%	LAB9	12281.2	0.05	1.9E−02	27.4%
LAB20	11131.1	0.64	4.3E−03	46.9%	LAB9	12284.1	0.05	2.6E−02	48.5%
LAB20	11134.4	0.53	2.5E−02	23.6%	LAB9	12286.1	0.05	3.9E−02	30.3%
LAB9	12284.1	0.79	1.8E−03	83.2%	LAB45	12364.2	0.05	4.7E−02	30.6%
LAB9	12286.1	0.56	4.3E−02	29.9%	Control		0.04		0.0%
LAB45	12365.1	0.75	2.5E−02	72.8%	LAB20	11131.1	0.07	5.9E−04	47.5%
LAB8	12425.4	0.68	5.9E−03	56.7%	LAB9	12284.1	0.08	1.0E−06	83.1%
Control		0.43		0.0%	LAB9	12286.1	0.06	1.3E−02	32.8%
LAB31	11423.4	0.60	6.8E−04	49.2%	LAB45	12361.1	0.06	4.5E−03	44.4%
LAB13	11481.5	0.57	2.1E−02	43.5%	LAB45	12365.1	0.08	3.8E−04	69.1%
LAB51	11561.2	0.55	2.5E−02	36.9%	LAB8	12422.3	0.06	2.6E−02	37.8%
LAB51	11561.5	0.55	3.3E−02	37.4%	LAB8	12425.4	0.07	8.1E−05	58.7%
Control		0.40		0.0%	Control		0.04		0.0%
LAB20	11131.1	0.55	2.1E−02	28.2%	LAB31	11423.4	0.06	2.7E−02	35.4%
LAB49	11283.5	0.58	3.7E−02	33.6%	Control		0.04		0.0%
LAB5	11444.5	0.54	2.6E−02	26.0%	LAB49	11283.5	0.06	2.8E−02	41.4%
Control		0.43		0.0%	Control		0.04		0.0%

Table 18: Analyses of leaf area and leaf area growth rate (leaf area growth rate) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under limiting nitrogen conditions [low nitrogen or nitrogen deficient conditions (0.75 mM N)] as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

The genes presented in Tables 19-26, hereinbelow, were found to improve plant performance by improving root performance, plant growth characteristic and plant biomass when grown under normal growth conditions, compared to control plants.
Tables 19-26 depict analyses of root growth (root length and root coverage; Tables 19 and 20); plant biomass (plant fresh, dry weight and leaf area; Tables 21 and 22); root growth rate (relative growth rate of root length and root coverage; Tables 23 and 24); leaf area and leaf area growth rate (Relative growth rate of leaf area; Tables 25 and 26) when grown under normal growth conditions (i.e., in the presence of 15 mM nitrogen) in plants overexpressing the polynucleotides of some embodiments of the invention under the regulation of a constitutive promoter [35S (SEQ ID NO:675) or At6669 (SEQ ID NO:674)]. Evaluation of each gene was performed by testing the performance of several events. Some of the genes were evaluated in more than one tissue culture assay and the second experiment confirmed the significant increment in plant biomass. Event with p-value <0.05 was considered statistically significant.

TABLE 19

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention under the regulation of the 35S
promoter exhibit improved plant roots under normal conditions

Gene

Plant root length [cm]

Gene

Plant root coverage [cm²]

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

BDL103	8033.4	5.29	7.6E−03	14.5%	BDL166	9985.2	7.21	3.1E−02	67.0%
Control		4.62		0.0%	Control		4.32		0.0%
CTF113	5871.3	2.51	2.7E−02	42.3%	CTF163	11012.7	6.41	1.6E−02	40.2%
Control		1.76		0.0%	Control		4.58		0.0%
CTF163	11012.7	6.38	9.6E−03	48.1%	CTF205	11972.3	4.64	2.8E−02	47.5%
Control		4.31		0.0%	Control		3.15		0.0%
CTF205	11972.3	5.26	5.5E−03	23.4%
Control		4.26		0.0%

Table 19: Analyses of plant roots (root length and root coverage) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 20

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention under the regulation of the At6669
promoter exhibit improved plant roots under normal conditions

Gene

Plant root length [cm]

Gene

Plant root coverage [cm²]

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB31	11421.5	6.55	1.15E−02	21.9%	LAB13	11482.2	8.17	1.1E−02	62.5%
LAB31	11423.2	6.44	4.87E−02	20.0%	LAB41	11551.2	6.90	2.7E−02	37.3%
LAB31	11423.4	6.32	4.20E−02	17.7%	LAB23	11572.6	7.11	9.5E−03	41.5%
LAB13	11482.2	6.90	2.98E−03	28.4%	Control		5.03		0.0%
LAB13	11484.2	6.69	3.18E−02	24.5%	LAB22	11062.3	5.77	7.7E−04	55.9%
LAB41	11551.2	6.89	2.57E−03	28.4%	LAB36	11585.5	5.21	6.3E−03	40.8%
LAB23	11572.6	6.72	4.99E−03	25.2%	Control		3.70		0.0%
Control		5.37		0.0%	LAB34	11175.1	5.97	6.2E−03	50.8%
LAB11	11022.1	5.79	8.49E−03	22.2%	LAB50	Control	4.86		0.0%
LAB11	11022.3	6.25	1.42E−03	31.9%	LAB4	11964.2	4.70	4.1E−02	41.8%
LAB11	11024.4	5.87	1.96E−02	23.9%	LAB18	Control	3.31		0.0%
LAB16	11032.2	5.84	1.06E−02	23.4%	LAB4	11964.1	5.30	1.5E−02	30.7%
LAB22	11062.3	6.09	2.15E−03	28.6%	Control		4.05		0.0%
LAB22	11063.4	5.66	2.93E−02	19.6%	LAB9	12281.2	5.45	2.5E−02	55.1%
LAB22	11064.6	5.78	1.99E−02	21.9%	LAB45	12365.1	5.91	3.5E−02	68.1%
LAB36	11585.5	5.77	3.64E−03	21.8%	Control		3.51		0.0%
Control		4.74		0.0%	LAB9	12284.1	8.02	6.4E−03	69.3%
LAB32	11162.2	5.27	2.10E−02	14.2%	LAB45	12365.1	7.92	1.8E−03	67.2%
LAB32	11163.2	5.91	2.14E−02	27.9%	LAB8	12423.1	7.10	6.2E−03	50.0%
LAB34	11175.1	5.55	1.21E−02	20.1%	LAB8	12425.4	7.47	3.8E−03	57.8%
LAB33	11272.4	5.07	4.85E−02	9.8%	Control		4.74		0.0%
LAB25	11341.1	5.79	3.40E−02	25.4%	LAB49	11281.4	5.43	3.3E−02	26.9%
LAB15	11642.2	5.74	3.61E−02	24.2%	Control		4.28		0.0%
LAB15	11644.2	5.15	3.29E−02	11.6%
Control		4.62		0.0%
LAB2	11234.2	5.18	4.51E−02	20.9%
LAB4	11962.1	5.15	2.22E−02	20.4%
LAB4	11963.2	4.98	1.41E−02	16.3%
Control		4.28		0.0%
LAB20	11131.1	5.52	3.31E−02	20.6%
LAB9	12281.2	5.22	6.31E−03	14.1%
LAB45	12365.1	5.84	1.27E−03	27.6%
Control		4.58		0.0%
LAB45	12361.1	6.10	2.44E−02	11.2%
LAB8	12425.4	6.31	8.97E−03	15.0%
Control		5.48		0.0%
LAB13	11482.2	5.86	4.74E−03	21.5%
Control		4.82		0.0%
LAB20	11131.2	6.81	8.98E−04	28.8%
LAB20	11132.7	6.58	1.90E−02	24.6%
LAB20	11134.4	6.12	1.60E−02	15.8%
LAB24	11193.5	6.10	2.51E−03	15.6%
LAB3	11333.1	6.45	1.02E−02	22.2%
LAB36	11584.5	6.53	3.45E−02	23.6%
Control		5.28		0.0%
LAB2	11234.2	5.17	0.045	7.81%
Control		4.80

Table 20: Analyses of plant roots (root length and root coverage) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 21

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention under the regulation of the 35S
promoter exhibit improved plant biomass under normal conditions

Gene

Plant Fresh Weight [g]

Gene

Plant Dry Weight [g]

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

CTF113	5872.1	0.15	2.1E−02	44.4%	CTF113	5872.1	0.0086	2.3E−02	0.44
Control		0.10		0.0%	Control	Control	0.0060		0.00
BDL14	5761.2	0.24	3.3E−02	44.5%	CTF175	8701.4	0.0054	4.5E−03	0.91
Control		0.16		0.0%	CTF175	8702.4	0.0042	4.9E−02	0.49
BDL17	6081.3	0.30	2.9E−02	63.5%	Control	Control	0.0028		0.00
Control		0.18		0.0%	BDL103	8033.12	0.0050	8.3E−03	0.13
BDL166	9985.2	0.29	5.9E−03	52.2%	Control	Control	0.0107		0.00
Control		0.19		0.0%	BDL166	9985.2	0.0163	8.5E−04	0.73
CTF226	10985.1	0.19	3.1E−02	40.0%	Control	Control	0.0094		0.00
CTF226	10985.5	0.17	2.2E−02	29.7%	CTF215	11072.1	0.0080	2.6E−02	0.29
Control		0.13		0.0%	Control	Control	0.0062		0.00
CTF205	11972.3	0.11	1.2E−02	32.1%	CTF226	10982.1	0.0097	2.5E−03	0.69
Control		0.09		0.0%	CTF226	10985.1	0.0081	1.5E−02	0.42
BDL17	6081.3	0.20	4.7E−03	78.5%	Control	Control	0.0057		0.00
BDL17	6081.5	0.20	2.2E−02	83.1%	CTF205	11972.3	0.0053	5.0E−03	0.20
BDL17	6083.2	0.21	1.0E−04	94.9%	Control	Control	0.0044		0.00
Control		0.11		0.0%	BDL17	6081.3	0.0088	4.0E−02	0.49
CTF180	11371.1	0.11	5.7E−04	63.2%	BDL17	6083.2	0.0106	2.1E−02	0.79
CTF180	11376.1	0.09	1.3E−02	29.5%	Control	Control	0.0059		0.00
CTF205	11972.3	0.10	1.6E−02	46.2%	CTF180	11371.1	0.0044	5.0E−02	0.49
CTF205	11973.2	0.09	3.7E−02	35.0%	CTF180	11376.1	0.0038	4.0E−02	0.28
Control		0.07		0.0%	Control	Control	0.0030		0.00

Table 21: Analyses of plant Biomass (fresh weight and dry weight) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 22

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention under the regulation of the At6669
promoter exhibit improved plant biomass under normal conditions

Plant Fresh Weight [g]

Plant Dry Weight [g]

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB23	11572.6	0.129	4.2E−02	31.2%	LAB23	11571.5	0.0057	1.0E−02	0.30
Control		0.098		0.0%	LAB23	11572.6	0.0062	1.1E−03	0.43
LAB11	11022.1	0.102	3.2E−03	43.8%	Control	Control	0.0044		0.00
LAB11	11022.3	0.099	5.7E−03	39.2%	LAB11	11022.1	0.0045	3.4E−02	0.53
LAB16	11032.2	0.126	1.5E−02	77.4%	LAB11	11023.4	0.0035	4.9E−02	0.18
LAB16	11033.2	0.098	2.1E−02	37.9%	LAB11	11024.4	0.0044	3.6E−02	0.52
LAB22	11064.6	0.125	3.7E−02	75.7%	LAB22	11064.6	0.0055	1.4E−02	0.89
LAB36	11585.5	0.091	1.7E−02	27.3%	LAB36	11584.2	0.0042	3.7E−02	0.43
Control		0.071		0.0%	LAB36	11584.5	0.0036	8.8E−03	0.24
LAB32	11163.2	0.116	8.2E−03	39.8%	Control	Control	0.0029		0.00
LAB33	11272.2	0.120	6.0E−03	43.7%	LAB32	11163.2	0.0057	1.9E−02	0.40
LAB17	11534.1	0.110	4.6E−02	31.9%	LAB33	11272.2	0.0051	1.9E−02	0.25
LAB15	11642.2	0.154	2.8E−02	85.2%	LAB15	Control	0.0041		0.00
Control		0.083		0.0%	LAB18	11653.7	0.0037	2.7E−02	0.24
LAB18	11653.7	0.076	3.3E−02	26.9%	Control	Control	0.0030		0.00
Control		0.060		0.0%	LAB20	11131.2	0.0063	3.8E−02	1.24
LAB20	11131.2	0.167	1.4E−04	146.4%	LAB9	12282.2	0.0039	2.4E−02	0.37
LAB20	11134.4	0.095	2.1E−02	40.7%	LAB9	12284.1	0.0057	4.2E−02	1.01
LAB9	12284.1	0.133	4.7E−02	95.8%	LAB45	12365.1	0.0047	2.6E−02	0.66
Control		0.068		0.0%	Control	Control	0.0028		0.00
LAB32	11163.1	0.089	3.8E−03	40.3%	LAB9	12281.2	0.0063	2.2E−02	0.30
LAB31	11422.5	0.099	1.4E−02	55.8%	LAB9	12284.1	0.0077	1.5E−03	0.58
LAB31	11423.1	0.106	8.6E−03	66.6%	Control	Control	0.0049		0.00
LAB13	11481.5	0.102	8.7E−05	60.6%	LAB32	11163.1	0.0046	9.9E−04	0.67
LAB13	11482.2	0.106	2.2E−02	65.9%	LAB31	11422.1	0.0041	2.5E−02	0.48
LAB13	11483.2	0.114	4.3E−02	79.4%	LAB31	11423.1	0.0050	4.3E−02	0.82
LAB13	11483.3	0.081	4.5E−02	27.2%	LAB31	11423.4	0.0053	3.5E−02	0.91
LAB13	11484.2	0.127	5.0E−04	99.6%	LAB13	11483.2	0.0051	3.2E−02	0.85
LAB51	11561.2	0.107	8.1E−03	67.3%	LAB13	11484.2	0.0063	8.4E−03	1.27
LAB51	11561.5	0.135	2.7E−02	112.1%	LAB51	11561.2	0.0039	3.5E−02	0.42
LAB51	11564.7	0.119	3.3E−02	86.9%	Control	Control	0.0028		0.00
LAB8	12422.3	0.079	3.6E−02	24.1%	LAB24	11193.6	0.0041	6.2E−03	0.44
LAB8	12423.3	0.081	2.5E−02	27.5%	LAB3	11334.1	0.0042	3.4E−03	0.47
Control		0.064		0.0%	LAB5	11443.4	0.0039	4.8E−03	0.36
LAB3	11333.5	0.128	1.6E−03	53.0%	LAB5	11444.5	0.0044	1.7E−02	0.54
Control		0.084		0.0%	LAB36	11583.1	0.0044	9.1E−04	0.57
					Control	.5	0.0028		0.00

Table 22: Analyses of plant biomass (fresh weight and dry weight) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 23

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention under the regulation of the 35S promoter
exhibit improved plant biomass and growth rate under normal conditions

Relative growth rate of root length	Relative growth rate of root coverage
(regression coefficient)	(regression coefficient)

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

BDL103	8033.4	0.509	2.23E−02	21.9%	CTF113	5871.3	0.27	2.3E−03	165.9%
Control		0.418		0.0%	Control		0.10		0.0%
CTF113	5871.3	0.192	3.31E−02	49.6%	BDL210	10831.5	0.42	4.9E−02	54.4%
Control		0.128		0.0%	Control		0.27		0.0%
BDL210	10831.5	0.356	3.29E−02	36.5%	BDL166	9985.2	0.83	1.2E−02	62.4%
Control		0.261		0.0%	Control		0.51		0.0%
CTF163	11012.7	0.604	8.70E−05	64.9%	CTF163	11012.7	0.75	4.0E−03	42.6%
Control		0.366		0.0%	Control		0.53		0.0%
					BDL17	6081.5	1.12	3.2E−02	52.6%
					Control		0.74		0.0%
					CTF205	11972.3	0.50	1.0E−02	38.7%
					Control		0.36		0.0%

Table 23: Analyses of root growth rate (relative growth rate of root length and root coverage) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 24

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention under the regulation of the At6669 promoter
exhibit improved plant biomass and growth rate under normal conditions

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB13	11482.2	0.594	1.86E−02	38.3%	LAB31	11423.4	0.83	1.6E−02	52.0%
LAB41	11551.2	0.618	6.18E−03	43.8%	LAB13	11482.2	0.97	4.6E−04	79.0%
Control		0.430		0.0%	LAB13	11484.2	0.79	2.6E−02	45.1%
LAB22	11062.3	0.498	1.22E−02	31.6%	LAB41	11551.2	0.80	1.7E−02	47.3%
LAB36	11585.5	0.487	1.84E−02	28.8%	LAB41	11552.4	0.77	4.7E−02	42.4%
Control		0.378		0.0%	LAB41	11554.3	0.94	4.9E−03	73.6%
LAB21	11144.1	0.423	2.99E−02	25.1%	LAB23	11572.6	0.82	7.6E−03	51.2%
LAB32	11162.2	0.412	3.64E−02	21.9%	Control		0.54		0.0%
LAB32	11163.2	0.455	1.59E−02	34.7%	LAB11	11022.1	0.59	1.7E−02	45.8%
LAB34	11175.1	0.432	1.57E−02	27.8%	LAB11	11022.3	0.58	1.4E−02	42.6%
LAB25	11341.1	0.457	8.49E−03	35.1%	LAB16	11032.3	0.56	2.1E−02	38.3%
LAB25	11342.2	0.464	3.22E−03	37.3%	LAB22	11062.3	0.67	2.1E−04	65.5%
LAB38	11434.4	0.431	2.27E−02	27.6%	LAB36	11585.5	0.59	6.3E−03	45.2%
LAB15	11641.1	0.480	2.38E−03	42.0%	Control		0.41		0.0%
LAB15	11642.2	0.464	7.31E−03	37.3%	LAB32	11163.2	0.60	6.9E−03	46.9%
Control		0.338		0.0%	LAB34	11175.1	0.64	8.6E−04	56.7%
LAB2	11231.1	0.430	4.51E−02	24.2%	LAB25	11341.1	0.55	3.7E−02	34.9%
LAB4	11962.1	0.439	1.79E−02	26.6%	LAB25	11342.2	0.59	3.3E−02	44.3%
LAB4	11964.2	0.480	5.38E−03	38.6%	LAB17	11533.7	0.57	4.9E−02	39.4%
Control		0.346		0.0%	LAB15	11642.2	0.73	1.5E−03	78.2%
LAB20	11131.1	0.476	3.64E−03	32.5%	LAB15	11644.1	0.58	2.1E−02	41.2%
LAB45	12364.2	0.439	4.72E−02	22.2%	LAB15	11644.2	0.58	1.4E−02	42.7%
LAB45	12365.1	0.462	5.73E−03	28.6%	Control		0.41		0.0%
Control		0.359		0.0%	LAB4	11964.2	0.55	3.0E−03	48.5%
LAB13	11482.2	0.505	2.79E−02	26.0%	Control		0.37		0.0%
Control		0.401		0.0%	LAB2	11231.1	0.58	2.6E−02	39.5%
LAB20	11131.2	0.543	1.00E−05	40.0%	LAB4	11963.2	0.65	4.0E−02	54.6%
LAB20	11132.7	0.527	1.08E−03	35.9%	LAB4	11964.1	0.58	1.8E−02	37.8%
LAB49	11281.4	0.482	3.02E−02	24.3%	Control		0.42		0.0%
LAB3	11333.1	0.519	4.80E−04	33.7%	LAB20	11131.1	0.49	3.3E−02	26.5%
LAB5	11444.1	0.470	9.27E−03	21.3%	LAB20	11131.2	0.55	2.9E−02	41.2%
LAB36	11584.2	0.485	1.39E−02	25.0%	LAB9	12281.2	0.60	4.2E−04	55.9%
LAB36	11584.5	0.546	2.23E−04	40.7%	LAB45	12361.2	0.62	7.1E−03	61.2%
Control		0.388		0.0%	LAB45	12364.2	0.54	8.5E−03	39.6%
LAB2	11231.1	0.43	0.04		LAB45	12365.1	0.63	8.7E−04	63.0%
Control		0.35		20.16%	Control		0.39		0.0%
					LAB9	12284.1	0.89	9.8E−04	68.3%
					LAB45	12361.2	0.75	2.9E−02	42.1%
					LAB45	12365.1	0.91	2.0E−04	71.0%
					LAB8	12423.1	0.80	5.6E−03	50.6%
					LAB8	12425.4	0.81	3.3E−03	53.1%
					Control		0.53		0.0%
					LAB13	11482.2	0.56	2.5E−02	47.4%
					LAB51	11561.5	0.63	1.3E−02	64.1%
					Control		0.38		0.0%
					LAB20	11131.2	0.58	4.1E−02	27.6%
					LAB20	11132.7	0.61	2.1E−02	32.9%
					LAB49	11281.4	0.59	1.9E−02	28.8%
					Control		0.46		0.0%
					LAB2	11231.1	0.58	0.025	39.5%
					Control		0.41

Table 24: Analyses of root growth rate (relative growth rate of root length and root coverage) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 25

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention under the regulation of the 35S promoter
exhibit improved plant biomass and growth rate under normal conditions

	Relative growth rate of leaf area
Leaf area (cm²)	(regression coefficient)

Gene					Gene
Name	Event #	Average	p-value	% incr.	Name	Event #	Average	p-value	% incr.

BDL17	6081.3	0.80	3.7E−03	35.7%	CTF113	5873.3	0.06	1.5E−02	46.6%
Control		0.59		0.0%	Control		0.04		0.0%
BDL103	8033.1	0.82	8.7E−03	39.5%	BDL17	6081.3	0.08	3.7E−02	34.6%
BDL103	8033.12	0.66	9.7E−04	12.3%	Control		0.06		0.0%
Control		1.16		0.0%	BDL103	8033.1	0.09	3.2E−02	38.2%
BDL166	9985.2	1.13	1.6E−03	40.7%	BDL103	8033.12	0.07	2.5E−03	8.1%
Control		0.80		0.0%	Control		0.13		0.0%
CTF226	10982.1	1.24	2.3E−02	37.6%	BDL166	9985.2	0.11	4.8E−03	41.5%
Control		0.90		0.0%	Control		0.08		0.0%
BDL17	6081.3	1.21	3.8E−03	54.7%	CTF215	11072.1	0.09	3.3E−02	34.4%
BDL17	6081.5	1.17	1.0E−02	50.0%	Control		0.07		0.0%
BDL17	6083.2	1.20	7.1E−03	54.0%	CTF205	11972.3	0.06	3.7E−02	25.3%
Control		0.78		0.0%	Control		0.05		0.0%
CTF180	11371.1	0.52	2.8E−02	39.5%	BDL17	6081.3	0.11	2.2E−02	43.1%
CTF180	11376.1	0.48	8.0E−03	27.8%	BDL17	6081.5	0.12	1.5E−02	46.9%
CTF205	11973.2	0.53	2.1E−02	41.3%	BDL17	6083.2	0.12	2.8E−03	59.2%
Control		0.38		0.0%	Control		0.08		0.0%
					CTF180	11371.1	0.05	1.1E−02	39.3%
					CTF180	11376.1	0.05	4.0E−02	28.6%
					CTF205	11973.2	0.05	4.5E−03	44.8%
					Control		0.04		0.0%

Table 25: Analyses of leaf area and leaf area growth rate (leaf area growth rate) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 26

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention under the regulation of the At6669 promoter
exhibit improved plant biomass and growth rate under normal conditions

Leaf area (cm²)

Relative growth rate of leaf area

LAB41	11554.3	0.66	4.8E−02	29.3%	LAB31	11421.5	0.08	1.0E−05	53.6%
LAB23	11572.6	0.76	1.9E−02	47.9%	LAB31	11423.4	0.07	2.7E−05	45.4%
Control		0.51		0.0%	LAB13	11482.2	0.06	2.1E−02	23.5%
LAB11	11022.3	0.60	5.0E−03	31.9%	LAB13	11484.2	0.06	1.7E−02	25.0%
LAB11	11024.4	0.69	2.8E−04	50.7%	LAB41	11551.2	0.07	2.0E−03	34.5%
LAB16	11032.2	0.62	4.4E−02	36.7%	LAB41	11554.3	0.07	9.9E−04	49.2%
LAB22	11064.6	0.63	1.9E−02	38.5%	LAB23	11572.6	0.09	0.0E+00	86.1%
LAB36	11584.2	0.61	2.2E−02	33.9%	Control		0.05		0.0%
Control		0.46		0.0%	LAB11	11024.4	0.08	4.4E−03	39.2%
LAB33	11272.2	0.65	1.2E−02	32.4%	Control		0.06		0.0%
LAB25	11341.2	0.64	3.8E−02	30.4%	LAB25	11341.1	0.07	4.9E−02	24.6%
LAB17	11534.1	0.70	8.2E−05	42.1%	LAB17	11534.1	0.08	1.8E−03	41.4%
LAB15	11642.2	0.82	4.6E−02	67.1%	Control		0.06		0.0%
Control		0.49		0.0%	LAB20	11131.2	0.05	1.3E−03	37.3%
LAB20	11131.2	0.58	2.0E−02	88.7%	LAB9	12281.2	0.05	1.9E−02	27.4%
LAB9	12284.1	0.51	3.3E−02	65.6%	LAB9	12284.1	0.05	2.6E−02	48.5%
LAB45	12361.1	0.44	1.8E−03	45.1%	LAB9	12286.1	0.05	3.9E−02	30.3%
Control		0.31		0.0%	LAB45	12364.2	0.05	4.7E−02	30.6%
LAB9	12284.1	0.85	1.3E−02	78.6%	Control		0.04		0.0%
LAB45	12365.1	0.83	1.3E−04	74.9%	LAB20	11131.1	0.07	5.9E−04	47.5%
LAB8	12423.1	0.62	2.9E−02	31.4%	LAB9	12284.1	0.08	1.0E−06	83.1%
Control		0.48		0.0%	LAB9	12286.1	0.06	1.3E−02	32.8%
LAB32	11163.1	0.41	4.0E−02	26.3%	LAB45	12361.1	0.06	4.5E−03	44.4%
LAB31	11422.5	0.40	3.4E−02	25.1%	LAB45	12365.1	0.08	3.8E−04	69.1%
LAB31	11423.1	0.48	4.0E−02	49.2%	LAB8	12422.3	0.06	2.6E−02	37.8%
LAB31	11423.4	0.55	3.3E−02	69.4%	LAB8	12425.4	0.07	8.1E−05	58.7%
LAB13	11481.5	0.54	4.3E−03	68.2%	Control		0.04		0.0%
LAB13	11482.2	0.43	1.5E−02	34.7%	LAB31	11423.4	0.06	2.7E−02	35.4%
LAB13	11483.2	0.58	3.3E−03	80.1%	Control		0.04		0.0%
LAB13	11484.2	0.69	1.8E−02	114.1%	LAB49	11283.5	0.06	2.8E−02	41.4%
LAB51	11561.2	0.45	1.5E−02	41.2%	Control		0.04		0.0%
LAB51	11561.5	0.58	3.4E−02	81.4%
LAB51	11563.2	0.42	1.2E−02	31.4%
LAB51	11564.5	0.48	3.1E−02	47.9%
LAB51	11564.7	0.53	1.0E−02	63.2%
LAB8	12422.3	0.42	2.9E−02	31.9%
LAB8	12423.1	0.40	4.7E−02	25.5%
Control		0.32		0.0%
LAB20	11132.1	0.47	2.2E−02	19.9%
LAB49	11281.4	0.50	2.4E−03	30.1%
LAB3	11333.5	0.50	1.0E−03	28.8%
LAB5	11444.5	0.58	1.3E−02	49.4%
LAB36	11583.1	0.47	1.8E−02	21.6%
LAB36	11585.5	0.48	7.4E−03	22.9%
Control		0.39		0.0%

Table 26: Analyses of leaf area and leaf area growth rate (leaf area growth rate) of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

Example 7

Evaluation of Transgenic Arabidopsis Plant Growth Under Abiotic Stress as Well as Under Favorable Conditions in

Greenhouse Assay Grown Until Seed Production
ABS Tolerance: Yield and Plant Growth Rate at High Salinity Concentration Under Greenhouse Conditions—
This assay follows the rosette area growth of plants grown in the greenhouse as well as seed yield at high salinity irrigation. Seeds were sown in agar media supplemented only with a selection agent (Kanamycin) and Hoagland solution under nursery conditions. The T₂transgenic seedlings were then transplanted to 1.7 trays filled with peat and perlite. The trays were irrigated with tap water (provided from the pots' bottom). Half of the plants were irrigated with a salt solution (40-80 mM NaCl and 5 mM CaCl₂) so as to induce salinity stress (stress conditions). The other half of the plants was irrigated with tap water (normal conditions). All plants were grown in the greenhouse until mature seeds, then harvested (the above ground tissue) and weighted (immediately or following drying in oven at 50° C. for 24 hours). High salinity conditions were achieved by irrigating with a solution containing 40-80 mM NaCl (“ABS” growth conditions) and compared to regular growth conditions.
Each construct was validated at its T₂generation. Transgenic plants transformed with a construct including the uidA reporter gene (GUS) under the At6669 promoter (SEQ ID NO:674) or with an empty vector including the At6669 promoter are used as control.
The plants were analyzed for their overall size, growth rate, flowering, seed yield, weight of 1,000 seeds, dry matter and harvest index (HI-seed yield/dry matter). Transgenic plants performance was compared to control plants grown in parallel under the same conditions. Mock-transgenic plants expressing the uidA reporter gene (GUS-Intron) or with no gene at all (empty vector, containing the Kan selection gene), under the same promoter were used as control.
The experiments were planned in nested randomized plot distribution. For each gene of the invention three to five independent transformation events were analyzed from each construct.
Digital Imaging—
A laboratory image acquisition system, which consists of a digital reflex camera (Canon EOS 300D) attached with a 55 mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which included 4 light units (4×150 Watts light bulb) was used for capturing images of plant samples.
The image capturing process was repeated every 2 days starting from day 1 after transplanting till day 16. Same camera, placed in a custom made iron mount, was used for capturing images of larger plants sawn in white tubs in an environmental controlled greenhouse. The tubs were square shape and include 1.7 liter trays. During the capture process, the tubs were placed beneath the iron mount, while avoiding direct sun light and casting of shadows.
An image analysis system was used, which consists of a personal desktop computer (Intel P4 3.0 GHz processor) and a public domain program—ImageJ 1.39 (Java based image processing program which was developed at the U.S. National Institutes of Health and freely available on the internet at Hypertext Transfer Protocol://rsbweb (dot) nih (dot) gov/). Images were captured in resolution of 10 Mega Pixels (3888×2592 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group standard) format. Next, analyzed data was saved to text files and processed using the JMP statistical analysis software (SAS institute).
Leaf Growth Analysis—
Using the digital analysis leaves data was calculated, including leaf number, rosette area, rosette diameter, leaf blade area, plot coverage, leaf petiole length.
Vegetative growth rate: is the rate of growth of the plant as defined by formulas VIII, IX, XI and XI
Relative growth rate of leaf blade area=Regression coefficient of leaf area along time course. Formula VIII:
Relative growth rate of rosette area=Regression coefficient of rosette area along time course. Formula IX:
Relative growth rate of rosette diameter=Regression coefficient of rosette diameter along time course. Formula X
Relative growth rate of plot coverage=Regression coefficient of plot coverage along time course. Formula XI
Seeds Average Weight (Seed Weight or 1000 Seed Weight)—
At the end of the experiment all seeds were collected. The seeds were scattered on a glass tray and a picture was taken. Using the digital analysis, the number of seeds in each sample was calculated.
Plant Dry Weight and Seed Yield—
On about day 80 from sowing, the plants were harvested and left to dry at 30° C. in a drying chamber. The biomass and seed weight of each plot were measured and divided by the number of plants in each plot.
Dry weight=total weight of the vegetative portion above ground (excluding roots) after drying at 30° C. in a drying chamber;
Seed yield per plant=total seed weight per plant (grams).
The Harvest Index can be calculated using Formula IV (as described above; Harvest Index=Average seed yield per plant/Average dry weight).
Statistical Analyses—
To identify genes conferring significantly improved ABST, nitrogen use efficiency, growth rate, biomass, oil content and yield production, the results obtained from the transgenic plants were compared to those obtained from control plants. To identify outperforming genes and constructs, results from the independent transformation events tested were analyzed separately. Data was analyzed using Student's t-test and results were considered significant if the p value was less than 0.1. The JMP statistics software package was used (Version 5.2.1, SAS Institute Inc., Cary, N.C., USA).
Experiment Results:
The genes presented in Tables 27-31, hereinbelow, have improved plant ABST when grown at high salinity irrigation levels (80-100 mM NaCl). These genes produced higher seed yield, harvest index, seed weight (expressed as 1000-seed weight) and plant biomass [(as expressed as plant dry weight (DW)] when grown under high salinity irrigation conditions, compared to control.
Tables 27-29 depict analyses of seed yield and weight (Table 27), harvest index (Table 28) and dry weight (Table 29) when grown under high salinity irrigation conditions in plants overexpressing the polynucleotides of some embodiments of the invention under the regulation of a constitutive (35S; SEQ ID NO:675). Evaluation of each gene was performed by testing the performance of several events. Some of the genes were evaluated in more than one tissue culture assay and the results obtained were repeated. Event with p-value <0.05 was considered statistically significant.

TABLE 27

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention under the regulation of the 35S promoter exhibit improved
seed yield and weight under high salinity irrigation growth conditions

Gene

Seed yield

Gene

Seeds weight (g)

Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

CTF113	5871.2	0.03	3.0E−03	75.5%	CTF113	5873.3	0.024	4.2E−02	13.5%
Control		0.02		0.0%	Control		0.021		0.0%

Table 27: Analyses of seed yield and weight [expressed as 1000-seed weight in grams (g)] of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under high salinity irrigation conditions (80-100 mM NaCl) as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 28

Transgenic Arabidopsis plants exogenously expressing the
polynucleotides of some embodiments of the invention under
the regulation of the 35S promoter exhibit improved harvest
index under nitrogen deficient growth conditions

Harvest Index

Gene Name	Event #	Average	p-value	% increment

CTF113	5871.1	0.05	2.4E−02	67.8%
CTF113	5871.2	0.04	4.9E−02	42.8%
Control		0.03		0.0%

Table 28: Analyses of harvest index of transgenic plants transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under high salinity irrigation conditions (80-100 mM NaCl) as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 29

Transgenic Arabidopsis plants exogenously expressing the
polynucleotides of some embodiments of the invention under
the regulation of the 35S promoter exhibit improved dry weight
under high salinity irrigationv growth conditions

Dry Weight

Gene Name	Event #	Average	p-value	% increment

CTF113	5871.2	0.82	4.3E−03	23.7%
CTF113	5873.3	0.83	2.9E−03	26.0%
Control		0.66		0.0%

Table 29: Analyses of dry weight of transgenic plants transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under high salinity irrigation conditions (80-100 mM NaCl) as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

The genes presented in Tables 30-31, hereinbelow, have improved plant performance and under regular growth conditions since they produced higher seed yield, harvest index, seed weight (expressed as 1000-seed weight) and plant biomass [(as expressed as plant dry weight (DW)] when grown under standard growth conditions, compared to control plants.
Tables 30-31 depict analyses of dry weight and seed yield (Table 30) and harvest index and seed weight (expressed as 1000-seed weight; Table 31) when grown under standard conditions (6 mM KNO₃, 1 mM KH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂and microelements) in plants overexpressing the polynucleotides of some embodiments of the invention under the regulation of a constitutive promoter (35S; SEQ ID NO:675). Evaluation of each gene was performed by testing the performance of several events. Some of the genes were evaluated in more than one tissue culture assay and the results obtained were repeated. Event with p-value <0.05 was considered statistically significant.

TABLE 30

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of
some embodiments of the invention under the regulation of the 35S promoter exhibit
improved plant biomass (dry weight) and seed yield under standard conditions

Gene

Dry Weight (g)

Gene

Seed Yield (g)

Name	Event #	Ave.	P-Value	% incr.	Name	Event #	Ave.	P-Value	% incr.

BDL103	7853.3	0.94	3.3E−02	37.0%	BDL103	7853.1	0.30	1.3E−02	32.4%
BDL103	8033.4	0.98	3.3E−02	42.3%	BDL103	8033.4	0.30	2.6E−02	36.2%
Control		0.69		0.0%	Control		0.22		0.0%
BDL103	8033.3	1.32	2.5E−02	31.8%	BDL103	8033.3	0.71	1.2E−04	43.7%
Control		1.00		0.0%	Control		0.49		0.0%

Table 30: Analyses of plant biomass (dry weight) and seed yield of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal growth conditions (6 mM KNO₃, 1 mM KH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂and microelements) as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 31

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of
some embodiments of the invention under the regulation of the 35S promoter exhibit
improved harvest index and seed weight under standard nitrogen conditions

Gene

Harvest Index

Gene

Seed Weight (g)

Name	Event#	Ave.	P-Value	% incr.	Name	Event #	Ave.	P-Value	% incr.

BDL14	5762.1	0.31	1.2E−02	45.1%	BDL103	7221.1	0.022	4.1E−03	22.3%
BDL14	5763.2	0.27	3.5E−02	29.6%	BDL103	7855.2	0.021	9.0E−03	18.3%
Control		0.21		0.0%	BDL103	8033.12	0.021	4.1E−02	19.5%
BDL14	5762.1	0.53	3.9E−02	8.4%	BDL103	8033.4	0.020	4.8E−02	13.3%
Control		0.49		0.0%	Control		0.018		0.0%

Table 31: Analyses of harvest index and seed weight of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under standard nitrogen conditions (6 mM KNO₃, 1 mM KH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂and microelements) as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

Example 8

Evaluation of Transgenic Arabidopsis Plant Growth Under Abiotic Stress as Well as Under Favorable Conditions in Greenhouse Assay Grown Until Bolting

This assay follows seed yield production, the biomass formation and the rosette area growth of plants grown in the greenhouse at high salinity and regular growth conditions. Transgenic Arabidopsis seeds were sown in agar media supplemented with ½ MS medium and a selection agent (Kanamycin). The T₂transgenic seedlings were then transplanted to 1.7 trays filled with peat and perlite. The trays were irrigated with tap water (provided from the pots' bottom). Half of the plants were irrigated with a salt solution (50-150 mM NaCl and 5 mM CaCl₂) so as to induce salinity stress (stress conditions). The other half of the plants was irrigated with tap water (normal conditions). All plants were grown in the greenhouse until 90% of plants reach bolting (inflorescent start to emerge). Plant biomass (the above ground tissue) was weighted immediately after harvesting the rosette (plant fresh weight [FW]). Following, plants were dried in an oven at 50° C. for 48 hours and weighted (plant dry weight [DW]).
Each construct was validated at its T₂generation. Transgenic plants transformed with a construct conformed by an empty vector carrying the 35S promoter and the selectable marker was used as control.
The plants were analyzed for their overall size, growth rate, fresh weight and dry matter. Transgenic plants performance was compared to control plants grown in parallel under the same conditions.
The experiment was planned in nested randomized plot distribution. For each gene of the invention three to five independent transformation events were analyzed from each construct.
Digital Imaging—
A laboratory image acquisition system, which consists of a digital reflex camera (Canon EOS 300D) attached with a 55 mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which includes 4 light units (4×150 Watts light bulb) was used for capturing images of plant samples.
The image capturing process was repeated every 2 days starting from day 1 after transplanting till day 15. Same camera, placed in a custom made iron mount, was used for capturing images of larger plants sawn in white tubs in an environmental controlled greenhouse. During the capture process, the tubes were placed beneath the iron mount, while avoiding direct sun light and casting of shadows.
An image analysis system was used, which consists of a personal desktop computer (Intel P4 3.0 GHz processor) and a public domain program—ImageJ 1.39 [Java based image processing program which was developed at the U.S. National Institutes of Health and freely available on the internet at Hypertext Transfer Protocol://rsbweb (dot) nih (dot) gov/]. Images were captured in resolution of 10 Mega Pixels (3888×2592 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group standard) format. Next, analyzed data was saved to text files and processed using the JMP statistical analysis software (SAS institute).
Leaf Analysis—
Using the digital analysis leaves data was calculated, including leaf number, rosette area, rosette diameter, leaf blade area, plot coverage and leaf petiole area.
Vegetative growth rate: is the rate of growth of the plant as defined by formula VIII, IX, X and XI as described in Example 7 hereinabove.
Plant Fresh and Dry Weight—
On about day 40 from sowing, the plants were harvested and directly weighted for the determination of the plant fresh weight (FW) and left to dry at 50° C. in a drying chamber for about 48 hours before weighting to determine plant dry weight (DW).
Statistical Analyses—
To identify genes conferring significantly improved ABST, the results obtained from the transgenic plants were compared to those obtained from control plants. To identify outperforming genes and constructs, results from the independent transformation events tested are analyzed separately. Data was analyzed using Student's t-test and results were considered significant if the p value was less than 0.1. The JMP statistics software package was used (Version 5.2.1, SAS Institute Inc., Cary, N.C., USA).
Experimental Results:
The genes presented in Tables 32-36, hereinbelow, were found to increase ABST when grown under high salinity irrigation conditions, compared to control plants. These genes produced larger plants with a larger photosynthetic capacity when grown under limiting nitrogen conditions.
Tables 32-36 depict analyses of plant biomass and photosynthetic area (fresh weight, dry weight, rosette diameter, rosette area and plot coverage) when grown under high salinity irrigation conditions (80-150 mM NaCl) in plants overexpressing the polynucleotides of some embodiments of the invention under the regulation of a constitutive promoter (At6669; SEQ ID NO:674). Evaluation of each gene was performed by testing the performance of several events. Some of the genes were evaluated in more than one tissue culture assay and the results obtained were repeated. Event with p-value <0.05 was considered statistically significant.

TABLE 32

Transgenic Arabidopsis plants exogenously expressing the
polynucleotides of some embodiments of the invention exhibit
improved plant biomass under high salinity conditions

Fresh weight (g)

Dry weight (g)

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB22	11064.6	0.57	3.0E−06	75.1%	LAB22	11064.6	0.07	1.0E−03	86.8%
LAB41	11552.1	0.40	1.8E−02	22.2%	LAB41	11551.2	0.07	2.4E−02	113.2%
Control		0.33		0.0%	Control		0.03		0.0%
LAB22	11062.1	0.57	1.5E−02	11.2%	LAB21	11144.1	0.07	6.4E−03	22.8%
LAB21	11144.1	0.63	9.3E−03	21.2%	LAB34	11175.1	0.07	1.8E−02	35.7%
LAB25	11341.2	0.64	3.4E−02	24.8%	LAB33	11272.4	0.06	2.0E−02	15.8%
LAB17	11533.1	0.58	8.1E−03	12.7%	LAB33	11273.1	0.06	2.0E−02	15.8%
LAB17	11533.6	0.66	1.5E−02	27.8%	LAB25	11341.2	0.06	1.3E−02	18.1%
LAB15	11641.1	0.56	3.9E−02	7.9%	LAB17	11533.6	0.07	1.9E−03	32.3%
Control		0.52		0.0%	LAB23	11571.2	0.08	1.2E−02	42.7%
LAB40	11154.1	0.75	1.2E−02	20.8%	LAB23	11571.5	0.07	5.7E−03	24.0%
LAB40	11154.4	0.86	8.2E−03	38.4%	LAB15	11642.2	0.07	3.8E−02	36.8%
LAB40	11154.5	0.76	2.0E−02	23.2%	Control		0.05		0.0%
LAB24	11193.1	0.84	3.9E−03	35.4%	LAB40	11151.1	0.09	1.4E−02	24.7%
LAB49	11281.2	0.84	1.0E−03	35.4%	LAB40	11154.5	0.08	4.2E−02	16.7%
LAB3	11333.9	0.83	5.0E−03	33.3%	LAB24	11193.1	0.09	3.9E−02	28.9%
LAB14	11471.1	0.92	2.9E−04	48.5%	LAB24	11193.5	0.09	2.5E−03	31.6%
LAB14	11474.1	0.87	7.6E−04	40.4%	LAB3	11333.9	0.08	4.6E−02	17.5%
LAB14	11474.3	0.76	4.5E−02	22.2%	LAB35	11461.2	0.08	3.1E−02	17.5%
LAB51	11563.1	0.83	1.4E−03	34.3%	LAB14	11471.1	0.10	9.4E−04	38.6%
Control		0.62		0.0%	LAB14	11474.1	0.09	1.3E−02	24.6%
LAB35	11462.3	0.77	1.7E−02	14.3%	LAB51	11561.2	0.10	5.7E−03	43.9%
LAB35	11462.5	0.75	3.9E−02	11.5%	LAB51	11563.1	0.09	9.7E−03	26.3%
LAB14	11472.1	0.88	7.8E−04	30.0%	Control		0.07		0.0%
Control		0.67		0.0%	LAB35	11462.3	0.09	2.3E−02	22.4%
					LAB35	11462.5	0.09	3.3E−02	18.9%
					LAB14	11472.1	0.09	1.2E−02	30.2%
					Control		0.07		0.0%

Table 32: Analyses of fresh weight and dry weight of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (6669) when grown under high salinity conditions as compared to control plants. “g” = grams. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 33

Transgenic Arabidopsis plants exogenously expressing the
polynucleotides of some embodiments of the invention exhibit
improved plant biomass under high salinity conditions

Rosette diameter (cm)

Rosette area (cm²)

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB22	11064.6	2.28	3.4E−02	25.5%	LAB22	11064.6	1.88	2.6E−03	49.0%
LAB41	11551.2	2.49	1.9E−02	37.3%	LAB41	11551.2	2.14	3.3E−02	70.0%
LAB41	11552.1	2.10	9.9E−03	15.8%	LAB41	11552.1	1.50	4.1E−02	18.8%
Control		1.81		0.0%	Control		1.26		0.0%
LAB21	11144.1	2.69	2.4E−02	12.6%	LAB21	11144.1	2.31	1.1E−02	27.4%
LAB25	11341.2	2.77	7.1E−03	16.2%	LAB25	11341.2	2.44	7.1E−03	34.4%
LAB17	11531.6	2.67	9.1E−03	12.1%	LAB17	11531.6	2.17	3.3E−02	19.7%
LAB15	11642.2	2.80	5.2E−03	17.4%	LAB17	11533.6	2.65	8.5E−03	45.7%
Control		2.39		0.0%	LAB15	11642.2	2.45	6.2E−03	35.1%
LAB40	11154.4	2.92	2.6E−03	24.3%	Control		1.82		0.0%
LAB40	11154.5	2.80	6.5E−03	19.2%	LAB40	11151.1	2.42	1.6E−02	44.8%
LAB24	11193.1	2.70	2.9E−02	15.3%	LAB40	11154.4	2.82	2.2E−02	68.2%
LAB24	11193.5	2.71	4.0E−02	15.7%	LAB40	11154.5	2.36	3.6E−03	40.7%
LAB49	11281.2	2.72	1.3E−02	16.0%	LAB24	11193.1	2.17	3.9E−02	29.9%
LAB5	11443.4	2.63	3.5E−02	12.2%	LAB49	11281.2	2.42	2.6E−03	44.8%
LAB5	11444.1	3.02	1.0E−03	28.8%	LAB3	11333.9	2.43	3.3E−02	45.4%
LAB5	11444.5	2.86	3.5E−03	21.9%	LAB5	11443.4	2.18	1.3E−02	30.2%
LAB51	11561.1	2.74	3.1E−02	17.0%	LAB5	11444.1	2.78	8.6E−03	66.1%
LAB51	11561.2	2.81	2.5E−02	19.8%	LAB5	11444.5	2.52	2.4E−02	50.3%
LAB51	11563.1	2.80	5.7E−03	19.3%	LAB51	11561.1	2.27	6.6E−03	35.5%
Control		2.34		0.0%	LAB51	11561.2	2.47	2.5E−03	47.6%
					LAB51	11563.1	2.37	2.1E−02	41.5%
					Control		1.67		0.0%

Table 33: Analyses of rosette diameter and area of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (6669) when grown under high salinity conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 34

Transgenic Arabidopsis plants exogenously expressing the
polynucleotides of some embodiments of the invention exhibit
improved plant biomass under high salinity conditions

Plot coverage (cm²)

Leaf number

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB22	11064.6	15.03	2.6E−03	49.0%	LAB16	11033.2	8.13	5.0E−04	12.3%
LAB41	11551.2	17.15	3.3E−02	70.0%	LAB16	11034.1	7.94	2.3E−03	9.7%
LAB41	11552.1	11.98	4.1E−02	18.8%	LAB22	11062.1	7.63	3.2E−02	5.4%
Control		10.09		0.0%	LAB22	11064.6	8.00	1.3E−02	10.6%
LAB21	11144.1	18.51	1.1E−02	27.4%	Control		7.23		0.0%
LAB25	11341.2	19.53	7.1E−03	34.4%	LAB5	11441.1	8.41	1.7E−02	13.1%
LAB17	11531.6	17.40	3.3E−02	19.7%	Control		7.44		0.0%
LAB17	11533.6	21.17	8.5E−03	45.7%
LAB15	11642.2	19.62	6.2E−03	35.1%
Control		14.53		0.0%
LAB40	11154.1	16.79	3.3E−02	25.4%
LAB40	11154.4	22.53	2.2E−02	68.2%
LAB40	11154.5	18.84	3.6E−03	40.7%
LAB24	11193.1	17.40	3.9E−02	29.9%
LAB49	11281.2	19.40	2.6E−03	44.8%
LAB3	11333.9	19.48	3.3E−02	45.4%
LAB5	11443.4	17.44	1.3E−02	30.2%
LAB5	11444.1	22.24	8.6E−03	66.1%
LAB5	11444.5	20.13	2.4E−02	50.3%
LAB51	11561.1	18.15	6.6E−03	35.5%
LAB51	11561.2	19.77	2.5E−03	47.6%
LAB51	11563.1	18.95	2.1E−02	41.5%
Control		13.39		0.0%

Table 34: Analyses of plot coverage and leaf number of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 32 above) under the regulation of a constitutive promoter (6669) when grown under high salinity conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 35

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention exhibit improved plant biomass
and growth rate under high salinity conditions

RGR of rosette area	RGR of rosette diameter
(Regression coefficient)	(Regression coefficient)

LAB11	11024.3	0.24	2.9E−02	46.4%	LAB11	11024.3	0.23	2.1E−02	32.4%
LAB16	11033.2	0.29	1.6E−03	75.5%	LAB16	11033.2	0.25	2.4E−03	44.8%
LAB16	11034.1	0.33	4.9E−05	101.2%	LAB16	11034.1	0.28	8.6E−05	58.1%
LAB22	11062.1	0.26	5.9E−03	59.3%	LAB16	11034.4	0.24	7.6E−03	39.2%
LAB22	11062.3	0.25	2.5E−02	51.6%	LAB22	11062.1	0.24	5.1E−03	37.9%
LAB22	11064.6	0.25	6.5E−03	55.1%	LAB22	11064.6	0.24	3.6E−03	37.4%
LAB41	11551.2	0.29	4.4E−04	76.7%	LAB41	11551.2	0.26	4.5E−04	47.8%
Control		0.16		0.0%	Control		0.17		0.0%
LAB21	11144.1	0.30	3.9E−02	27.0%	LAB25	11341.2	0.28	4.7E−02	15.9%
LAB25	11341.2	0.32	1.4E−02	34.6%	LAB13	Control	0.24		0.0%
LAB17	11533.6	0.34	2.5E−03	43.8%	LAB3	11333.9	0.30	3.9E−02	25.6%
LAB17	11534.1	0.31	4.4E−02	29.0%	Control		0.24		0.0%
LAB23	11571.2	0.34	1.4E−02	40.4%
LAB23	11571.5	0.32	1.1E−02	34.6%
LAB15	11642.2	0.32	1.2E−02	33.1%
Control		0.24		0.0%
LAB40	11151.1	0.31	2.5E−02	45.7%
LAB40	11154.4	0.36	2.7E−03	65.8%
LAB49	11281.2	0.31	4.4E−02	41.6%
LAB49	11281.4	0.35	6.3E−03	62.6%
LAB3	11333.9	0.31	3.3E−02	45.8%
LAB5	11444.1	0.36	3.5E−03	65.6%
LAB5	11444.5	0.32	1.8E−02	50.1%
LAB51	11561.2	0.32	2.6E−02	47.4%
LAB51	11563.1	0.31	3.9E−02	42.9%
Control		0.22		0.0%

Table 35: Analyses of relative growth rate (RGR) of rosette area and diameter of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (6669) when grown under high salinity conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 36

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention exhibit improved plant biomass
and growth rate under high salinity conditions

RGR of plot coverage

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB11	11024.3	1.90	2.9E−02	46.4%	LAB40	11151.1	2.80	6.3E−03	62.6%
LAB16	11033.2	2.28	1.6E−03	75.5%	LAB40	11154.4	2.51	3.3E−02	45.8%
LAB16	11034.1	2.61	4.9E−05	101.2%	LAB49	11281.2	2.85	3.5E−03	65.6%
LAB22	11062.1	2.07	5.9E−03	59.3%	LAB49	11281.4	2.59	1.8E−02	50.1%
LAB22	11062.3	1.97	2.5E−02	51.6%	LAB3	11333.9	2.54	2.6E−02	47.4%
LAB22	11064.6	2.02	6.5E−03	55.1%	LAB5	11444.1	2.46	3.9E−02	42.9%
LAB41	11551.2	2.30	4.4E−04	76.7%	LAB5	11444.5	1.90	2.9E−02	46.4%
Control		2.43	3.9E−02	27.0%	LAB51	11561.2	2.28	1.6E−03	75.5%
LAB21	11144.1	2.58	1.4E−02	34.6%	LAB51	11563.1	2.61	4.9E−05	101.2%
LAB25	11341.2	2.76	2.5E−03	43.8%	Control		2.07	5.9E−03	59.3%
LAB17	11533.6	2.47	4.4E−02	29.0%	LAB40	11151.1	2.80	6.3E−03	62.6%
LAB17	11534.1	2.69	1.4E−02	40.4%	LAB40	11154.4	2.51	3.3E−02	45.8%
LAB23	11571.2	2.58	1.1E−02	34.6%	LAB49	11281.2	2.85	3.5E−03	65.6%
LAB23	11571.5	2.55	1.2E−02	33.1%	LAB49	11281.4	2.59	1.8E−02	50.1%
LAB15	11642.2	2.86	2.7E−03	65.8%	LAB3	11333.9	2.54	2.6E−02	47.4%
Control		2.44	4.4E−02	41.6%	LAB5	11444.1	2.46	3.9E−02	42.9%
					LAB5	11444.5	1.90	2.9E−02	46.4%
					LAB51	11561.2	2.28	1.6E−03	75.5%
					LAB51	11563.1	2.61	4.9E−05	101.2%
					Control		2.07	5.9E−03	59.3%

Table 36: Analyses of relative growth rate (RGR) of plot coverage of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (6669) when grown under high salinity conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

Tables 37-41 depict analyses of plant biomass, growth rate and photosynthetic area (fresh weight, dry weight, rosette diameter, rosette area and plot coverage) when grown under normal conditions in plants overexpressing the polynucleotides of some embodiments of the invention under the regulation of a constitutive promoter (At6669; SEQ ID NO:674). Evaluation of each gene was performed by testing the performance of several events. Some of the genes were evaluated in more than one tissue culture assay and the results obtained were repeated. Event with p-value <0.05 was considered statistically significant.

TABLE 37

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention exhibit improved plant biomass under normal conditions

Fresh weight (g)

Dry weight (g)

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB16	11033.2	1.13	2.4E−02	125.3%	LAB16	11033.2	0.11	0.0E+00	111.5%
LAB22	11064.6	1.19	0.0E+00	139.1%	LAB22	11062.1	0.09	4.0E−06	72.2%
LAB11	Control	0.50		0.0%	LAB22	11064.6	0.11	0.0E+00	114.0%
LAB38	11434.3	2.59	4.5E−02	28.7%	Control		0.05		0.0%
Control		2.02		0.0%	LAB40	11151.1	0.16	6.7E−03	18.8%
LAB40	11151.1	1.81	2.9E−02	16.9%	LAB40	11154.5	0.15	1.7E−02	13.1%
LAB40	11154.5	1.81	2.9E−02	16.9%	LAB39	11182.1	0.17	5.6E−03	27.2%
LAB39	11182.1	1.98	1.4E−03	27.4%	LAB24	11193.1	0.16	6.3E−03	16.9%
LAB24	11193.1	1.84	1.2E−02	19.0%	LAB49	11281.6	0.18	1.8E−04	36.6%
LAB49	11281.6	2.19	2.6E−05	41.1%	LAB5	11444.1	0.16	5.2E−03	23.0%
LAB3	11331.1	1.74	1.4E−02	12.1%	LAB35	11461.2	0.19	1.4E−02	46.0%
LAB5	11444.1	2.07	8.1E−03	33.5%	LAB14	11471.1	0.15	1.8E−02	13.6%
LAB35	11461.2	2.30	1.0E−05	48.4%	LAB51	11561.5	0.15	2.3E−02	15.5%
LAB35	11462.5	1.91	1.2E−03	23.0%	Control		0.13		0.0%
LAB14	11474.1	1.80	3.3E−02	16.1%
Control		1.55		0.0%
LAB49	11281.6	1.84	3.8E−02	9.9%
Control		1.68		0.0%

Table 37: Analyses of fresh weight and dry weight of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under noraml conditions as compared to control plants, “g” = grams. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 38

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention exhibit improved plant biomass normal conditions

Rosette diameter (cm)

Rosette area (cm²)

Gene					Gene
Name	Event #	Ave.	p-value	% Incr.	Name	Event #	Ave.	p-value	% Incr.

LAB16	11033.2	3.00	3.5E−02	73.6%	LAB16	11033.2	2.91	9.5E−03	150.9%
LAB22	11064.6	2.84	0.0E+00	64.4%	LAB22	11064.6	2.85	0.0E+00	145.9%
LAB41	11551.4	1.95	3.7E−02	12.8%	Control		1.16		0.0%
Control		1.73		0.0%	LAB38	11434.3	4.48	4.1E−02	46.7%
LAB40	11151.1	3.31	6.5E−03	17.4%	Control		3.05		0.0%
LAB40	11154.1	3.82	2.3E−02	35.7%	LAB40	11151.1	3.19	2.4E−04	42.6%
LAB39	11182.1	3.30	1.0E−03	17.2%	LAB39	11182.1	3.05	5.8E−03	36.0%
LAB24	11192.1	3.23	2.4E−03	14.7%	LAB24	11192.1	2.90	1.9E−03	29.5%
LAB24	11193.1	3.21	2.8E−03	13.9%	LAB24	11193.1	2.77	1.2E−02	23.6%
LAB49	11281.4	3.33	2.0E−03	18.1%	LAB49	11281.4	2.91	1.7E−03	29.7%
LAB49	11281.6	3.57	1.5E−04	26.6%	LAB49	11281.6	3.26	1.8E−04	45.4%
LAB3	11333.1	3.22	2.4E−03	14.3%	LAB3	11333.1	2.76	2.3E−02	23.1%
LAB3	11333.9	3.24	5.2E−03	15.1%	LAB3	11333.9	2.92	4.1E−02	30.4%
LAB5	11444.1	3.42	3.6E−04	21.3%	LAB5	11443.3	3.19	1.8E−02	42.4%
LAB35	11461.2	3.81	1.8E−02	35.3%	LAB5	11444.1	3.24	2.5E−04	44.7%
LAB51	11561.5	3.30	1.3E−03	17.0%	LAB35	11461.2	4.25	1.8E−02	89.8%
Control		2.82		0.0%	LAB51	11561.5	2.77	9.7E−03	23.6%
					Control		2.24		0.0%

Table 38: Analyses of rosette diameter and area of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 39

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention exhibit improved plant biomass under normal conditions

Plot coverage (cm²)

Leaf number

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB16	11033.2	23.25	9.5E−03	150.9%	LAB16	11032.5	8.44	4.1E−02	10.9%
LAB22	11064.6	22.79	0.0E+00	145.9%	LAB22	11064.6	8.69	9.0E−04	14.2%
Control		9.27		0.0%	Control		7.61		0.0%
LAB38	11434.3	35.81	4.1E−02	46.7%	LAB39	11182.1	8.25	3.2E−02	8.5%
Control		24.41		0.0%	LAB49	11283.6	8.31	1.1E−02	9.3%
LAB40	11151.1	25.56	2.4E−04	42.6%	LAB35	11461.2	9.00	1.3E−03	18.4%
LAB39	11182.1	24.38	5.8E−03	36.0%	Control		7.60		0.0%
LAB24	11192.1	23.21	1.9E−03	29.5%
LAB24	11193.1	22.15	1.2E−02	23.6%
LAB49	11281.4	23.25	1.7E−03	29.7%
LAB49	11281.6	26.05	1.8E−04	45.4%
LAB3	11333.1	22.06	2.3E−02	23.1%
LAB3	11333.9	23.38	4.1E−02	30.4%
LAB5	11443.3	25.53	1.8E−02	42.4%
LAB5	11444.1	25.93	2.5E−04	44.7%
LAB35	11461.2	34.01	1.8E−02	89.8%
LAB51	11561.5	22.16	9.7E−03	23.6%
Control		17.92		0.0%

Table 39: Analyses of plot coverage and leaf number of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 40

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention exhibit improved plant biomass
and growth rate under normal conditions

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB16	11032.5	0.33	9.2E−04	121.8%	LAB16	11032.5	0.27	1.7E−03	65.7%
LAB16	11033.2	0.39	0.0E+00	163.5%	LAB16	11033.2	0.32	0.0E+00	97.3%
LAB22	11062.1	0.30	4.3E−05	101.9%	LAB22	11062.1	0.29	3.0E−06	77.5%
LAB22	11063.4	0.29	7.0E−03	93.7%	LAB22	11063.4	0.25	8.5E−03	55.1%
LAB22	11064.1	0.21	4.9E−02	43.5%	LAB22	11064.1	0.21	3.2E−02	29.1%
LAB22	11064.6	0.39	0.0E+00	161.4%	LAB22	11064.6	0.31	0.0E+00	92.7%
Control		0.15		0.0%	LAB41	11551.4	0.20	4.0E−02	22.1%
LAB17	11534.1	0.66	4.6E−02	60.4%	Control		0.16		0.0%
Control		0.41		0.0%	LAB40	11154.1	0.39	5.1E−03	31.3%
LAB40	11151.1	0.42	2.6E−02	42.0%	LAB49	11281.6	0.38	9.4E−03	29.2%
LAB40	11154.1	0.51	7.6E−04	75.6%	LAB35	11461.2	0.39	6.0E−03	31.7%
LAB39	11182.1	0.41	3.6E−02	38.3%	Control		0.29		0.0%
LAB49	11281.6	0.43	2.0E−02	45.4%
LAB5	11443.3	0.42	2.1E−02	43.5%
LAB5	11444.1	0.42	2.2E−02	44.5%
LAB5	11444.5	0.45	1.9E−02	53.3%
LAB35	11461.2	0.56	1.6E−04	89.8%
Control		0.29		0.0%

Table 40: Analyses of relative growth rate (RGR) of rosette area and diameter of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 41

Transgenic Arabidopsis plants exogenously expressing the polynucleotides
of some embodiments of the invention exhibit improved plant biomass
and growth rate under normal conditions

RGR of plot coverage

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

LAB16	11032.5	2.64	9.2E−04	121.8%	LAB40	11151.1	3.33	2.6E−02	42.0%
LAB16	11033.2	3.14	0.0E+00	163.5%	LAB40	11154.1	4.12	7.6E−04	75.6%
LAB22	11062.1	2.40	4.3E−05	101.9%	LAB39	11182.1	3.24	3.6E−02	38.3%
LAB22	11063.4	2.30	7.0E−03	93.7%	LAB49	11281.6	3.41	2.0E−02	45.4%
LAB22	11064.1	1.71	4.9E−02	43.5%	LAB5	11443.3	3.36	2.1E−02	43.5%
LAB22	11064.6	3.11	0.0E+00	161.4%	LAB5	11444.1	3.39	2.2E−02	44.5%
Control		1.19		0.0%	LAB5	11444.5	3.59	1.9E−02	53.3%
LAB17	11534.1	5.31	4.6E−02	60.4%	LAB35	11461.2	4.45	1.6E−04	89.8%
Control		3.31		0.0%	Control		2.34		0.0%

Table 41: Analyses of relative growth rate (RGR) of plot coverage of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (At6669; SEQ ID NO: 674) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

Tables 42-46 depict analyses of plant biomass and photosynthetic area (fresh weight, dry weight, rosette diameter, rosette area and plot coverage) when grown under normal conditions in plants overexpressing the polynucleotides of some embodiments of the invention under the regulation of a constitutive promoter (35S; SEQ ID NO:675). Evaluation of each gene was performed by testing the performance of several events. Some of the genes were evaluated in more than one tissue culture assay and the results obtained were repeated. Event with p-value <0.05 was considered statistically significant.

TABLE 42

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention exhibit improved plant biomass under normal conditions

Fresh weight (g)

Dry weight (g)

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

BDL210	10834.3	1.83	4.6E−02	17.1%	BDL210	10831.3	0.16	3.9E−02	16.4%
Control		1.56		0.0%	BDL210	10833.1	0.19	4.5E−02	32.0%
CTF226	10985.2	1.90	4.3E−02	10.7%	BDL210	10834.3	0.17	8.8E−03	23.1%
Control		1.72		0.0%	Control		0.14		0.0%

Table 42: Analyses of fresh and dry weight of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under noraml conditions as compared to control plants. “g” = grams. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 43

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention exhibit improved plant biomass normal conditions

Rosette diameter (cm)

Rosette area (cm²)

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

BDL210	10831.3	3.65	2.7E−02	6.5%	BDL210	10831.3	4.21	1.5E−02	16.9%
Control		3.43		0.0%	BDL210	10834.2	4.21	4.8E−02	17.0%
					Control		3.60		0.0%

Table 43: Analyses of rosette diameter and area of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 44

Transgenic Arabidopsis plants exogenously expressing the polynucleotides of some
embodiments of the invention exhibit improved plant biomass under normal conditions

Plot coverage (cm²)

Leaf number

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

BDL210	10831.3	33.67	1.5E−02	16.9%	BDL210	10833.1	9.31	6.4E−03	6.2%
BDL210	10834.2	33.70	4.8E−02	17.0%	Control		8.77		0.0%
		28.80		0.0%

Table 44: Analyses of plot coverage and leaf number of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 45

Transgenic plants exogenously expressing the polynucleotides of some embodiments
of the invention exhibit improved plant biomass under normal conditions

RGR of rosette area

RGR of rosette diameter

Gene					Gene
Name	Event #	Ave.	p-value	% incr.	Name	Event #	Ave.	p-value	% incr.

BDL210	10833.1	0.81	2.4E−03	66.5%	BDL210	10833.1	0.46	3.8E−02	21.9%
Control		0.49		0.0%	Control		0.38		0.0%

Table 45: Analyses of relative growth rate (RGR) of rosette area and diameter of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

TABLE 46

Transgenic Arabidopsis plants exogenously expressing the
polynucleotides of some embodiments of the invention
exhibit improved plant biomass under normal conditions
RGR of plot coverage

	Gene Name	Event #	Ave.	p-value	% incr.

BDL210	10833.1	6.50	2.4E−03	66.5%
Control		3.90		0.0%

Table 46: Analyses of relative growth rate (RGR) of plot coverage of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Incr.” = increment with respect to a control plant which has been transformed with an empty vector. Ave. = Average calculated from several transgenic events. “Event #” = number of event (transgenic transformation).

Example 9

Improved Transgenic Plant Performance Under Normal Conditions

To analyze whether the transgenic plants has performed better, plants were grown in pots with an adequate amount of nutrient and water. The plants were analyzed for their overall size, growth rate, time to inflorescence emergence (bolting) and flowering, seed yield, oil content of seed, weight of 1,000 seeds, dry matter and harvest index (HI-seed yield/dry matter). Transgenic plants performance was compared to control plants grown in parallel under the same conditions. Mock-transgenic plants expressing the uidA reporter gene (GUS-Intron) under the same promoter were used as control.
Parameters were measured as described in Examples 6, 7 and 8 above.
Statistical Analyses—
To identify genes conferring significantly improved plant performance, the results obtained from the transgenic plants were compared to those obtained from control plants. Plant growth rate, plant area, time to bolt, time to flower, weight of 1,000 seeds, seed yield, total yield, oil yield, oil percent in seeds, dry matter, harvest index, rosette area and growth rate data were analyzed using one-way ANOVA. To identify outperforming genes and constructs, results from mix of transformation events or independent events tested were analyzed. The Least Mean Squares were calculated for each experiment. For gene versus control analysis T-test was applied, using significance of p<0.05. The JMP statistics software package was used (Version 5.2.1, SAS Institute Inc., Cary, N.C., USA).
Experimental Results
The polynucleotide sequences of the invention were assayed for a number of commercially desired traits.
Tables 47-57 depict analyses of seed yield (Table 47), oil yield (Table 48), dry matter (Table 49), harvest index (HI) (Tables 50 and 51), growth rate (Table 52), rosette area (Table 53), oil % in seed (Table 54), weight of 1000 seeds (Tables 55 and 56) and total yield (Table 57) in plants overexpressing the polynucleotides of some embodiments of the invention under the regulation of a constitutive (35S; SEQ ID NO:675) or a seed specific (napin; SEQ ID NO:676) promoter. Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B) are significantly different (p<0.05) from the control.

TABLE 47

Genes showing improved plant performance
Arabidopsis: Seed yield

Seed yield per plant (g)

	Under		Significance	%
	regula-	Least	(t-Test compare	improve-
Gene Id	tion of	Mean Sq	to control)	ment

BDL11	35S	0.420	A	4.2
BDL17	35S	0.426	A	5.8
CONTROL	35S	0.403	A	0.0
(GUS Intron)
BDL12	35S	0.319	B	9.7
BDL14	35S	0.378	A	30.3
CONTROL	35S	0.290	B	0.0
(GUS Intron)

Table 47: Analyses of seed yield per plant of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant seed yield as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 48

Genes showing improved plant performance Arabidopsis: Oil yield

Oil yield per plant (gr)

BDL11	35S	0.12	A	7.0
BDL17	35S	0.12	A	6.5
CONTROL (GUS	35S	0.12	A	0.0
Intron)
BDL12	35S	0.100	B	14.2
BDL14	35S	0.114	A	31.1
CONTROL (GUS	35S	0.087	B	0.0
Intron)

Table 48: Analyses of oil yield per plant of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant oil yiel as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 49

Genes showing improved plant performance
Arabidopsis: Dry matter

Dry matter per plant (gr)

	Under		Significance	%
	regula-	Least	(t-Test compare	improve-
Gene Id	tion of	Mean Sq	to control)	ment

BDL14	35S	1.0444	A	9.7
CONTROL (GUS	35S	0.9523	A	0.0
Intron)
BDL11	35S	1.3638	A	1.2
CONTROL (GUS	35S	1.3474	A	0.0
Intron)

Table 49. Analyses of dry matter per plant of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant dry matter as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 50

Genes showing improved plant performance
Arabidopsis: harvest index (HI)

HI

BDL11	35S	0.3063	B	2.0
BDL17	35S	0.3526	A	17.5
CONTROL (GUS	35S	0.3002	B	0.0
Intron)

Table 50. Analyses of harvest index of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic harvest index as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 51

Genes showing improved plant performance
Arabidopsis: Harvest index

Harvest index

	Under		Significance	%
	regula-		(t-Test compare	improve-
Gene Id	tion of	Mean	to control)	ment

BDL103	35S	0.341	A	16.8
CONTROL (GUS	35S	0.292	B	0
Intron)

Table 51. Analyses of harvest index of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant harvest index as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 52

Genes showing improved plant performance
Arabidopsis: Growth rate

Growth rate (cm²/day)

BDL14	35S	2.48	A	6.4
CONTROL (GUS	35S	2.33	A	0.0
Intron)
BDL11	35S	1.80	A	15.4
CONTROL (GUS	35S	1.56	A	0.0
Intron)
BDL12	35S	1.58	B	2.0
BDL14	35S	1.95	A	26.3
CONTROL (GUS	35S	1.55	B	0.0
Intron)

Table 52. Analyses of growth rate of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant growth rate as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 53

Genes showing improved plant performance
Arabidopsis: Rossete area

Rosette area (cm²)

BDL14	35S	11.83	A	9.2
CONTROL (GUS	35S	10.83	B	0.0
Intron)
BDL11	35S	14.09	A	13.2
CONTROL (GUS	35S	12.44	A	0.0
Intron)
BDL12	35S	7.92	B	−2.5
BDL14	35S	9.96	A	22.7
CONTROL (GUS	35S	8.12	B	0.0
Intron)

Table 53: Analyses of rosette area of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant rosette area as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter. It should be noted that an increase in rosette area means better soil coverage and reduced water loss from soil. Decrease in rosette area means more plants could be put per area increasing yield.

TABLE 54

Genes showing improved plant performance
Arabidopsis: oil % in seed

Oil % in seed

BDL14	35S	31.31	A	3.1
CONTROL (GUS	35S	30.355	A	0.0
Intron)
BDL11	35S	29.216	A	1.5
BDL17	35S	28.904	A	0.4
CONTROL (GUS	35S	28.78	A	0
Intron)
BDL12	35S	31.30	A	3.7
BDL14	35S	30.27	A	0.3
CONTROL (GUS	35S	30.19	A	0.0
Intron)

Table 54. Analyses of oil percent in seed of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant oil percent in seed as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 55

Genes showing improved plant performance
Arabidopsis: weight of 1,000 seeds

Weight of 1000 seeds (gr)

BDL14	35S	0.019	B	6.1
CONTROL (GUS	35S	0.018	B	0.0
Intron)
BDL11	35S	0.0235	A	15.7
CONTROL (GUS	35S	0.0203	B	0
Intron)
BDL12	35S	0.0234	A	0.1
CONTROL (GUS	35S	0.0234	A	0.0
Intron)

Table 55. Analyses of weight of 1,000 seeds of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant weight of 1,000 seeds as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 56

Genes showing improved plant performance
Arabidopsis: weight of 1,000 seeds

Weight of 1000 seeds (gr)

	Under		Significance	%
	regula-	Least	(t-Test compare	improve-
Gene Id	tion of	Mean Sq	to control)	ment

BDL14	Napin	0.0227	A	2.3
CONTROL (GUS	Napin	0.0222	A	0.0
Intron)
BDL12	Napin	0.0206	A	0.2
CONTROL (GUS	Napin	0.0205	A	0.0
Intron)

Table 56. Analyses of weight of 1,000 seeds of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a seed specific napin promoter (SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant weight of 1,000 seeds as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

TABLE 57

Genes showing improved plant performance
Arabidopsis: total yield

total yield (gr/plant)

BDL103	35S	0.305	A	10.1
CONTROL (GUS	35S	0.277	B	0
Intron)

Table 57. Analyses of total yield per plant of transgenic plants overexpressing the exogenous polynucleotides of some embodiments of the invention (using the cloned or synthetic genes listed in Table 10 above) under the regulation of a constitutive promoter (35S promoter; SEQ ID NO: 675) when grown under normal conditions as compared to control plants. “Least Mean Sq” = Least Mean Square. “% improvement” relates to improvement of transgenic plant total yield as compared to control plants that have been transformed with a vector comprising GUS intron under the transcriptional regulation of the same promoter.

Example 10

Transgenic Arabidosis which Exogenously Express BDL103 Exhibit Increased Commercially Desired Traits in a Tissue Culture Assay

Nitrogen Use Efficiency—
Tissue culture assays were performed as described in Example 6 hereinabove for determining plant performance under normal (i.e., 15 mM nitrogen) or nitrogen deficiency (i.e., 0.75 mM nitrogen) conditions.
Abiotic Stress Tolerance—
To determine whether the transgenic plants exhibit increased tolerance to abiotic stress such as drought, an osmotic stress was induced by adding sorbitol or polyethylene glycol (PEG 8000) to the culturing medium. Control and transgenic plants were germinated and grown in plant-agar plates for 10 days, after which they were transferred to plates containing either 1.5% PEG8000 or 500 mM of sorbitol. Plants were grown under the osmotic stress conditions or the normal conditions for about additional 10 days, during which various parameters which indicate plant characteristics were measured. The measured parameters [e.g., plant weight (fresh and dry), yield, growth rate] were compared between the control and transgenic plants.
Tables 58-60 depict analyses of root coverage, root length, growth rate of root coverage, growth rate of root length and biomass in plants overexpressing the BDL103-short (SEQ ID NO:671) and BDL103-long (SEQ ID NO:670) polynucleotides under the regulation of a constitutive (35S; SEQ ID NO:675) when grown under normal conditions (Table 58), under nitrogen limiting conditions (Table 59), or under osmotic stress (15% PEG). Each Table includes data of several transformation events per gene. Results were considered significant if p-value was lower than 0.1 when compared to control plants (which were transformed with a vector containing GUS reporter gene).

TABLE 58

Improved growth rate, root coverage, root length and biomass in transgenic
Arabidopsis plants exogenously expressing BDL103 under normal conditions

BDL103 Long or Short/Event No.

Long/	Long/	Long/	Long/	Long/	Short/	Short/	Short/	Short/	Short/
3054	3055	3056	3057	3058	3060	3061	3062	3063	3064

Roots	P			0.10
Coverage	A			1.21
(time point 1)
Roots	P	0.08		0.25
Coverage	A	1.36		1.12
(time point 6)
Roots	P	0.07
Coverage	A	1.23
(time point 9)
Roots Length	P			0.03
(time point 1)	A			1.19
Roots Length	P	0.05		0.22
(time point 6)	A	1.17		1.10
Roots Length	P	0.01
(time point 9)	A	1.15
GR (growth	P	0.07		0.46
rate) of Roots	A	1.47		1.10
Coverage
(time point 6)
GR of Roots	P	0.04
Length (time	A	1.31
point 6)
RGR of	P	0.06	0.70			0.13	0.02	0.10
Roots	A	1.56	1.16			1.31	1.49	1.28
Coverage
(time point 6)
RGR of	P				0.23
Roots	A				1.87
Coverage
(time point 9)
RGR of	P	0.08	0.74				0.21	0.11
Roots Length	A	1.37	1.11				1.16	1.13
(time point 6)
RGR of	P				0.23		0.41
Roots Length	A				1.62		1.12
(time point 9)
DW [gr]	P	0.56
(time point 1)	A	1.13
DW [gr]	P	0.56
(time point 6)	A	1.13
DW [gr]	P	0.56
(time point 9)	A	1.13

Table 58. Analysis of growth parameters in tissue culture conditions of transgenic plants overexpressing BDL103-Short polynucleotide (SEQ ID NO: 671) or BDL103-Long polynucleotide (SEQ ID NO: 670) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under normal conditions (15 mM nitrogen). Each event number refers to an independent transformation event in a plant (i.e., generation of a transgenic plant expressing the polynucleotide of choice). “A” = average; “P” = p-value; “GR” = growth rate; “RGR” = relative growth rate; “DW” = dry weight; “gr” = grams; Root coverage is presented in cm²; root length is presented in cm; GR of root length is presented in cm/day; RGR of root length is presented in cm/day; RGR of root coverage is presented in cm²/day. The various time points indicate days from beginning of experiment in which parameters were measured.

TABLE 59

Improved growth rate, root coverage, root length and biomass in transgenic
plants exogenously expressing BDL103 under nitrogen limiting conditions

BDL103 Long or Short/Event No.

Roots	P		0.34			0.03
Coverage	A		1.10			1.26
(time point 6)
Roots	P		0.05			0.00			0.27
Coverage	A		1.19			1.40			1.18
(time point 9)
Roots Length	P					0.04
(time point 1)	A					1.11
Roots Length	P					0.00
(time point 6)	A					1.20
Roots Length	P		0.01			0.00
(time point 9)	A		1.14			1.25
GR of Roots	P		0.21			0.03
Coverage	A		1.19			1.31
(time point 6)
GR of Roots	P	0.53	0.09		0.37	0.01			0.14	0.34
Coverage	A	1.19	1.37		1.27	1.70			1.66	1.26
(time point 9)
GR of Roots	P		0.11			0.01
Length (time	A		1.21			1.25
point 6)
GR of Roots	P		0.06		0.09	0.01			0.07	0.37
Length (time	A		1.28		1.28	1.39			1.39	1.11
point 9)
RGR of	P		0.08		0.00	0.21		0.06	0.04		0.00
Roots	A		1.55		1.69	1.20		1.39	1.57		1.91
Coverage
(time point 6)
RGR of	P	0.04	0.33	0.11	0.22	0.23	0.14	0.45	0.13	0.02	0.31
Roots	A	2.06	1.23	1.95	1.43	1.32	1.52	1.23	1.71	2.53	1.50
Coverage
(time point 9)
RGR of	P		0.07		0.01	0.32		0.31	0.12		0.05
Roots Length	A		1.34		1.39	1.11		1.14	1.24		1.39
(time point 6)
RGR of	P	0.21	0.38	0.21	0.06	0.33	0.19	0.33	0.06	0.08	0.55
Roots Length	A	1.35	1.15	1.27	1.32	1.13	1.29	1.22	1.40	1.67	1.22
(time point 9)
DW [gr]	P		0.13
(time point 1)	A		1.22
DW [gr]	P		0.13
(time point 6)	A		1.22
DW [gr]	P		0.13
(time point 9)	A		1.22

Table 59. Analysis of growth parameters in tissue culture conditions of transgenic plants overexpressing BDL103-Short polynucleotide (SEQ ID NO: 671) or BDL103-Long polynucleotide (SEQ ID NO: 670) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under nitrogen limiting conditions (N 0.75 mM; see example 6). Each event number refers to an independent transformation event in a plant (i.e., generation of a transgenic plant expressing the polynucleotide of choice). “A” = average; “P” = p-value; “RGR” = relative growth rate; “DW” = dry weight; “gr” = grams; Root coverage is presented in cm²; root length is presented in cm; GR of root length is presented in cm/day; RGR of root length is presented in cm/day; RGR of root coverage is presented in cm²/day. The various time points indicate days from beginning of experiment in which parameters were measured.

TABLE 60

Improved growth rate, root coverage, root length and biomass in transgenic
plants exogenously expressing BDL103 under osmotic stress conditions

BDL103 Long or Short/Event No.

Roots	P						0.08	0.08
Coverage	A						1.25	1.34
(time point 9)
Roots Length	P						0.08	0.03
(time point 9)	A						1.13	1.26
GR of Roots	P	0.32					0.06	0.13
Coverage	A	1.19					1.23	1.23
(time point 6)
GR of Roots	P	0.39		0.28			0.04	0.04
Coverage	A	1.22		1.20			1.65	1.96
(time point 9)
GR of Roots	P	0.11					0.03	0.05
Length (time	A	1.25					1.27	1.40
point 6)
GR of Roots	P	0.09		0.02			0.01	0.02
Length (time	A	1.23		1.28			1.45	1.80
point 9)
RGR of	P	0.00					0.01	0.05	0.68
Roots	A	2.12					1.84	2.14	1.16
Coverage
(time point 6)
RGR of	P	0.46	0.04	0.27	0.24	0.56	0.02	0.01	0.63
Roots	A	1.26	1.42	1.20	1.67	1.24	1.50	1.80	1.26
Coverage
(time point 9)
RGR of	P	0.00					0.00	0.05
Roots Length	A	1.64					1.57	1.76
(time point 6)
RGR of	P	0.18	0.09	0.03	0.57	0.15	0.01	0.02
Roots Length	A	1.26	1.28	1.21	1.23	1.32	1.40	1.65
(time point 9)
DW [gr]	P		0.04				0.05	0.41
(time point 1)	A		1.41				1.30	1.22
DW [gr]	P		0.04				0.05	0.41
(time point 6)	A		1.41				1.30	1.22
DW [gr]	P		0.04				0.05	0.41
(time point 9)	A		1.41				1.30	1.22
FW [gr]	P	0.47					0.17	0.30
(time point 1)	A	1.32					1.23	1.36
FW [gr]	P	0.47					0.17	0.30
(time point 6)	A	1.32					1.23	1.36
FW [gr]	P	0.47					0.17	0.30
(time point 9)	A	1.32					1.23	1.36

Table 60. Analysis of growth parameters in tissue culture conditions of transgenic plants overexpressing BDL103-Short polynucleotide (SEQ ID NO: 671) or BDL103-Long polynucleotide (SEQ ID NO: 670) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) when grown under osmotic stress condition in the presence of 15% PEG (polyethylene glycol). Each event number refers to an independent transformation event in a plant (i.e., generation of a transgenic plant expressing the polynucleotide of choice). “A” = average; “P” = p-value; “GR” = growth rate; “RGR” = relative growth rate; “DW” = dry weight; “FW” = fresh weigh; “gr” = grams; Root coverage is presented in cm²; root length is presented in cm; GR of root length is presented in cm/day; RGR of root length is presented in cm/day; RGR of root coverage is presented in cm²/day. The various time points indicate days from beginning of experiment in which parameters were measured.

Example 11

Transgenic Arabidopsis Plants which Exogenously Express BDL103 Exhibit Increased Commercially Desired Traits in a Greenhouse Assay

Greenhouse assays were performed as described in Example 7 hereinabove for determining plant performance under normal conditions (i.e., irrigation with tap water).
Tables 61-62 depict analyses of growth rate, biomass, rosette diameter, rosette area, plot coverage, leaf number, petiole relative area, leaf blade area, blade relative area and harvest index in plants overexpressing the BDL103-long (SEQ ID NO:670; Table 61) and the BDL103-short (SEQ ID NO:671; Table 62) polynucleotides under the regulation of a constitutive (35S; SEQ ID NO:675) when grown in a greenhouse under normal conditions until seed production. Each Table includes data of several transformation events per gene. Results were considered significant if p-value was lower than 0.1 when compared to control plants (transformed with an empty vector).

TABLE 61

Improved growth rate, biomass, rosette diameter, rosette area, plot coverage,
leaf number, petiole relative area, leaf blade area, blade relative area
and harvest index in transgenic arabidopsis plants exogenously expressing
BDL103-long (SEQ ID NO: 670) under favorable conditions

Event No.

Parameter	2541	2541	2542	2542	2543	2543	2545	2545	2546	2546
(time point)	A	P	A	P	A	P	A	P	A	P

Yield	1.76	0.40	1.18	0.43	1.51	0.05	1.36	0.03
Rosette Diameter	1.11	0.18	1.37	0.03	1.33	0.10	1.14	0.41	1.12	0.65
(time point 8)
Rosette Diameter	1.13	0.00	1.31	0.22	1.25	0.10	1.11	0.43	1.13
(time point 5)
Rosette Diameter			1.59	0.01	1.52	0.07	1.29	0.25	1.16	0.55
(time point 3)
Rosette Diameter			1.20	0.08	1.19	0.30
(time point 1)
Rosette Area	1.37	0.00	1.65	0.24	1.53	0.14	1.24	0.35
(time point 8)
Rosette Area	1.23	0.12	1.48	0.17	1.25	0.19			1.21
(time point 5)
Rosette Area			1.97	0.16	1.68	0.06	1.37	0.38	1.23	0.60
(time point 3)
Rosette Area	1.17	0.36	1.23	0.04
(time point 1)
RGR of Rosette			1.13	0.54	1.14	0.07			1.38
Diameter
(time point 8)
RGR of Rosette	9.61	0.11
Diameter
(time point 5)
RGR of Rosette			2.20	0.18	2.00	0.11	2.00	0.11	2.41	0.04
Diameter
(time point 3)
RGR of Rosette Area	1.18	0.36	1.15	0.30	1.32	0.00	1.19	0.00	1.19
(time point 8)
RGR of Rosette Area	6.59	0.29
(time point 5)
RGR of Rosette Area			2.22	0.28	2.66	0.00	2.67	0.26	2.11	0.12
(time point 3)
RGR of Plot	1.18	0.36	1.15	0.30	1.32	0.00	1.19	0.00	1.19
Coverage
(time point 8)
RGR of Plot	6.59	0.29
Coverage
(time point 5)
RGR of Plot			2.22	0.28	2.66	0.00	2.67	0.26	2.11	0.12
Coverage
(time point 3)
RGR of Leaf Number	8.30	0.21
(time point 5)
RGR of Leaf Number			1.39	0.44	2.59	0.01	3.00	0.33	2.37	0.02
(time point 3)
Plot Coverage	1.40	0.00	1.59	0.34	1.55	0.13	1.26	0.32
(time point 8)
Plot Coverage	1.24	0.10	1.42	0.32	1.27	0.17	1.11	0.53
(time point 5)
Plot Coverage			1.90	0.26	1.71	0.05	1.39	0.36	1.15	0.64
(time point 3)
Plot Coverage	1.19	0.33	1.17	0.09
(time point 1)
Petiole Relative Area	1.31	0.81			1.23	0.66
(time point 8)
Petiole Relative Area	2.69	0.02
(time point 3)
Petiole Relative Area					1.18	0.43	1.42	0.00	1.39	0.26
(time point 1)
Leaf Petiole Area	1.95	0.64			1.67	0.22
(time point 8)
Leaf Petiole Area	3.05	0.00
(time point 3)
Leaf Petiole Area					1.11	0.61	1.33	0.01	1.18	0.49
(time point 1)
Leaf Number			1.10	0.02	1.10	0.03
(time point 8)
Leaf Number	1.07	0.05	1.12	0.15	1.11	0.28			1.12
(time point 5)
Leaf Number			1.50	0.02	1.43	0.12	1.25	0.40	1.28	0.29
(time point 3)
Leaf Number	1.28	0.00	1.37	0.00
(time point 1)
Leaf Blade Area	1.43	0.00	1.53	0.32	1.37	0.15	1.28	0.00
(time point 8)
Leaf Blade Area	1.18	0.17	1.36	0.14	1.14	0.11			1.11
(time point 5)
Leaf Blade Area			1.59	0.20	1.41	0.02	1.31	0.14	1.14	0.59
(time point 3)
Harvest index	1.19	0.01	1.18	0.38					1.13	0.08
Blade Relative Area							1.03	0.07
(time point 8)
Blade Relative Area	1.03	0.05
(time point 5)
Blade Relative Area			1.19	0.08	1.18	0.09	1.18	0.10	1.18	0.09
(time point 3)
Blade Relative Area			1.11	0.01
(time point 1)

Table 61. Analysis of growth parameters in a greenhouse assay of transgenic plants overexpressing BDL103-long polynucleotide (SEQ ID NO: 670) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) which were grown until seed production under normal conditions (as described in Example 7 above). Each event number refers to an independent transformation event in a plant (i.e., generation of a transgenic plant expressing the polynucleotide of choice). “A” = average; “P” = p-value; “RGR” = relative growth rate; “gr” = grams; yield is presented in mg/plant; Rosette Diameter is presented in cm/plant; Rosette Area is presented in cm²/plant; RGR of Rosette Diameter is presented in cm/plantday; RGR of Rosette Area is presented in cm²/plantday; RGR of Plot Coverage is presented in cm²/plant*day; RGR of Leaf Number is presented in 1/day; Plot Coverage is presented in cm²; Petiole Relative Area is presented in percent; Leaf Petiole Area is presented in cm²; Leaf Number is presented as number of leaves per plant; Leaf Blade Area is presented in cm²; Harvest Index is presented in g/DW (dry weight); Blade Relative Area is presented in percent; The various time points indicate days from beginning of experiment in which parameters were measured.

TABLE 62

Improved growth rate, biomass, rosette diameter, rosette area, plot coverage,
leaf number, petiole relative area, leaf blade area, blade relative area and
harvest index in transgenic arabidopsis plants exogenously expressing
BDL103-short (SEQ ID NO: 671) under favorable conditions

Event No.

Parameter	2353	2353	2357	2357	2359	2359	2360	2360	2361	2361
(time point)	A	P	A	P	A	P	A	P	A	P

Yield	1.32	0.26	1.32	0.01	1.14	0.18
Rosette Diameter	1.28		1.24	0.00	1.13	0.44	1.21	0.31	1.15	0.05
(time point 8)
Rosette Diameter	1.26	0.07	1.15	0.13
(time point 5)
Rosette Diameter	1.43	0.01	1.28	0.06	1.21	0.13	1.29	0.41	1.10	0.52
(time point 3)
Rosette Diameter	1.26	0.29
(time point 1)
Rosette Area	1.46		1.36	0.00	1.12	0.10	1.32	0.39	1.15	0.23
(time point 8)
Rosette Area	1.32	0.00	1.27	0.00			1.16	0.61
(time point 5)
Rosette Area	1.36	0.10	1.56	0.02			1.31	0.63
(time point 3)
Rosette Area	1.18	0.06
(time point 1)
RGR of Rosette	1.13		1.19	0.32	1.26	0.52	1.29	0.22	1.37	0.14
Diameter
(time point 8)
RGR of Rosette	1.52	0.46	2.09	0.08	1.94	0.12	1.64	0.42	1.66	0.46
Diameter
(time point 3)
RGR of Rosette Area	1.15				1.18	0.07	1.21	0.03	1.23	0.01
(time point 8)
RGR of Rosette Area	1.34	0.34	2.01	0.04	1.58	0.34	1.77	0.11	2.15	0.25
(time point 3)
RGR of Plot	1.15				1.18	0.07	1.21	0.03	1.23	0.01
Coverage
(time point 8)
RGR of Plot	1.34	0.34	2.01	0.04	1.58	0.34	1.77	0.11	2.15	0.25
Coverage
(time point 3)
RGR of Leaf Number	1.19
(time point 8)
RGR of Leaf Number	1.22	0.63	1.42	0.56	2.51	0.13	1.75	0.25	3.70	0.00
(time point 3)
Plot Coverage	1.48		1.29	0.10			1.35	0.37	1.17	0.19
(time point 8)
Plot Coverage	1.34	0.00	1.21	0.06			1.18	0.58
(time point 5)
Plot Coverage	1.39	0.09	1.49	0.06			1.33	0.61	1.11	0.75
(time point 3)
Plot Coverage	1.20	0.06
(time point 1)
Petiole Relative Area					1.21	0.71	1.92	0.25	3.37	0.44
(time point 8)
Petiole Relative Area									1.20	0.25
(time point 5)
Petiole Relative Area					1.31	0.20			1.75	0.01
(time point 1)
Leaf Petiole Area					1.32	0.60	2.47	0.32	3.96	0.43
(time point 8)
Leaf Petiole Area									1.20	0.38
(time point 5)
Leaf Petiole Area					1.24	0.02			1.96	0.21
(time point 1)
Leaf Number	1.15
(time point 8)
Leaf Number	1.12	0.01	1.21	0.00	1.08	0.05
(time point 5)
Leaf Number	1.31	0.09	1.32	0.11	1.19	0.29	1.28	0.45	1.15	0.63
(time point 3)
Leaf Number	1.26	0.01	1.21	0.23
(time point 1)
Leaf Blade Area	1.29		1.33	0.05	1.08	0.05	1.22	0.39
(time point 8)
Leaf Blade Area	1.21	0.01	1.10	0.03			1.11	0.63
(time point 5)
Leaf Blade Area	1.22	0.16	1.42	0.02
(time point 3)
Harvest index	1.30	0.44
Blade Relative Area			1.04	0.07
(time point 5)
Blade Relative Area	1.15	0.16	1.19	0.09	1.10	0.28
(time point 3)

Table 62. Analysis of growth parameters in a greenhouse assay of transgenic plants overexpressing BDL103-short polynucleotide (SEQ ID NO: 671) under the regulation of a constitutive promoter (35S; SEQ ID NO: 675) which were grown until seed production under normal conditions (as described in Example 7 above). Each event number refers to an independent transformation event in a plant (i.e., generation of a transgenic plant expressing the polynucleotide of choice). “A” = average; “P” = p-value; “RGR” = relative growth rate; “gr” = grams; yield is presented in mg/plant; Rosette Diameter is presented in cm/plant; Rosette Area is presented in cm²/plant; RGR of Rosette Diameter is presented in cm/plantday; RGR of Rosette Area is presented in cm²/plantday; RGR of Plot Coverage is presented in cm²/plant*day; RGR of Leaf Number is presented in 1/day; Plot Coverage is presented in cm²; Petiole Relative Area is presented in percent; Leaf Petiole Area is presented in cm²; Leaf Number is presented as number of leaves per plant; Leaf Blade Area is presented in cm²; Harvest Index is presented in g/DW (dry weight); Blade Relative Area is presented in percent; The various time points indicate days from beginning of experiment in which parameters were measured.

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 increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide which exhibits at least 80% sequence identity to SEQ ID NO: 82, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, or 672, thereby increasing the abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of the plant.

2. The method of claim 1, wherein said polypeptide exhibits at least 90% sequence identity to the polypeptide selected from the group consisting of SEQ ID NOs: 82, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672.

3. The method of claim 1, wherein said polypeptide exhibits at least 95% sequence identity to the polypeptide selected from the group consisting of SEQ ID NOs: 82, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672.

4. The method of claim 1, wherein said polypeptide is selected from the group consisting of SEQ ID NOs: 82, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672.

5. The method of claim 1, wherein said nucleic acid sequence encoding said polypeptide exhibits at least 80% sequence identity to SEQ ID NO: 625, 23, 617, 606, 615, 629, 1-15, 17-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650, 651, 670, or 671.

6. The method of claim 1, wherein said nucleic acid sequence encoding said polypeptide is selected from the group consisting of SEQ ID NOs: 625, 23, 617, 606, 615, 629, 1-15, 17-49, 51-59, 113-343, 345-351, 353-358, 605, 607-614, 616, 618, 620-628, 630-638, 641, 642, 644, 644-646, 648-651, 670, and 671.

7. An isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence which exhibits at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 82, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, or 672, wherein said amino acid sequence is capable of increasing abiotic stress tolerance, yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency of a plant.

8. The isolated polynucleotide of claim 7, wherein said amino acid sequence exhibits at least 90% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672.

9. The isolated polynucleotide of claim 7, wherein said amino acid sequence exhibits at least 95% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-98, 100-109, 111, 112, 359-589, 591-597, 600-604, 653-662, 664, 666-669, and 672.

10. The isolated polynucleotide of claim 7, wherein said nucleic acid sequence encoding said polypeptide exhibits at least 80% sequence identity to SEQ ID NO: 625, 23, 617, 606, 615, 629, 1-15, 17-36, 40, 41, 43-45, 49, 52-56, 58, 113-343, 351, 354-358, 605, 607-614, 616, 618, 620-628, 630-638, 642, 645, 650-651, 670, or 671.

11. The isolated polynucleotide of claim 7, wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 625, 23, 617, 606, 615, 629, 1-15, 17-49, 51-59, 113-343, 345-351, 353-358, 605, 607-614, 616, 618, 620-628, 630-638, 641, 642, 644, 644-646, 648-651, 670, and 671.

12. A nucleic acid construct comprising the isolated polynucleotide of claim 7, and a promoter for directing transcription of said nucleic acid sequence in a host cell.

13. A plant cell transformed with the nucleic acid construct of claim 12.

14. The plant cell of claim 13, wherein said plant cell forms part of a plant.

15. A transgenic plant comprising the nucleic acid construct of claim 12.

16. The method of claim 1, further comprising growing the plant expressing said exogenous polynucleotide under the abiotic stress.

17. The method of claim 1, 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.

18. The method of claim 1, further comprising selecting the plant expressing said exogenous polynucleotide for an increased abiotic stress tolerance, yield, biomass, growth rate, oil content, fiber yield, fiber quality, and/or nitrogen use efficiency as compared to a non-transformed plant which is grown under the same growth conditions.

19. The method of claim 18, wherein said selecting is performed under non-stress conditions.

20. A method of producing seeds of a crop, comprising:

(a) selecting a parent plant being transformed with an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence which exhibits at least 80% sequence identity to the polypeptide selected from the group consisting of SEQ ID NOs: 82, 73, 652, 71, 86, 60-70, 72, 74, 76-85, 87-95, 108-109, 112, 359-589, 602-604, 653-660, 665, 668, and 672, said parent plant exhibits an increased trait selected from the group consisting of: increased abiotic stress tolerance, increased yield, increased biomass, increased growth rate, increased oil content, increased fiber yield, increased fiber quality, or increased nitrogen use efficiency as compared to a non-transformed plant which is grown under the same growth conditions, and;

(b) growing a seed producing plant from said parent plant resultant of step (a), wherein said seed producing plant which comprises said exogenous polynucleotide has said increased trait, and;

(c) producing seeds from said seed producing plant resultant of step (b),

thereby producing seeds of the crop.