WO2011021190A1 - Plants producing high crop yield - Google Patents

Abstract

The present invention relates to transgenic plants over-expressing Tobacco aquaporin NtAQP1, producing higher crop yield compared to corresponding non-transgenic plants when grown under normal and abiotic stress conditions.

Description

PLANTS PRODUCING HIGH CROP YIELD

FIELD OF THE INVENTION

The present invention relates to transgenic plants over-expressing tobacco aquaporin NtAQPl, producing higher crop yield compared to corresponding non- transgenic plants, particularly when grown under water and salt stress conditions.

BACKGROUND OF THE INVENTION

Aquaporins (AQPs) are integral membrane proteins that increase the permeability of membranes to water, as well as to other small molecules including CO₂, H₂O₂, glycerol and boron. Of all the kingdoms, plants contain the largest aquaporin family, consisting of over 30 members (e.g. 35 members in Ar abidopsis, 36 in maize and 37 in tomato). The plant aquaporins were shown to have a role in regulating plant water balance and water-use efficiency (Maurel C, 2007. FEBS Lett. 581 :2227-2236; Kaldenhoff R and Fischer M, 2006. Acta Physiol. 187: 169-176).

Raising ambient CO₂ levels can potentially improve the growth rates of certain C₃ plants under optimal growth conditions, suggesting that membrane permeability to CO₂ is a growth-limiting factor in these plants. Impaired CO₂ conductance in the mesophyll following treatment with the non-specific aquaporin inhibitor HgCl₂ was one of the first pieces of evidence suggesting the ability of aquaporins to conduct CO₂ (Terashima I and Ono K, 2002. Plant Cell Physiol. 43:70-78.). The first direct evidence of this ability was reported for tobacco aquaporin 1 (NtAQPl) expressed in Xenopus oocytes (Uehlein N et al., 2003. Nature 425:734-737). Those authors also induced NtAQPl expression in tobacco plants and reported significant increase in photosynthetic rate, stomatal opening and leaf growth rate. A role for NtAQPl in the photosynthetic mechanism has been recently reported in tobacco plants overexpressing NtAQPl, which showed a 20% increase in photosynthetic rate, while plants expressing the NtAQP 1-anύsense showed a decrease of 13% relative to control plants (Flexas J et al., 2006. Plant Cell Environ. 31 :602-621). However, no morphological, growth-rate or yield parameters were reported for the NtAQP 1 -overexpressing plants.

The presence of NtAQPl has been reported in both the plasma membrane (PM) and the chloroplast inner membrane (CIM) in mesophyll and epidermal guard cells (Uehlein N et al., 2008. Plant Cell 20:648-657). However, these two membranes showed opposite permeability coefficients with regard to water and CO₂ transport: while the water permeability of the CIM was threefold higher compared to the PM, its CO₂ permeability was about five times lower than that of the PM. Moreover, plants in which the NtAQPl was silenced showed about 10-fold decrease in the CIM permeability to CO₂ with no significant decreased in the PM permeability, and vice versa effect for water permeability (Uehlein N et al., 2008. ibid). These results indicate that NtAQPl cellular activity is location-related.

In addition to its role in regulating CO₂ and water permeability of mesophyll tissues and stomatal conductance, it was suggested that NtAQPl controls root hydraulic conductivity (Lp), based on its high abundance in the roots, especially around the xylem vessels, its impact on increasing the water permeability of Xenopus oocytes (Biela A et al., 1999. Plant J. 18:565-570) and the decrease in root Lp of NtAQPl -silenced plants

(Siefritz F et al., 2002. Plant Cell 14:869-876), NtAQPl was suggested to play a role in controlling root Lp. This activity of NtAQPl might be related to abiotic stress conditions since NtAQPl is a stress-induced gene, it is regulated by an ABA-sensitive promoter (Siefritz F et al., 2001. J. Exp. Bot. 52:1953-1957) and it shows a significant increase in root transcript level during drought stress (Mahdieh M et al., 2008. Plant

Cell Physiol. 49:801-813). It was also shown that NtAQPl -silenced plants revealed higher drought sensitivity than control plants (Siefritz et al., 2002. ibid).

Research on the role of aquaporins in water and CO₂ transport and stress- resistance activities has progressed in parallel, but without much interaction. Aharon R et al. (2003. The Plant Cell 15:439-447) found that constitutive overexpression of the Arabidopsis plasma membrane aquaporin, PIPIb, enhanced plant growth and transpiration under favorable growth conditions, but negatively affected plant growth under water stress conditions.

A paper by inventors of the present invention and co-workers published after the priority date of the present application describes the role of tobacco aquaporing-1 in improving water use efficiency, hydraulic conductivity, and yield production in plants transformed with this aquaporin (Sade N. et al., 2010. Plant Physiol. 152:245-254).

Crop production is affected by numerous abiotic environmental factors with soil salinity and drought having the most detrimental effects. Approximately 70% of the genetic yield potential in major crops is lost due to abiotic stresses, and most major agricultural crops are susceptible to drought stress. In water-limited environments, crop yield is a function of water use, water use efficiency (WUE; defined as aerial biomass yield/water use) and the harvest index (HI; the ratio of yield biomass to the total cumulative biomass at harvest). WUE is a complex trait that involves water and CO2 uptake, transport and exchange at the leaf surface (transpiration). Improved WUE has been proposed as a criterion for yield improvement under drought. Water deficit can also have adverse effects in the form of increased susceptibility to disease and pests, reduced plant growth and reproductive failure.

There is a continuing need for, and it would be highly advantageous to have means and methods for improving the water use efficiency and yield of crop plants.

SUMMARY OF THE INVENTION

The present invention answers the above-described need by providing transgenic plants expressing a Nicotiana tabacum aquaporin-1 aquaporin (NtAQPl), which show increase in crop yield production compared to reference plants, particularly under drought and salt stress conditions.

The present invention is based in part on the discovery that constitutive expression of NtAQPl unexpectedly results in higher leaf conductance (g_s), higher root hydraulic conductivity (Lp) and higher CO₂ assimilation rates under optimal water availability as well as under drought and salt stress, leading to higher yield production.

In hitherto known systems, CO₂ assimilation and yield production are compromised for maintaining the root hydraulic conductivity, particularly under stress conditions. Without wishing to be bound by any particular theory or mechanism of action the ability of the transgenic plants of the invention to produce higher yield may be attributed to the NtAQPl -mediated enhancement of water transport both in the roots and in the leaves in parallel to enhancement of CO₂ transport in the leaves. This dual activity, which increases the plant water use and photosynthetic rate under optimal conditions and improves its water-use efficiency under stress conditions, contributes to the plant yield production under all tested conditions.

Thus, according to one aspect, the present invention provides a transgenic crop plant comprising at least one root cell and at least one leaf cell transformed with a DNA construct comprising a polynucleotide encoding the Nicotiana tabacum aquaporin-1 (NtAQPl), wherein the plant has increase yield compared to a corresponding non- transgenic plant.

According to typical embodiments, the NtATQPl comprises the amino acids sequence set forth in SEQ ID NO:1 (accession number (AJ001416). According to other typical embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:2.

The DNA construct typically comprises all necessary elements for transcription and translation of the polynucleotide encoding NtAQPl, such that an active protein is encoded. According to certain embodiments, the expression of the NtAQPl is controlled by a constitutive promoter. According to other embodiments, the constitutive promoter is tissue specific. According to currently typical embodiments, the promoter is root specific or shoot specific. According to other typical embodiments, the promoter is selected from the group consisting of guard cell specific promoter (shoot); endodermis (root) and bundle sheath (shoot) 'scarecrow' promoter; bundle sheath OSTMTl promoter (shoot); and the green tissue Fbpase promoter (shoot).

According to other embodiments, the expression vector further comprises a regulatory element selected from the group consisting of an enhancer, an origin of replication, a transcription termination sequence, a polyadenylation signal and the like.

According to further embodiments, the transgenic crop plant has an increase yield compared to a corresponding non-transgenic plant when the plants are grown under optimal water availability conditions. According to other embodiments, the transgenic crop plant has an increase yield compared to a corresponding non-transgenic plant when the plants are grown under abiotic stress conditions. According to certain embodiments, the abiotic stress condition is selected from the group consisting of water stress (drought), high soil salinity, extreme temperatures, low oxygen levels or presence of heavy metals. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, optimal water availability refers to soil water content of at least 85%. According to other embodiments, drought conditions refer to soil water content of less than 70%.

Soil salinity is typically measured as soil electric conductivity (EC). According to certain embodiments, low soil salinity refers to soil electric conductivity of less than 4 dS/m, medium soil salinity refers to EC of from about 4 dS/m to 8dS/m and high salinity to EC of above 8 dS/m.

According to certain embodiments, the crop plant is selected from the group consisting of plants producing fruit; flower and ornamental plants; grain producing plants crops (wheat, oats, barely, rye , rice, maize); legumes (peanuts, peas soybean lentil etc); forage crops used for hay or pasture; root crops (sweet potatoes etc), fiber crops (cotton, flax etc); trees for wood industry; tuber crops (potato), sugar crops (sugar beet, sugar came), oil crops (canola, sunflower, sesame etc), wherein each possibility represents a separate embodiment of the invention.

According to typical embodiments, the crop plant is a plant producing a fruit crop

(including vegetables). According to other embodiments, the crop plant in other than tobacco. According to certain specific embodiments, the plant is a tomato plant.

According to certain embodiments, the transgenic plant has an increase of at least 50%, typically at least 60%, more typically at least 70%, 80% or 90% or more in the yield. As used herein, the term "increase in yield" refers to increase in the quantity of the desired product, its weight or a combination thereof.

The present invention also encompasses seeds of the transgenic plant, wherein plants grown from said seeds comprise at least one root cell and at least one leaf cell transformed with a polynucleotide encoding NtAQPl, and have increase yield compared to plants grown from seeds of corresponding non-transgenic plant. The present invention further encompasses fruit, leaves or any part of the transgenic plant, as well as tissue cultures derived thereof and plants regenerated therefrom.

According to another aspect, the present invention provides a method for increasing the yield of a crop plant, comprising (a) transforming a plant cell with a DNA construct comprising a polynucleotide encoding NtAPQl and (b) regenerating the transformed cell into a transgenic plant comprising at least one root cell and at least one leaf cell expressing NtAQPl having an increased yield compared to a corresponding non-transgenic plant.

The DNA constructs comprises all the necessary elements for expression of NtAQPl as described hereinabove. According to certain embodiments, the expression of NtAQPl is controlled by a constitutive, tissue specific promoter. Transformation of plants with an expression vector may be performed by various means, as is known to one skilled in the art. Common methods are exemplified by, but are not restricted to, Agrobacterium-mediated transformation, microprojectile bombardment, pollen mediated transfer, plant RNA virus mediated transformation, liposome mediated transformation, direct gene transfer (e.g. by microinjection) and electroporation of compact embryogenic calli. According to one embodiment, the transgenic plants of the present invention are produced using Agrobacterium mediated transformation.

Transgenic plants comprising the polynucleotides of the present invention may be selected employing standard methods of molecular genetics, as are known to a person of ordinary skill in the art. According to certain embodiments, the presence of the transformed polynucleotide is verified by Polymerase Chain Reaction (PCR) using appropriate primers.

According to yet additional aspect, the present invention provides a method of screening for a plant capable of producing high yield when grown under abiotic stress conditions comprising: (a) obtaining a plurality of samples from a plurality of plant lines and a control sample from a reference plant, the samples comprising genetic material; (b) measuring the expression level of a polynucleotide encoding NtAQPl or an ortholog thereof in the samples; (c) comparing the expression level of the polynucleotide encoding NtAQPl or the ortholog thereof in the plurality of samples to the control sample; wherein a plant line overexpressing said polynucleotide encoding

NtAQPl or ortholog thereof is capable of producing high yield when grown under abiotic stress conditions.

According to certain embodiments, the abiotic stress condition is selected from the group consisting of water stress (drought), high soil salinity, extreme temperatures, low oxygen levels or presence of heavy metals. Each possibility represents a separate embodiment of the invention.

According to certain typical embodiments, the abiotic stress is water stress. According to other typical embodiments the abiotic stress is high soil salinity.

According to typical embodiments, the polynucleotide encodes NtATQPl protein having at least 75%, typically at least 85% or more homology to the amino acids sequence set forth in SEQ ID NO:1. According to other typical embodiments the polynucleotide comprises a nucleic acids sequence having at least 75%, typically at least 85% or more homology to the nucleic acid sequence set forth in SEQ ID NO:2.

Methods for determining the expression level of polynucleotide having particular sequence are known to a person skilled in the art. According to certain embodiments, expression level of the polynucleotide is measured using NAT (nucleic acid technology)-based assays. According to typical embodiments, the NAT-based assay is selected from the group consisting of a quantitative PCR and Real-Time PCR, Northern blot and the like.

According to certain embodiments, the expression level of the polynucleotide is measured by quantitative PCR using a primer pair having the nucleic acid sequence set forth in SEQ ID NO:3 and SEQ ID NO:4.

According to further embodiments, the method further comprises (a) planting the plant line overexpressing the polynucleotide encoding NtAQPl or ortholog thereof and a corresponding control plant having lower expression of said polynucleotide under abiotic stress conditions; (b) comparing the crop yield of the plant line to the crop yield of the control plant; and selecting plant lines having increased crop yield compared to said control plant.

According to certain embodiments, the plant lines are of the same plant species. According to other embodiments, the plant limes are of different species.

According to currently typical embodiment, the reference plant is Tobacco

(Nicotiana tabacuni).

Other objects, features and advantages of the present invention will become clear from the following description and drawings. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates that NtAQPl expression increases the tobacco mesophyll membrane water permeability coefficient (P_f). Columns represent the mean P_f (±SE) of tobacco mesophyll protoplasts expressing NtAQPl (white bar; n = 15) and control protoplasts (black bar; n = 9). * denotes significant difference between means (t-test P < 0.05).

FIG. 2 shows the presence of NtAQPl DNA, RNA and protein in tomato plants regenerated following co-cultivation of explants with Agrobacterium (To generation). Fig. 2 A: DNA of selected plants was subjected to PCR using NtAQPl -specific primers; transgenic plants yielded the expected 930-bp product. M, 100-bp ladder. Fig. 2B: cDNA of selected plants was subjected to RT-PCR using NtAQPl -specific primers; transgenic plants yielded the expected 830-bp product. M, 100-bp ladder. Fig.2C: Western blot analysis of selected regenerated plants using an NtAQPl -specific antibody (upper panel); Ponceau red staining of the membrane (lower panel).

FIG. 3 demonstrates the response of net photosynthesis (A_N) to substomatal CO₂ concentration (Ci) in control (black line) and TOM-NtAQPl transgenic (gray line) plants. Fig. 3 A: Plants under normal irrigation regime (n NtAQPl = 20, n Control = 10). Fig. 3B: Plants after treatment with 100 mM NaCl irrigation (n NtAQPl= 13, n Control = 9). Measurements were performed in a controlled greenhouse on young, fully expanded leaves. Data are given as mean ± SE. When not seen, SE is smaller than the symbol.

FIG. 4 demonstrates instantaneous water use efficiency (IWUE) of transgenic TOM- NtAQPl (white bars) and control plants (black bars). Columns represent the mean IWUE (±SE) of plants under normal irrigation (n NtAQPl = 9, n Control = 4), water- deficient irrigation (n NtAQPl = 14, n Control = 6) and 100 mM NaCl in the irrigation water (n NtAQPl = 13, n Control = 9). * denotes significant difference between means (t-test P < 0.05).

FIG. 5 shows root system sap exudation discharge, measured from de-topped plants under vacuum, before and following application of 50 mM NaCl. Fig. 5A: Normal irrigation treatment and Fig. 5B: 50 mM NaCl treatment. TOM- NtAQPl plants (white bars, n = 4), control plants (black bars, n = 4). Data are given as mean ± SE. Different letters indicate significant difference (t test, P < 0.05).

FIG. 6 shows daily transpiration rate and relative transpiration of TOM-NtAQPl (gray line and white bar, respectively) vs. control plants (black line and black bar, respectively) grown under normal irrigation in a commercial greenhouse. Fig. 6A: mean daily transpiration rate (±SE; normalized to leaf area) of TOM-NtAQPl (n = 3) and control plants (n = 3). Fig. 6B: The normalized (to vapor pressure deficit (VPD); atmospheric demand; and leaf area) mean daily relative transpiration (±SE; n NtAQPl = 3, n Control = 3). SE is marked for each 10th sampling point; when not visible, SE is smaller than symbol. * denotes significant difference between means (t-test P < 0.05).

FIG. 7 demonstrates the impact of salt and drought stress on the daily transpiration rate and relative transpiration (inserts) of TOM-NtAQPl (gray line, white bar) vs. control (black line, black bar). Plants were grown in pots in a commercial greenhouse. Fig. 7A and 7B show the mean daily transpiration rate, normalized to leaf area, on days 1 and 3, respectively, of plants subjected to 100 mM NaCl salinity stress, and Fig. 7C shows their first day of recovery (n NtAQPl = 3, n Control = 3). Fig 7D and 7E show the mean daily transpiration rate on days 1 and 2 of drought stress, respectively (n NtAQPl = 3, n Control = 3), and Fig. 7F shows their recovery after 2 days of normal irrigation. All inserts represent mean daily relative transpiration of plants presented in the graphs normalized to VPD and leaf area. Data are given as mean ± SE for each 1 Oth sampling point. When not visible, SE is smaller than the symbol. * denotes significant difference between means (t-test P < 0.05).

FIG. 8 shows daily transpiration rate, gs and A^N under normal and 10OmM NaCl irrigation. Fig.8A: Whole plant mean daily transpiration rate normalized to leaf area of all reciprocal grafted TOM-NtAQPl and control plants measured simultaneously under normal irrigation and (Fig. 8B) 3 days of 10OmM NaCl irrigation (T/T n=3, C/C N=3, C/T n=8, T/C n=6). SE is given for each 10th sampling point, when not seen the SE bar in smaller than the line. Morning hours are 08:00-11 :00, and noon hours are 11:00- 14:00. At these hours and under the same salt conditions, a parallel measurement of reciprocal grafted scions were conducted for stomata conductance (gs) and leaf net photosynthesis (A_N), during the morning hours and during noon hours (Fig. 8C and 8E; 8D and 8F, respectively). Bars represents the mean ± SE of grafted plants (white for TOM-NtAQPl part and black for control part; T\T n=3 for morning and noon; C/C n=4 for morning and n=l 1 for noon; C/T n=6 for morning and n=5 for noon; T/C n=5 for morning and n=6 for noon). - a, b, c, d different letters indicates significant difference (t test P<0.05). All measurements were performed in a semi-controlled greenhouse (see material and methods), gas exchange measurements was conducted using portable apparatuses (Li-cor 6400) on young, fully expanded leaves.

FIG. 9 compares the yield parameters of TOM-NtAQPl and control plants. Plants grown for 3 months under normal (n NtAQPl = 8, n Control = 3), 100 mM NaCl (n NtAQPl = 5, n Control = 5) and drought (n NtAQPl = 12, n Control = 6) irrigation conditions. Columns represent the mean (±SE) of individual fruit weight (Fig. 9A); fruit number (Fig. 9B); shoot fresh weight (Fig. 9C); and harvest index (Fig. 9D). a, b, c, d: different letters indicate significant difference (t-test P < 0.05).

FIG. 10 shows NtAQPl's impact on Arabidopsis plant dry weight under normal and 100 mM NaCl irrigation. Fig.1OA: 45-day-old Arabidopsis plants constitutively expressing AtNtAQPl (upper panel) and control plants (lower panel) grown under normal irrigation regime (left panel) and under 100 mM NaCl irrigation regime for 33 days (right panel). Fig. 1OB: Mean (±SE) of shoot dry weight of Arabidopsis plants overexpressing NtAQPl and control plants under normal (n NtAQPl = 11, n Control = 8) and 100 mM NaCl (n NtAQPl = 12, n Control = 6) irrigation conditions, a, b, c, d: different letters indicate significant difference (t-test P < 0.05).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides means and method to answer a long lasting need of crop plants the produce high yield when grown under sub-optimal conditions, particularly under water and/or salt stress. Unexpectedly, the present invention now shows that expression of the Nicotiana tabacum aquaporin (NtAQPl) enhances transpiration and CO₂ assimilation under stress conditions that typically lead to stomatal closure and reduction in CO₂ assimilation. Furthermore, the present invention shows for the first time that NtAQPl acts as active water channel in mesophyll cells, and thus has a significant contribution to the water transport throughout the plant, enabling the plant to efficiently use the water resources. In addition, expression of NtAQPl in crop plants increased the stomatal pore area, CO₂ conductivity and overall yield production.

Definitions

The term "plant" is used herein in its broadest sense. It includes, but is not limited to, any species of woody, herbaceous, perennial or annual plant. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at a stage of the plant development capable of producing crop.

As used herein, the term "crop plant" refers to a plant with at least one part having commercial value. The term encompasses plants producing edible fruit (including vegetables), plants producing grains (as a food, feed and for oil production), plant producing flowers and ornamental plants, legumes, root crops, tuber crops, leafy crops and the like.

The term "increased yield" as used herein refers to an increase in the overall production of the commercially valuable plant part. The term encompasses increase in the plant part mass, number or both.

The term Nicotiana tabacum aquaporin -1 (NtAQPl) refers to the tobacco aquaporin denoted by accession number AJ001416 having the amino acid sequence set forth in SEQ ID NO: 1 and encoded by the polynucleotide having SEQ ID NO:2.

The term "abiotic stress conditions" as used herein refers to conditions where water is the limiting factor for plant growth. These include water stress (drought) high soil salinity, extreme temperatures, low oxygen levels or presence of heavy metals.

As used herein, the term "soil salinity" refers to the salt concentration of the soil solution in terms of g/1 or electric conductivity (EC) in dS/m. EC of 5 is about 60 mM NaCl; EC of 10 is about 120 mM NaCl and of EC 12.5 is about 25OmM NaCl. Sea water may have a salt concentration of 30 g/1 (3%) and an EC of 50 dS/m. Soils are considered saline when the EC > 4. When 4 < EC < 8, the soil is called moderately saline and when 8 < EC the soil is called highly saline.

The terms "water stress", "drought conditions" and "low soil water content" are used herein interchangeably and refer to sub-optimal soil hydration conditions for the growth of a particular plant species. Soil hydration can be measured by various methods as is known to a person skilled in the art, depending on the soil type. According to certain embodiments, the soil water content is measured relative to the maximum amount of water that a given soil can retain ("filed capacity") as weight/weight percentage. According to these embodiments, drought conditions refer to soil water content of less than 70%.

It is to be understood that different plant species show different response to a certain abiotic stress, particularly to soil salinity and soil water content. Accordingly, as used herein the term "a plant having an increased crop yield" refers to a detectable change in the crop yield of the transgenic plant of the invention compared to a corresponding non-transgenic plant of the same species, wherein both plants are grown under the same normal or stress conditions.

The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of RNA or a polypeptide. A polypeptide can be encoded by a full-length coding sequence or by any part thereof. The term "parts thereof when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleic acid sequence comprising at least a part of a gene" may comprise fragments of the gene or the entire gene.

The term "gene" also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.

The terms "polynucleotide", "polynucleotide sequence", "nucleic acid sequence", and "isolated polynucleotide" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA or hybrid thereof, that is single- or double-stranded, linear or branched, and that optionally contains synthetic, non-natural or altered nucleotide bases. The terms also encompass RNA/DNA hybrids.

The term "DNA construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. The construct may further include a marker gene which in some cases can also be the gene of interest. In certain embodiments, the DNA construct is an expression vector further comprising appropriate regulatory sequences, operably linked to the gene of interest. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. According to certain typical embodiments, the DNA construct of the present invention comprises a constitutive promoter. The term construct includes vectors (including expression vectors and transformation vectors) but should not be seen as being limited thereto. According to certain typical embodiments, the DNA construct of the present invention is an expression vector. According to these embodiments, the expression vector comprises a constitutive promoter operably linked to the polynucleotide encoding NtAQPl.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. According to typical embodiments of the present invention, the polynucleotide encoding NtAQPl is operably linked to the regulatory sequences in a sense orientation.

The term "transgenic" when used in reference to a plant or seed (i.e., a "transgenic plant" or a "transgenic seed") refers to a plant or seed that contains at least one heterologous transcribeable gene in one or more of its cells. The term "transgenic plant material" refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous gene in at least one of its cells.

The terms "transformants" or "transformed cells" include the primary transformed cell and cultures derived from that cell regardless to the number of transfers. All progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants.

Transformation of a cell may be stable or transient. The term "transient transformation" or "transiently transformed" refers to the introduction of one or more exogenous polynucleotides into a cell in the absence of integration of the exogenous polynucleotide into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA), which detects the presence of a polypeptide encoded by one or more of the exogenous polynucleotides. Alternatively, transient transformation may be detected by detecting the activity of the protein (e.g. β -glucuronidase) encoded by the exogenous polynucleotide.

The term "transient transformant" refers to a cell which has transiently incorporated one or more exogenous polynucleotides. In contrast, the term "stable transformation" or "stably transformed" refers to the introduction and integration of one or more exogenous polynucleotides into the genome of a cell. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the exogenous polynucleotides. Alternatively, stable transformation of a cell may also be detected by enzyme activity of an integrated gene in growing tissue or by the polymerase chain reaction of genomic DNA of the cell to amplify exogenous polynucleotide sequences. The term "stable transformant" refers to a cell which has stably integrated one or more exogenous polynucleotides into the genomic or organellar DNA. It is to be understood that a plant or a plant cell transformed with the nucleic acids, constructs and/or vectors of the present invention can be transiently as well as stably transformed.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

According to one aspect, the present invention provides a transgenic crop plant comprising at least one root cell and at least one leaf cell transformed with a DNA construct comprising a polynucleotide encoding the Nicotiana tabacum aquaporin-1

(NtAQPl), wherein the plant has increase yield compared to a corresponding non- transgenic plant.

According to typical embodiments, the NtATQPl comprises the amino acids sequence set forth in SEQ ID NO:1 (accession number (AJOOl 416). According to other typical embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:2.

The impact of NtAQPl on whole-plant water-use efficiency in general and under stress in particular is shown in the present invention for the first time, although separate studies of the role of this aquaporin in water and CO₂ transport have been reported.

Although NtAQPl was shown to be an active water channel in Xenopus oocytes (Biela et al., 1999. ibid), its contribution to the water permeability of mesophyll cells was not obvious. The present invention shows for the first time that NtAQPl is an active water channel in mesophyll protoplasts, significantly increasing the cell water permeability coefficient (P_f) level relative to controls (Figure 1). This activity is additional to the activity of NtAQPl as CO₂ channel. Interestingly, NtAQPl has a dual effect not only at the cellular level but also at the whole plant level, where its expression increases both transpiration and net photosynthesis fluxes.

According to further embodiments, the transgenic crop plant has an increase yield compared to a corresponding non-transgenic plant when the plants are grown under optimal water availability conditions. According to other embodiments, the transgenic crop plant has an increase yield compared to a corresponding non-transgenic plant when the plants are grown under abiotic stress conditions. According to certain embodiments, the abiotic stress condition is selected from the group consisting of water stress (drought), high salt conditions, extreme temperatures, low oxygen levels or presence of heavy metals.

Reduced plant production under stress conditions can be explained and evaluated by the crop's transpiration-use efficiency (biomass produced per unit of water transpired) ratio. This ratio is linear and depends on crop characteristics.

As exemplified hereinbelow, expression of NtAQPl in tomato plant under constitutive promoter (TOM-BtAQPl plants) increased both transpiration rate and CO₂ assimilation in the plant. TOM-NtAQPl plants showed an increased response of photosynthetic rate (AN) to substomatal CO₂ concentration (Ci) under normal growth conditions, and retained this photosynthetic advantage under stress conditions (Figure 3). In addition, the leaf gs of the transgenic plants and whole-plant transpiration rate were significantly increased O due to wider stomatal aperture (Table 1, Figure 7), resulting in improved yield parameters in all treatments relative to controls (Figure 9).

Table 1: Photosynthetic characteristics including transpiration and root hydraulic characteristics of TOM-NtAQPl and control plants treated with normal irrigation and 10O mM NaCl in a controlled greenhouse

* For the root hydraulic measurements (Lp and Na discharge), plants were treated with 5O mM NaCl

Under normal growth conditions, the above changes yielded a major improvement in productivity due to the high photosynthetic rate; however, this process was accompanied by a proportionally higher transpiration rate, thereby maintaining the same

IWUE (Figure 4). On the other hand, under stress conditions, the ratio between CO₂ assimilation and transpiration increased, as reflected by the improved IWUE of TOM-

NtAQPl plants (Figure 4), which eventually led to higher biomass and/or yield (in comparison to controls) under stress, in both tomato and arabidopsis plants (Figure 9 and Figure 10).

The involvement of NtAQPl in the mechanism controlling stomatal and CO₂ conductance and in photosynthetic rate has been reported previously (Uehlein et al., 2003. ibid; Flexas et al., 2006. ibid). Those studies showed increased stomatal conductance (gs) in NtAQP 1-overexpressing tobacco plants and decreased gs in NtAQPl antisense plants. This impact on the guard cells might be related to a direct effect of NtAQPl in transporting CO₂ or water, as demonstrated in this study, although other indirect effects cannot be rule out. Nevertheless, as exemplified herein by grafting experiments, the impact of NtAQPl on stomatal conductance (gs) and photosynthetic rate (A_N) is independent of the conventional root-to-shoot signal.

Signals initiated under water stress (salinity or drought) are sensed by the roots, transported to the leaf, and ultimately reduce gs. These root-to-shoot signals may be either chemical or hydraulic. A hydraulic signal may form due to the sharp decrease in root hydraulic conductance (Lp) in response to abiotic stress. Such a decrease has been reported as a general reaction in plants to many abiotic stresses (Steudle E, 2000. J. Exp. Bot. 51:1531-1542) and was recorded also in the control plants (more than 3-fold reduction in Lp) in response to 50 mM NaCl. In contrast, TOM-NtAQPl plants reduced their Lp by less than 40% under the same salt stress (Table 1). A reduced root hydraulic signal might explain TOM-NtAQPl 's higher transpiration, gs, and AN under stress conditions compared with stressed control plants. Yet, a TOM-NtAQPl scion grafted on a control rootsfock (T/C) and exposed to salt stress still exhibited higher gs and A_N, similar to T/T plants (Figure 8C-8F). This suggests that NtAQP l's activity in controlling gs and A_N is dominant and nearly independent of root signals.

However, T/C plants showed a midday drop in transpiration rate under both normal and stressed conditions (Figure 8A and 8B). This "drop" came just after these plants had reached their daily peak transpiration rate (assumed to be coupled with peak xylem tension). Replacing the control rootstock with TOM-NtAQPl (T/T) revealed a mirror image of these results (i.e. a peak instead of a drop in midday transpiration rate), thereby indicating the roots involvement in this process. Moreover, since gs of T/C plants remained high during the midday drop in transpiration Figure 8C-8F), it is concluded that this drop is not due to stomatal closure and that it might be due to hydraulic failure resulting from the combination of high gs and low Lp, leading to a decrease in plant water content.

Without wishing to be bound by any specific theory or mechanism of action, the stress resistance of the transgenic TOM-NtAQPl plants of the invention may be tightly related to NtAQPl water transport activity in the roots. Accordingly, NtAQPl might act as the root's "emergency" hydraulic valve (i.e. release hydraulic tension by increasing root Lp under higher transpiration rate or other stress, which in turn decreases xylem tension), thereby preventing hydraulic failure in the xylem system.

According to yet additional aspect, the present invention provides a method of screening for a plant capable of producing high yield when grown under abiotic stress conditions comprising: (a) obtaining a plurality of samples from a plurality of plant lines and a control sample from a reference plant, the samples comprising genetic material; (b) measuring the expression level of a polynucleotide encoding NtAQPl or an ortholog thereof in the samples; (c) comparing the expression level of the polynucleotide encoding NtAQPl or the ortholog thereof in the plurality of samples to the control sample; wherein a plant line overexpressing said polynucleotide encoding NtAQPl or ortholog thereof is capable of producing high yield when grown under abiotic stress conditions.

According to certain embodiments, the abiotic stress condition is selected from the group consisting of water stress (drought), high soil salinity, extreme temperatures, low oxygen levels or presence of heavy metals. Each possibility represents a separate embodiment of the invention.

According to certain typical embodiments, the abiotic stress is water stress.

According to other typical embodiments the abiotic stress is high soil salinity.

According to typical embodiments, the polynucleotide encodes NtATQPl protein having amino acids sequence set forth in SEQ ID NO:1. According to other typical embodiments the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:2.

Methods for detecting a particular polynucleotide sequence and measuring its expression level are known in the art. According to certain embodiments, the assay is a nucleic acid technology (NAT)-based assay, typically quantitative PCR, employing primers specific to the target DNA.

As used herein, the term "quantitative" when in reference to differences in expression levels of a polynucleotide as described herein, refers to absolute differences in quantity of expression, as determined by any means known in the art, or in other embodiments, relative differences, which may be statistically significant.

As used herein, a "primer" defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.

According to certain embodiments, the expression level of the polynucleotide is measured by quantitative PCR using a primer pair having the nucleic acid sequence set forth in SEQ ID NO:3 and SEQ ID NO:4. Preferred Modes for Carrying Out the Invention

Cloning of a polynucleotide encoding the NtAQPl can be performed by any method as is known to a person skilled in the art. Various DNA constructs may be used to express the NtAQPl in a desired plant.

According to certain embodiments, the present invention provides an expression vector comprising all necessary elements for transcription and translation of the polynucleotide encoding NtAQPl, such that the encoded protein is active.

According to certain embodiments, the expression of the NtAQPl is controlled by a constitutive promoter. According to other embodiments, the constitutive promoter is tissue specific. According to currently typical embodiments, the promoter is root specific or mesophyll specific.

The terms "promoter element," "promoter," or "promoter sequence" as used herein, refer to a DNA sequence that is located at the 5' end (i.e. precedes) the protein coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in Okamuro J K and Goldberg R B (1989) Biochemistry of Plants 15:1-82.

Among the most commonly used promoters are the nopaline synthase (NOS) promoter (Ebert et al., 1987 Proc. Natl. Acad. Sci. U.S.A. 84:5745-5749), the octapine synthase (OCS) promoter, caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987 Plant MoI Biol. 9:315-324), the CaMV 35S promoter (Odell et al., 1985 Nature 313:810-812), and the figwort mosaic virus 35S promoter, the light inducible promoter from the small subunit of rubisco, the Adh promoter (Walker et al., 1987 Proc. Natl. Acad. Sci. U.S.A. 84:6624-66280), the sucrose synthase promoter (Yang et al., 1990 Proc. Natl. Acad. Sci. U.S.A. 87:4144- 4148), the R gene complex promoter (Chandler et al., 1989 Plant Cell 1:1175-1183), the chlorophyll a/b binding protein gene promoter, etc. Other commonly used promoters are, the promoters for the potato tuber ADPGPP genes, the sucrose synthase promoter, the granule bound starch synthase promoter, the glutelin gene promoter, the maize waxy promoter, Brittle gene promoter, and Shrunken 2 promoter, the acid chitinase gene promoter, and the zein gene promoters (15 kD, 16 kD, 19 kD, 22 kD, and 27 kD; Perdersen et al. 1982 Cell 29:1015-1026). A plethora of promoters is described in International Patent Application Publication No. WO 00/18963. According to certain currently preferred embodiments, the expression vector of the present invention comprises the constitutive CaMV 35S promoter. According to other currently preferred embodiments, the expression vector comprises root specific or shoot specific promoters selected from the group consisting of the guard cell specific promoter KSTl (Plesch G et al., 2001. Plant Journal 28:455-464); endodermis and bundle sheath 'scarecrow' promoter (Wysocka-Diller J W et al., 2000. Development 127:595-603); bundle sheath OSTMTl promoter (Cho J L et al. 2010. New Phytologist 186:657-668) and the green tissue Fbpase (Lloyd J C et al., 1991. Molecular & General Genetics 225:209-216).

According to certain embodiments, the expression vector further comprises regulatory elements at the 3¹ non-coding sequence. As used herein, the "3¹ non-coding sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht I L et al. (1989 Plant Cell 1 :671-680).

Those skilled in the art will appreciate that the various components of the nucleic acid sequences and the transformation vectors described in the present invention are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the constructs and vectors of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.

According to another aspect, the present invention provides a method for increasing the yield of a crop plant, comprising (a) transforming a plant cell with an expression vector comprising a polynucleotide encoding NtAPQl and (b) regenerating the transformed cell into a transgenic plant comprising at least one root cell and at least one leaf cell expressing NtAQPl having an increased yield compared to a corresponding non-transgenic plant.

Methods for transforming a plant cell with nucleic acids sequences according to the present invention are known in the art. As used herein the term "transformation" or "transforming" describes a process by which a foreign DNA, such as a DNA construct, including expression vector, enters and changes a recipient cell into a transformed, genetically modified or transgenic cell. Transformation may be stable, wherein the nucleic acid sequence is integrated into the plant genome and as such represents a stable and inherited trait, or transient, wherein the nucleic acid sequence is expressed by the cell transformed but is not integrated into the genome, and as such represents a transient trait. According to preferred embodiments the nucleic acid sequence of the present invention is stably transformed into a plant cell.

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

The principal methods of the stable integration of exogenous DNA into plant genomic DNA includes two main approaches:

Agrobacterium-mediated gene transfer: The Agrobacterium-medi&ted system includes the use of plasmid vectors that contain defined DNA segments which 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 (Horsch et al., 1988. Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht). The floral dip transformation method is typically used to transform the model plant Arabidopsis (Clough S J and Bent A F, 1998. Plant J 16:735-743). A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially useful in the generation of transgenic dicotyledenous plants.

Direct DNA uptake: There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field, opening up mini-pores to allow DNA to enter. In microinjection, the DNA is mechanically injected directly into the cells using 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.

According to certain embodiments, transformation of the DNA constructs of the present invention into a plant cell is performed using Agrobacterium system.

The transgenic plant is then grown under conditions suitable for the expression of the recombinant DNA construct or constructs. Expression of the recombinant DNA construct results in the presence of active NtAQPl within the plant cell, particularly within the root and mesophyll cells.

The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art

(Weissbach and Weissbach, In.: Methods for Plant Molecular Biology, (Eds.), 1988

Academic Press, Inc., San Diego, CA). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

Selection of transgenic plants transformed with a nucleic acid sequence of the present invention as to provide transgenic plants expressing NtAQPl is performed employing standard methods of molecular genetic, known to a person of ordinary skill in the art. For example, the expression vector further comprises a nucleic acid sequence encoding a product conferring resistance to antibiotic, and thus transgenic plants are selected according to their resistance to the antibiotic. Antibiotic typically serving as a selectable marker is one of the aminoglycoside group consisting of paromomycin and kanamycin.

Alternatively, the presence the NtAQPl gene is confirmed using PCR with

NtAQPl specific primers. The expression of the NtAQPl may be monitored by conventional methods known to a person skilled in the art, for example by extracting proteins from tissues of the transgenic plants, particularly root and leaf tissue and testing with antibodies directed against the NtAQPl, as exemplified hereinbelow.

The development or regeneration of plants containing the foreign, exogenous gene that encodes a protein of interest is well known in the art. There is a variety of methods in the art for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines, or pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one of skill in the art.

As exemplified hereinbelow, the transgenic plants of the present invention produced higher yield in a soil salinity range of 6 dS/m to 11 dS/m compared to the non-transgenic plants grown under the same conditions.

As used herein, the term "salt concentration" refers particularly to "NaCl concentration". However, it is to be understood that the teachings of present invention encompasses any equivalent salt that may be present in a plant growth medium, including, for example, KCl, and CaCl₂.

According to other embodiment, the transgenic plants of the present invention show an enhanced tolerance to drought stress compared to unmodified plants. Plants having increased tolerance to drought can easily adjust to growth under semi-dry and dry conditions, a trait which is highly desirable due to the growing process of desertification in agricultural areas all over the world. Drought treatment irrigation consisted of ca. 700 ml per pot of 3.9-liter once a day.

The transgenic plants of the present invention produces higher crop yield compared to corresponding non-transgenic plants. The yield is measured according to the crop type and typically includes total crop mass. When appropriate, crop yield is also measured by number, for example for fruit, flowers, tubers and the like. Harvest index is calculated by dividing total weight of fruit per plant (fruit number x individual fruit weight) by fresh weight per plant.

Also within the scope of this invention are seeds or plant parts obtained from the transgenic plants. Plant parts include differentiated and undifferentiated tissues, including but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture such as single cells, protoplasts, embryos, and callus tissue. The plant tissue may be in plant or in organ, tissue or cell culture.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Material and Methods

Construction of Transgenic Plants

The full-length cDNA of the NtAQPl gene was digested from pCRII TOPO (Invitrogen) using BamHl and Xhol restriction enzymes. NtAQPl was then cloned, using the same restriction enzymes, into binary plasmid pBIN203 (courtesy of Dr. Orit

Edelbaum) under the regulation of the 35S constitutive promoter. M82 tomato (Solanum

Iy coper sicuπi) lines were genetically transformed using disarmed Agrobacterium tumefaciens transformation methods (Barg R et al., 1997. J. Exp. Bot. 48:1919-1923). Arabidopsis {Arabidopsis thaliana) plants were genetically transformed using the floral dip transformation method (Clough and Bent, 1998. ibid). Plants were assayed for the presence of the NtAQPl gene using PCR (4 min initial denaturation at 94⁰C, followed by 33 cycles of 94°C for 30s, 58⁰C for 30s, 72°C for 1 min, and a final step at 72°C for

15 min). Primers: sense primer 35Sprom-FWD 5'-TATCCTTCGCAAGACCCTCC-S' (SEQ ID NO:3), and the reverse complementary primer NtAQPl-REV 5'-

TGCCTGGTCTGTGTTGTAGAT-3' (SEQ ID NO:4), amplifying a 930-bp NtAQPl

DNA fragment. mRNA Extraction and cDNA Construction

Leaf tissue (100 mg) was taken from transgenic and control plants and total RNA was extracted using Tri-Reagent (Sigma-Aldrich) according to the manufacturer's protocol. To rule out the effect of any residual genomic DNA in the preparation, RNA was treated with TURBO ONA-free™ (Ambion) according to the manufacturer's instructions. Total RNA (1 μg) was taken for RT-PCR using ReverseTranscriptase

Moloney murine leukemia virus RevertAid according to the manufacturer's protocol

(Fermentas). cDNA amplification was performed using the sense primer NtAQPl-RNA

SENSE 5'- CCGGGCAGGTGTACTATCC 3' (SEQ ID NO:5), and the reverse complementary primer NtAQPl-REV 5'- TGCCTGGTCTGTGTTGTAGAT-3' (SEQ

ID NO:4), amplifying an 830-bp NtAQPl cDNA fragment.

Protein Extraction and Detection

Leaf tissue (100 mg) was taken from transgenic and control plants and homogenized in three volumes of homogenization buffer (330 mM sucrose, 100 mM KCl, 1 mM EDTA, 50 mM Tris/0.05% MES pH 7.5, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF)). The sample was then centrifuged twice for 15 min, once at lOOOg (supernatant collected) and then at 10,000g (supernatant collected). Finally the sample was centrifuged at 48,00Og for 75 min to extract the microsomal phase. The pellet (microsomal fraction) was resuspended in membrane buffer (330 mM sucrose, 20 mM DTT, 50 mM Tris/0.05% MES pH 8.5).

Protein extracts were diluted in sample buffer (10% [v/v] glycerol, 5% [v/v] mercaptoethanol, 0.125 M Tris-HCl pH 6.8, 3% [w/v] SDS, 0.05% [w/v] Bromophenol blue) and subjected to 10% SDS-PAGE. After electrophoresis, proteins were electroblotted onto a Hybond-C Extra membrane (Amersham Life Science) at 4°C for 2 h at 110 V, using transfer buffer (25 mM Tris HCl pH 8.3, 192 mM glycine) supplemented with 10% (v/v) methanol. The membranes were blocked for 1 h at 22⁰C to 25°C with 2% (w/v) bovine serum albumin (BSA) in 10 mM Tris HCl pH 7.5, 150 mM NaCl containing 0.1% Tween 20 (TBS-T). Briefly, membranes were incubated for 18 h at 4°C with primary antibody (1 :5000 dilution, kind gift from Prof. RaIf Kaldenhoff). All subsequent steps were performed at 22°C to 25⁰C. Following five washes of 10 min each in TBS-T, membranes were incubated for 1 h with horseradish peroxidase-linked secondary antibody. After intensive washes with TBS-T, immobilized conjugates were visualized by enhanced chemiluminescence (ECL, Amersham Life Science, Buckinghamshire, UK), followed by exposure to X-ray film.

Plant Material and Growth Conditions

The experiments were conducted on T2 generation plants from three independent transgenic M82 tomato lines overexpressing the tobacco PM AQP gene NtAQPl: TOM-

NtAQPl and nontransgenic plants as controls. The plants were transplanted to 3.9-liter pots with ready mixed growing substrate and were grown for approximately 3 months

(May to August) in a controlled greenhouse at 25⁰C and 50% to 60% relative humidity.

The experimental design was completely randomized. Fertilization was added to the irrigation system automatically. Normal fertigation consisted of approximately 500 ml, three times a day. Drought treatment irrigation consisted of ca. 700 ml once a day. Salt treatment was applied by treating the plants with 1.5 1 of 100 mMNaCl solution in the fertigation solution, given once a day.

The Arabidopsis experiment consisted of two independent T2 transgenic Arabidopsis lines overexpressing NtAQPl and nontransgenic plants as controls. All plants were grown in a controlled growth chamber at 22⁰C under short-day conditions (10 h of light) in 200-ml pots with commercial growing medium containing slow- release fertilizers. Plants were irrigated with tap water or 100 mM NaCl solution until shoot harvesting (45 days from transplanting). Yield Parameters

Total number and weight of fruits from the transgenic TOM-NtAQPl and control plants were measured for each plant under normal, drought and salt stress (100 mM NaCl) conditions. Average fruit weight was calculated by dividing the total weight of the fruits by their number. The fresh weight of the above ground shoots was measured. Harvest index was calculated by dividing total weight of fruits per plant (fruit number x individual fruit weight) by fresh weight per plant.

Gas-Exchange Measurements

The response of net photosynthesis (A_N) to sub-stomatal CO₂ concentration (Cj),

A_N-CJ measurements, were performed on three independent T₂ transgenic TOM- NtAQPl and control plants inside a commercial green house on fully expanded leaves, under all tested irrigation conditions, using Li-6400 portable gas-exchange system (Li- Cor Inc.). Photosynthesis was induced in saturating light (1200 μmol m^"2 s^"1) and 370 μmol mol^'1 CO₂ surrounding the leaf (C₃). The amount of blue light was set to 15% PFD (photosynthetically active photon flux density) to optimize stomatal aperture. The leaf- to-air vapor pressure deficit (VPD) was kept around 1-2.5 kPa during all measurements. Leaf temperature for all measurements was approximately 26⁰C (ambient temperature). Once steady state was reached, a CO₂-response curve was measured and finally, the A_N- Cj curve was plotted.

Osmotic Water Permeability Coefficient (Pf) Measurements

Protoplasts were isolated from tobacco leaf mesophyll (Uehlein et al., 2003. ibid) and subjected to 10 mg/liter tetracycline for about 1 h to induce NtAQPl gene expression. P_f was measured from the initial (videotaped) rate of volume increase in a single protoplast in response to hypotonic solution. The P_f was determined by a numerical approach (off-line curve-fitting procedure using several algorithms), which has been proven to yield accurate P_f values over a large range of water permeability values. The analyses were performed with the P_fFit program incorporating these equations, as described in detail in Moshelion M et al. (2002. Plant Physiol. 128: 34-

642) and Volkov V et al. (2007. J. Exp. Bot. 58: 377-390).

Measurements of whole-plant transpiration rate

The calculation of whole-plant transpiration rate was based on the rate of the plant's weight loss. The examined plants were planted in 3.9-liter pots. Each pot was placed on a temperature-compensated load cell with digital output. To monitor the temporary variation in water demand in the greenhouse, a vertical wet wick was used, made of 0.14-m² cotton fibers, that was partially submerged in a 1 -liter water tank. The wick system was located on a load cell. Evaporation from the growth-medium surface was prevented by covering the pot surface with aluminum foil. Each pot was immersed in a non-transparent plastic container (13 x 21.5 x 31.5 cm [Height X Width X Length) through a hole in its upper cover. The container was sealed to prevent evaporation. The load cell output was monitored every 10 s and the average readings over 3 min were logged in a data logger for further analysis. The whole-plant transpiration rate was calculated by a numerical derivative of the load cell output after a data-smoothing process. The plants' daily transpiration rate was normalized by their total leaf area

(measure by Li-Cor area meter, model Li-3100, Li-Cor Inc.) and the neighboring submerged wick (i.e. mean daily evaporation was averaged for a given treatment, over all plants; wick daily amount = 100%).

The plants were fertigated once a day by adding a commercial fertilizer solution to the container. Two stress treatments were applied to the transgenic and control plants - salinity and drought. The salinity stress included a solution of 100 mM NaCl to which the normal dosage of nutrients was added. The salinity treatment was applied for 3 consecutive days. Drought was imposed by stopping the irrigation until the plant showed significant turgor loss. Normal irrigation was resumed at the end of the stress treatments to examine the plants' recovery patterns. Stomatal aperture and density

Abaxial leaf stomata were imprinted on glass as detailed in Geisler M and Sack F D (2002. New Phytol. 153:469-476). Counting and photographing were performed under a bright-field inverted microscope (1M7100; Zeiss) mounted with a Hitachi HV- D30 CCD camera. Stomatal images were later analyzed to determine aperture using the Grafting experiment

Seedlings from three independent T₂ transgenic TOM-NtAQPl plants (2 weeks after germination) were used as scions on control rootstocks and vice versa. As controls, both TOM-NtAQPl plants and control plants grafted on corresponding rootstock were used (control/control; TOM-NtAQP 1/TOM-NtAQPl). Two weeks after grafting, the plants were transplanted to 3.9-liter pots and grown in a semi-controlled greenhouse, cooled by compact evaporative air cooling unit that allows lowering the temperature of the air passing through the evaporative pad (approximately 30⁰C). 8-week-old plants were exposed to a series of measurements including whole-plant transpiration rate and gas exchange (leaf temperature was ambient temperature, about 30⁰C) as described above.

Root conductivity (Lp)

Two independent T₂ transgenic TOM-NtAQPl plants and control plants were used. On the night before the experiment, the main stem was cut with a razor 5 cm aboveground and the stump was sleeved with a silicone tube sealed air-tight. The plants were then irrigated with fresh nutrient solution until drainage. The next morning (08:00 a.m.), the plants were irrigated again and a vacuum pump (RK 400, Today's Instruments Co.) was connected to the sleeve via a custom-made liquid trap and vacuum was adjusted to a suction of 80 kPa. The first 15 min of exuded sap was discarded and thereafter the sap was collected every 30 min. After 3 h, excess salt solution was added (50 mM NaCl) and sap collection was continued for additional 3 h. Sap fractions were weighed and plant discharge was calculated. Samples were then stored in the cold (5°C) for later mineral element analysis. Stem cross sections of 250 μm width were taken using a Vibratom (VTlOOOS, Leica, http://www.leica.com) and photographed under bright-field inverted microscope (Olympus-IX8 CeIl-R) mounted with an Orca-AG CCD camera (Hamamatsu). Image analysis of the sections was used to determine the area of the xylem elements using the ImageJ software area measurement tool. A microscopic ruler (Olympus) was used for the size calibration. Sap discharge was normalized to flux (average velocity) based on conductive area.

Lp was calculated using the general flow equation and accounted for both hydrostatic and osmotic pressure gradients (JoIy R J, 1989. Plant Physiol 91 :1262— 1265). The osmotic component included only sodium concentrations, as the osmotic component calculated for the other cations in the sap was relatively small; therefore, it was neglected. The reflection coefficient of the entire root system was assumed to be 0.5 (based on Steudle E, 2001. Plant Cell Physiol 43:70-78).

Sap ion concentration

Inductively coupled plasma mass spectrometry analysis (ARCOS-SOP, Spectro

Analytical Instruments) was used to determine sodium (Na⁺) sap concentration and Na⁺- uptake rate was quantified by multiplying Na⁺ concentration by sap discharge.

Statistical analysis

Statistically significant differences between plants with altered NtAQPl expression and their respective controls were analyzed using Student's t-test for comparison of the means. In all cases, the level of significance used was not more than

5% (P < 0.05). P-value was computed using Fisher's Exact Test statistic. All of the results, unless otherwise specified, are presented as mean ± SE. Root conductivity measurement (Lp) and sap ion concentration results were grouped into pre- and post-salt treatment analyzed for significance using EMS procedure followed by Student's t test with α = 0.05 (JMP, SAS Institute). Results

Example 1: NtAQPl increases the osmotic water permeability of tobacco mesophyll cells

The impact of NtAQPl on the Pf value of tobacco mesophyll protoplasts was measured by cell-swelling assay. Mesophyll protoplasts were isolated from tobacco (Nicotiana tabacum line Ho 20.20, Uehlein et al., 2003. ibid) expressing NtAQPl under a 35S tetracycline-inducible promoter. The induced cells had three times higher P_f values than the control non-induced cells (Figure 1), indicating NtAQP l's activity as a functional water channel in mesophyll cells.

Example 2: The Impact of NtAOPl Water Conductivity and Photosynthesis in Tomato Plants under Normal and Salt Stress Conditions

To determine NtAQP l's impact on whole-plant water conductivity (A_N) and abiotic-stress resistance (which was defined as fruit yield under stress relative to controls), the NtAQPl gene was introduced into tomato {Solarium lycopersicum), producing TOM-NtAQP 1.

Three different independent transgenic tomato lines expressing NtAQPl under the constitutive promoter 35S were identified at the genomic DNA, mRNA and membrane protein levels (Figure 2).

The impact of NtAQPl on leaf photosynthesis and stomatal conductivity was tested using a gas-exchange measuring system that enables monitoring CO₂ assimilation rate. TOM-NtAQPl plants showed significantly higher A_N and gs, while the Ci concentration remained the same relative to control plants under normal conditions

(Table 1; Figure 3).

Higher gs can result from changes in stomatal density and/or stomatal pore area. An anatomical characterization of the leaf epidermis of both TOM-NtAQPl and control plants was performed to explore this issue. Imprints of the abaxial leaf surface revealed no significant change in stomatal density among plants, whereas a significantly larger stomatal pore area was measured in TOM-NtAQPl plants under both normal and salt stress conditions (Table 1). The higher A_N was maintained under 10OmM NaCl, suggesting that NtAQPl ability to transport CO₂ provides a substantial benefit in improving carbon's diffusive permeability under stress conditions. Calculating the instantaneous WUE (IWUE) revealed improvement in TOM-NtAQPl plants under normal, drought and salt-stress growth conditions compared with the control plants, with the highest improvement seen in TOM-NtAQPl plants under salt stress (Figure 4).

The rate of tension-induced root exudation from the de-topped root system of TOM-NtAQPl plants differed from that of controls under normal irrigation. Moreover, salt application caused a considerable decrease in root exudation in control plants, while

TOM-NtAQPl plants retained their original rate of sap exudation (Figure 5). Root Lp accounting for the total cross-sectional area of the xylem, in TOM-NtAQPl plants did not differ from that in control plants under normal irrigation. However, when irrigated with water containing 50 mM NaCl, the TOM-NtAQPl plants decreased their Lp only by about 40%, while Lp of control plants decreased more than 3-fold (Table 1).

Both plant types yielded a low and stable sodium concentration in their xylem sap of around 2mM Na⁺ during the 3 h prior to irrigation with water containing 50 mM Naci. Within a short time after irrigation with the salt-containing water, the sodium concentration rose dramatically in the xylem sap of both plants, with control plants reaching over 26 mM Na⁺ almost twice the Na⁺ concentration in the xylem sap of TOM-NtAQPl plants. However, when the lower exudate volume collected from the control plants following the salt treatment relative to the TOM-NtAQPl plants was taken into account, the amount of Na⁺ that was taken up by the de-topped root systems (Na+ discharge) did not differ between the two treatments (Table 1 ).

The higher gs in TOM-NtAQPl compared with control plants was supported by the wider stomatal aperture measured; however, under salinity stress, the gs values in both plants were similar, while TOM-NtAQPl plants continued to show significantly wider stomatal aperture (Table 1). This phenomenon suggested that the timing of the stomatal conductivity measurements may be of importance. Therefore, a continuous daily monitoring of plant transpiration was conducted.

Using a multiple lysimeter system normalized for vapor-pressure deficit (VPD) and plant leaf area, the daily transpiration rates was measured in all TOM-NtAQPl and control plants simultaneously. Daily transpiration rate in the former was higher than in control plants under normal growth conditions (Figure 6A), mainly during the hours of high water demand (11 :00 a.m. to 3:00 p.m.), resulting in significantly higher relative transpiration levels for the entire day (Figure 6B). Total daily transpiration was defined by the area under the daily transpiration rate curve. The differences in transpiration between TOM-NtAQPl and control plants were significant during the salinity stress treatment (Figure 7 A and 7B) and during subsequent recovery from salinity stress (Figure 7D). Despite the fact that transpiration levels in all plants were dramatically decreased following the drought treatment, the relative transpiration level of TOM- NtAQPl remained significantly higher than that of control plants as long as the plants did not reach severe loss of leaf turgor (Figure 7D and 7E).

Example 3: NtAQPTs Role in Preventing Root-Shoot Hydraulic Failure and Improving Whole-Plant Stress Resistance Increasing gs and transpiration, on the one hand, while maintaining normal root

Lp under osmotic stress conditions, on the other hand, suggests dual independent roles for the NtAQPl channel in whole-plant hydraulic control. To estimate the relations between these activities of NtAQPl and the relative importance of each in the whole plant's response to stress, a reciprocal grafting experiment was conducted. Whole-plant transpiration rates and relative daily transpiration of all grafted plants were simultaneously measured (using the multiple lysimeter system and normalization of each plant to VPD and leaf area; see Materials and Methods section hereinabove). The grafting process did not affect the plants' behavior, as reflected by the fact that the control grafted plants, TOM-NtAQPl over TOM-NtAQPl (T/T) and control over control (C/C), maintained similar transpiration rate patterns as their non-grafted counterparts, that is, higher transpiration rate and relative daily transpiration of the transgenic plants under both normal and salt treatments (Figure 8A and 8B). Interestingly, the T/C grafted plants exhibited a considerable reduction, starting at midday, in the rate of the whole-plant daily course of transpiration. This midday "break" in transpiration rate in T/C plants (clearly seen under both normal and salt stress conditions) might be explained by stomatal closure. Another explanation for the break might be a failure in Lp resulting from their higher gs and lower LP (as was demonstrated previously in the non-grafted plants).

To estimate the relative impact of root hydraulic signals on shoot gs and A_N under salt stress in the reciprocal grafted plants, both were measured in the grafted plants.

These measurements were taken during two time intervals: morning (8:00-11 :00 a.m.) and noontime (11 :00 a.m. to 2:00 p.m.). During the morning period, no change in whole-plant transpiration rate (Figure 8B) or in either gs or AN could be detected in the grafted plants (Figure 8C and 8E). During the noon period, however, a break in transpiration rate was clearly seen in all but the T/T plants (Figure 8B). While the reduction in whole-plant transpiration rate could be explained by stomatal closure for the C/C and C/T plants, this could not explain the high gs and A_N values of T/C plants, which remained higher than controls and similar to T/T plants (Figure 8D and 8F): both T/T and T/C plants maintained similar significantly higher gs and A_N values than C/C or than C/C or C/T plants.

Example 4: TOM-NtAQPl Plants Show Higher Yields than Control under Normal and Salt Treatments

The fact that TOM-NtAQPl plants showed significant increases in whole-plant transpiration and A_N, resulting in increased water use efficiency, suggests that NtAQPl may contribute to plant vigor, biomass, and yield parameters under both optimal and stress conditions. Thus, a greenhouse experiment was conducted to determine the impact of NtAQPl on plant productivity under normal, drought and salinity conditions.

Three independent transgenic TOM-NtAQPl plants were grown in a controlled greenhouse under optimal, water-deficient or 100 ΓΠM NaCl conditions for an entire growing season. In each trial, the transgenic genotypes were compared with non- transformed plants as controls. In the salt stress trial, all of the plants were continuously irrigated with water containing 100 rriMNaCl. TOM-NtAQPl plants did not appear to be more vigorous than control plants under either control or stress irrigation; nevertheless, TOM-NtAQPl plants showed improved yield parameters, relative to controls, under both favorable and stressed (salt and drought) growth conditions (Figure 9).

Under drought conditions, TOM-NtAQPl plants developed significantly higher fruit number (57%) and fruit weight (90%), with no difference in plant fresh weight compared to controls. Consequently, a significantly higher harvest index was revealed for the TOM-NtAQPl plants under drought conditions (Figure 9D). The salt-stressed

TOM-NtAQPl plants showed significant improvement only in fresh plant weight and fruit weight (Figure 9A and 9C). To rule out the possibility that NtAQPl 's impact is unique to tomato or to the Solanaceae, a complementary experiment with transgenic

Arabidopsis plants expressing NtAQPl was conducted. These plants showed increased tolerance to a salt stress of 100 HIM NaCl compared to control plants, as reflected by their 125% higher dry biomass under salt stress and 81% higher dry biomass under control irrigation (Figure 10).

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A transgenic crop plant comprising at least one root cell and at least one leaf cell transformed with a DNA construct comprising a polynucleotide encoding a Nicotiana tabacum aquaporin-1 (NtAQPl), wherein the plant has increase yield compared to a corresponding non-transgenic plant.

2. The transgenic crop plant of claim 1, wherein the polynucleotide encodes an NtATQPl comprising the amino acids sequence set forth in SEQ ID NO:1.

3. The transgenic crop plant of claim 2, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:2.

4. The transgenic crop plant of claim 1, wherein the DNA construct further comprises a regulatory element selected from the group consisting of a promoter, an enhancer, a termination sequence and a polyadenylation signal.

5. The transgenic crop plant of claim 4, wherein the promoter is a constitutive promoter.

6. The transgenic crop plant of claim 4, wherein the promoter is a tissue specific promoter selected from the group consisting of root specific promoter and shoot specific promoter.

7. The transgenic crop plant of claim 6, wherein the tissue specific promoter is selected from the group consisting of guard cell specific promoter (shoot); endodermis (root) and bundle sheath (shoot) 'scarecrow' promoter; bundle sheath OSTMTl promoter (shoot); and the green tissue Fbpase promoter (shoot).

8. The transgenic crop plant of claim 1, wherein said plant is grown under optimal water availability conditions.

9. The transgenic crop plant of claim 1 , wherein said plant is grown under abiotic stress conditions.

10. The transgenic crop plant of claim 9, wherein the abiotic stress is selected from the group consisting of water stress (drought), high soil salinity, extreme temperatures, low oxygen levels or presence of heavy metals.

11. The transgenic crop plant of claim 10, wherein the abiotic stress is water content of less than 70%.

12. The transgenic crop plant of claim 10, wherein the abiotic stress is soil salinity ofabove 4 dS/m.

13. The transgenic crop plant of claim 1, said plant is selected from the group consisting of a plant producing fruit; flower and ornamental plant; grain producing plant: wheat, oats, barely, rye, rice, maize; legumes: peanuts, peas soybean lentil; plant producing forage; plant producing fiber: cotton, flax; a tree for wood industry; plant producing tuber or root crop; sugar beet; sugar came; plant producing oil: canola, sunflower, sesame.

14. The transgenic crop plant of claim 13, wherein said plant is a tomato plant.

15. The transgenic crop plant of claim 1, said plant has a yield increase of at least 60% compared to the corresponding non-transgenic plant.

16. A seed of the plant of claim 1, wherein a plant grown from said seed comprises at least one root cell and at least one leaf cell comprising a DNA construct comprising polynucleotide encoding NtAQPl, and has increased yield compared to a plant grown from a seed of corresponding non-transgenic plant.

17. A tissue culture comprising at least one transgenic cell of the plant of claim 1 or a protoplast derived therefrom.

18. The tissue culture of claim 17, wherein said tissue culture regenerates a plant having at least one root cell and at least one leaf cell comprising a DNA construct comprising polynucleotide encoding NtAQPl, and has increased yield compared to a plant grown from a seed of corresponding non-transgenic plant.

19. A plant regenerated from the tissue culture of claim 18.

20. A method for increasing the yield of a crop plant, comprising (a) transforming a plant cell with a DNA construct comprising a polynucleotide encoding NtAPQl and (b) regenerating the transformed cell into a transgenic plant comprising at least one root cell and at least one leaf cell expressing NtAQPl having an increased yield compared to a corresponding non-transgenic plant.

21. The method claim 20, wherein the polynucleotide encodes an NtATQPl comprising the amino acids sequence set forth in SEQ ID NO:1.

22. The method claim 21, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:2.

23. The method claim 20, wherein the DNA construct further comprises a regulatory element selected from the group consisting of a promoter, and enhancer, a termination sequence and a polyadenylation signal.

24. The method of claim 23, wherein the promoter is a constitutive promoter.

25. The method of claim 23, wherein the promoter is tissue specific promoter selected from the group consisting of root specific promoter and shoot specific promoter.

26. The method of claim 25, wherein the tissue specific promoter is selected from the group consisting of guard cell specific promoter (shoot); endodermis (root) and bundle sheath (shoot) 'scarecrow' promoter; bundle sheath OSTMTl promoter (shoot); and the green tissue Fbpase promoter (shoot).

21. A method of screening for a plant capable of producing high yield when grown under abiotic stress conditions comprising: (a) obtaining a plurality of samples from a plurality of plant lines and a control sample from a reference plant, the samples comprising genetic material; (b) measuring the expression level of a polynucleotide encoding NtAQPl or an ortholog thereof in the samples; (c) comparing the expression level of the polynucleotide encoding NtAQPl or the ortholog thereof in the plurality of samples to the control sample; wherein a plant line overexpressing said polynucleotide encoding NtAQPl or ortholog thereof is capable of producing high yield when grown under abiotic stress conditions.

28. The method of claim 27, wherein the abiotic stress condition is selected from the group consisting of water stress (drought), high soil salinity, extreme temperatures, low oxygen levels or presence of heavy metals.

29. The method of claim 28, wherein the abiotic stress is water content of less than 70%.

30. The method of claim 28, wherein the abiotic stress is soil salinity of above 4 dS/m.

31. The method of claim 27, wherein the polynucleotide encodes NtATQPl having at least 75%, 85% or more homology to the protein having the amino acids sequence set forth in SEQ ID NO: 1.

32. The method of claim 27, wherein the polynucleotide comprises a nucleic acids sequence having at least 75%, 85% or more homology to the nucleic acid sequence set forth in SEQ ID NO:2.

33. The method of claim 27, wherein the expression level of the polynucleotide is measured using NAT (nucleic acid technology)-based assays selected from the group consisting of quantitative PCR. Quantitative real time PCR and Northern Blot.

34. The method of claim 33, wherein the NAT assay is PCR employing a primer pair having the nucleic acid sequence set forth in SEQ ID NO: 3 and SEQ ID NO:4.

35. The method of claim 27, further comprising (a) planting the plant line overexpressing the polynucleotide encoding NtAQPl or ortholog thereof and a corresponding control plant having lower expression of said polynucleotide under abiotic stress conditions; (b) comparing the crop yield of the plant line to the crop yield of the control plant; and selecting plant lines having increased crop yield compared to the control plant.

36. The method of claim 27, wherein the plant lines are selected from plants of the same species and plants of different species.

37. The method of claim 27, wherein the control plant is tobacco (Nicotiana tabacum).