WO2011021190A1 - Plantes produisant un haut rendement de culture - Google Patents

Plantes produisant un haut rendement de culture Download PDF

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WO2011021190A1
WO2011021190A1 PCT/IL2010/000665 IL2010000665W WO2011021190A1 WO 2011021190 A1 WO2011021190 A1 WO 2011021190A1 IL 2010000665 W IL2010000665 W IL 2010000665W WO 2011021190 A1 WO2011021190 A1 WO 2011021190A1
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plant
ntaqpl
plants
promoter
transgenic
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PCT/IL2010/000665
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Nir Sade
Menachem Moshelion
Michaele Gebretsadik
Ralf Kaldenhoff
Rony Wallach
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
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Publication of WO2011021190A1 publication Critical patent/WO2011021190A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • 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.
  • Aquaporins are integral membrane proteins that increase the permeability of membranes to water, as well as to other small molecules including CO 2 , H 2 O 2 , 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).
  • 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-an ⁇ sense showed a decrease of 13% relative to control plants (Flexas J et al., 2006. Plant Cell Environ. 31 :602-621).
  • no morphological, growth-rate or yield parameters were reported for the NtAQP 1 -overexpressing plants.
  • NtAQPl 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 CO 2 transport: while the water permeability of the CIM was threefold higher compared to the PM, its CO 2 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 CO 2 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • NtAQPl Nicotiana tabacum aquaporin-1 aquaporin
  • 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 CO 2 assimilation rates under optimal water availability as well as under drought and salt stress, leading to higher yield production.
  • 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.
  • 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.
  • the NtATQPl comprises the amino acids sequence set forth in SEQ ID NO:1 (accession number (AJ001416).
  • 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.
  • the expression of the NtAQPl is controlled by a constitutive promoter.
  • the constitutive promoter is tissue specific.
  • the promoter is root specific or shoot specific.
  • 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).
  • 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.
  • 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.
  • the transgenic crop plant has an increase yield compared to a corresponding non-transgenic plant when the plants are grown under abiotic stress conditions.
  • 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.
  • optimal water availability refers to soil water content of at least 85%.
  • 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.
  • EC soil electric conductivity
  • 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.
  • the crop plant is a plant producing a fruit crop
  • the crop plant in other than tobacco.
  • the plant is a tomato plant.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR Polymerase Chain Reaction
  • 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.
  • 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.
  • water stress rought
  • high soil salinity high soil salinity
  • extreme temperatures high temperatures
  • low oxygen levels low oxygen levels
  • the abiotic stress is water stress. According to other typical embodiments the abiotic stress is high soil salinity.
  • 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.
  • 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.
  • expression level of the polynucleotide is measured using NAT (nucleic acid technology)-based assays.
  • the NAT-based assay is selected from the group consisting of a quantitative PCR and Real-Time PCR, Northern blot and the like.
  • 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.
  • 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.
  • the plant lines are of the same plant species. According to other embodiments, the plant limes are of different species.
  • the reference plant is Tobacco
  • FIG. 1 demonstrates that NtAQPl expression increases the tobacco mesophyll membrane water permeability coefficient (P f ).
  • 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 CO 2 concentration (Ci) in control (black line) and TOM-NtAQPl transgenic (gray line) plants.
  • FIG. 4 demonstrates instantaneous water use efficiency (IWUE) of transgenic TOM- NtAQPl (white bars) and control plants (black bars).
  • IWUE instantaneous water use efficiency
  • 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
  • Fig. 5B 50 mM NaCl treatment.
  • 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. 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. 8 shows daily transpiration rate, gs and A N under normal and 10OmM NaCl irrigation.
  • 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).
  • 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. 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).
  • 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.
  • the present invention now shows that expression of the Nicotiana tabacum aquaporin (NtAQPl) enhances transpiration and CO 2 assimilation under stress conditions that typically lead to stomatal closure and reduction in CO 2 assimilation.
  • 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.
  • expression of NtAQPl in crop plants increased the stomatal pore area, CO 2 conductivity and overall yield production.
  • 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.
  • 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.
  • increased yield 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.
  • Nicotiana tabacum aquaporin -1 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.
  • abiotic stress conditions 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.
  • oil 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.
  • soil stress water stress
  • drought conditions low soil water content
  • 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%.
  • 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.
  • 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.
  • 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.
  • polynucleotide polynucleotide sequence
  • nucleic acid sequence nucleic acid sequence
  • 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.
  • DNA construct 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.
  • 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.
  • 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.
  • the DNA construct of the present invention is an expression vector.
  • the expression vector comprises a constitutive promoter operably linked to the polynucleotide encoding NtAQPl.
  • 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.
  • 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.
  • the polynucleotide encoding NtAQPl is operably linked to the regulatory sequences in a sense orientation.
  • 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.
  • 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.
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • transient transformation may be detected by detecting the activity of the protein (e.g. ⁇ -glucuronidase) encoded by the exogenous polynucleotide.
  • transient transformant refers to a cell which has transiently incorporated one or more exogenous polynucleotides.
  • 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.
  • 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.
  • polypeptide peptide
  • protein protein
  • 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.
  • 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
  • the NtATQPl comprises the amino acids sequence set forth in SEQ ID NO:1 (accession number (AJOOl 416).
  • the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:2.
  • 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 CO 2 channel.
  • 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.
  • 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.
  • the transgenic crop plant has an increase yield compared to a corresponding non-transgenic plant when the plants are grown under abiotic stress conditions.
  • 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.
  • 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
  • NtAQPl The involvement of NtAQPl in the mechanism controlling stomatal and CO 2 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 CO 2 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.
  • 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.
  • Lp root hydraulic conductance
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • water stress rought
  • high soil salinity high soil salinity
  • extreme temperatures high temperatures
  • low oxygen levels low oxygen levels
  • the abiotic stress is water stress.
  • the abiotic stress is high soil salinity.
  • 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.
  • the assay is a nucleic acid technology (NAT)-based assay, typically quantitative PCR, employing primers specific to the target DNA.
  • NAT nucleic acid technology
  • 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.
  • 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.
  • 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.
  • 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.
  • the expression of the NtAQPl is controlled by a constitutive promoter.
  • the constitutive promoter is tissue specific.
  • the promoter is root specific or mesophyll specific.
  • promoter element refers 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.
  • nopaline synthase (NOS) promoter (Ebert et al., 1987 Proc. Natl. Acad. Sci. U.S.A. 84:5745-5749)
  • OCS octapine synthase
  • caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987 Plant MoI Biol.
  • 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.
  • the expression vector of the present invention comprises the constitutive CaMV 35S promoter.
  • 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).
  • the expression vector further comprises regulatory elements at the 3 1 non-coding sequence.
  • the "3 1 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • microprojectiles such as magnesium sulfate crystals or tungsten particles
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • salt concentration refers particularly to "NaCl concentration”.
  • teachings of present invention encompasses any equivalent salt that may be present in a plant growth medium, including, for example, KCl, and CaCl 2 .
  • 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.
  • 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 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
  • Iy coper sicu ⁇ i 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 94 0 C, followed by 33 cycles of 94°C for 30s, 58 0 C for 30s, 72°C for 1 min, and a final step at 72°C for
  • 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-freeTM (Ambion) according to the manufacturer's instructions. Total RNA (1 ⁇ g) was taken for RT-PCR using ReverseTranscriptase
  • 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)).
  • 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.
  • 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.
  • 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
  • the membranes were blocked for 1 h at 22 0 C 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 25 0 C. 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.
  • ECL enhanced chemiluminescence
  • 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
  • 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 22 0 C 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.
  • a N -CJ measurements were performed on three independent T 2 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 ⁇ mol m "2 s "1 ) and 370 ⁇ mol mol '1 CO 2 surrounding the leaf (C 3 ). 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 26 0 C (ambient temperature). Once steady state was reached, a CO 2 -response curve was measured and finally, the A N - Cj curve was plotted.
  • VPD photosynthetically active photon flux density
  • 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 f Fit program incorporating these equations, as described in detail in Moshelion M et al. (2002. Plant Physiol. 128: 34-
  • 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-m 2 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
  • 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
  • T 2 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.
  • a vacuum pump RK 400, Today's Instruments Co.
  • 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).
  • Example 1 NtAQPl increases the osmotic water permeability of tobacco mesophyll cells
  • NtAQPl 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.
  • NtAQP l was introduced into tomato ⁇ Solarium lycopersicum), producing TOM-NtAQP 1.
  • 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).
  • 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
  • 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).
  • 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).
  • TOM-NtAQPl plants Three independent transgenic TOM-NtAQPl plants were grown in a controlled greenhouse under optimal, water-deficient or 100 ⁇ M 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).
  • 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

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Abstract

La présente invention porte sur des plantes transgéniques surexprimant l'aquaporine du tabac NtAQP1, produisant un rendement de culture supérieur par comparaison avec des plantes non-transgéniques correspondantes lorsqu'elles poussent dans des conditions normales et de stress abiotique.
PCT/IL2010/000665 2009-08-17 2010-08-17 Plantes produisant un haut rendement de culture WO2011021190A1 (fr)

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WO2014209790A1 (fr) * 2013-06-24 2014-12-31 North Carolina State University Expression transgénique de superoxyde réductase archéenne
WO2016011179A3 (fr) * 2014-07-15 2016-03-10 Ceres, Inc. Procédés pour augmenter le rendement de cultures sous stress abiotique
NL2033935B1 (en) * 2022-04-29 2023-11-13 Univ Anhui Agricultural Tea plant aquaporin gene csaqp95 and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018963A1 (fr) 1998-10-01 2000-04-06 Monsanto Company Procedes de culture et de criblage de feves de soja permettant d'ameliorer la productivite et feves de soja produites en quantites ameliorees
WO2009083958A2 (fr) * 2007-12-27 2009-07-09 Evogene Ltd. Peptides isolés, polynucléotides utiles pour modifier l'efficacité d'utilisation de l'eau, l'efficacité d'utilisation d'engrais, la tolérance aux stress biotiques/abiotiques, le rendement et la biomasse dans des plantes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018963A1 (fr) 1998-10-01 2000-04-06 Monsanto Company Procedes de culture et de criblage de feves de soja permettant d'ameliorer la productivite et feves de soja produites en quantites ameliorees
WO2009083958A2 (fr) * 2007-12-27 2009-07-09 Evogene Ltd. Peptides isolés, polynucléotides utiles pour modifier l'efficacité d'utilisation de l'eau, l'efficacité d'utilisation d'engrais, la tolérance aux stress biotiques/abiotiques, le rendement et la biomasse dans des plantes

Non-Patent Citations (44)

* Cited by examiner, † Cited by third party
Title
AHARON R ET AL., THE PLANT CELL, vol. 15, 2003, pages 439 - 447
AHARON REFAEL ET AL: "Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress.", PLANT CELL, vol. 15, no. 2, February 2003 (2003-02-01), pages 439 - 447, XP002606013, ISSN: 1040-4651 *
BARG R ET AL., J. EXP. BOT., vol. 48, 1997, pages 1919 - 1923
BIELA A ET AL., PLANT J., vol. 18, 1999, pages 565 - 570
CHANDLER ET AL., PLANT CELL, vol. 1, 1989, pages 1175 - 1183
CHO J L ET AL., NEW PHYTOLOGIST, vol. 186, 2010, pages 657 - 668
CLOUGH S J; BENT A F, PLANT J, vol. 16, 1998, pages 735 - 743
EBERT ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 84, 1987, pages 5745 - 5749
FLEXAS J ET AL., PLANT CELL ENVIRON., vol. 31, 2006, pages 602 - 621
FLEXAS J ET AL: "Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO 2 in vivo", PLANT JOURNAL, vol. 48, no. 3, November 2006 (2006-11-01), BLACKWELL PUBLISHING LTD GB, pages 427 - 439, XP002606010, DOI: 10.1111/J.1365-313X.2006.02879.X *
FLEXAS JAUME ET AL: "Mesophyll conductance to CO2: current knowledge and future prospects", PLANT CELL AND ENVIRONMENT, vol. 31, no. 5, May 2008 (2008-05-01), pages 602 - 621, XP002606015, ISSN: 0140-7791 *
GEISLER M; SACK F D, NEW PHYTOL., vol. 153, 2002, pages 469 - 476
HORSCH ET AL.: "Plant Molecular Biology Manual", vol. A5, 1988, KLUWER ACADEMIC PUBLISHERS, pages: 1 - 9
INGELBRECHT I L ET AL., PLANT CELL, vol. 1, 1989, pages 671 - 680
JOLY R J, PLANT PHYSIOL, vol. 91, 1989, pages 1262 - 1265
KALDENHOFF R; FISCHER M, ACTA PHYSIOL., vol. 187, 2006, pages 169 - 176
LAWTON ET AL., PLANT MOL BIOL., vol. 9, 1987, pages 315 - 324
LLOYD J C ET AL., MOLECULAR & GENERAL GENETICS, vol. 225, 1991, pages 209 - 216
MAHDIEH M ET AL., PLANT CELL PHYSIOL., vol. 49, 2008, pages 801 - 813
MAHDIEH MAJID ET AL: "Drought stress alters water relations and expression of PIP-type aquaporin genes in Nicotiana tabacum plants", PLANT AND CELL PHYSIOLOGY, vol. 49, no. 5, May 2008 (2008-05-01), pages 801 - 813, XP002606011, ISSN: 0032-0781 *
MAUREL C, FEBS LETT., vol. 581, 2007, pages 2227 - 2236
MOSHELION M ET AL., PLANT PHYSIOL., vol. 128, 2002, pages 34 - 642
ODELL ET AL., NATURE, vol. 313, 1985, pages 810 - 812
OKAMURO J K; GOLDBERG R B, BIOCHEMISTRY OF PLANTS, vol. 15, 1989, pages 1 - 82
PERDERSEN ET AL., CELL, vol. 29, 1982, pages 1015 - 1026
PLESCH G ET AL., PLANT JOURNAL, vol. 28, 2001, pages 455 - 464
POTRYKUS I, ANNU REV PLANT PHYSIOL PLANT MOL BIOL, vol. 42, 1991, pages 205 - 225
SADE N. ET AL., PLANT PHYSIOL., vol. 152, 2010, pages 245 - 254
SADE NIR ET AL: "The Role of Tobacco Aquaporin1 in Improving Water Use Efficiency, Hydraulic Conductivity, and Yield Production Under Salt Stress", PLANT PHYSIOLOGY (ROCKVILLE), vol. 152, no. 1, January 2010 (2010-01-01), pages 245 - 254, XP002606012, ISSN: 0032-0889 *
SHIMAMOTO K. ET AL., NATURE, vol. 338, 1989, pages 274 - 276
SIEFRITZ F ET AL., J. EXP. BOT., vol. 52, 2001, pages 1953 - 1957
SIEFRITZ F ET AL., PLANT CELL, vol. 14, 2002, pages 869 - 876
SIEFRITZ FRANKA ET AL: "PIP1 plasma membrane aquaporins in tobacco: From cellular effects to function in plants", PLANT CELL, vol. 14, no. 4, April 2002 (2002-04-01), pages 869 - 876, XP002606014, ISSN: 1040-4651 *
SIEFRITZ FRANKA ET AL: "The tobacco plasma membrane aquaporin NtAQP1", JOURNAL OF EXPERIMENTAL BOTANY, vol. 52, no. 363, October 2001 (2001-10-01), pages 1953 - 1957, XP002606016, ISSN: 0022-0957 *
STEUDLE E, J. EXP. BOT., vol. 51, 2000, pages 1531 - 1542
STEUDLE E, PLANT CELL PHYSIOL, vol. 43, 2001, pages 70 - 78
TERASHIMA I; ONO K, PLANT CELL PHYSIOL., vol. 43, 2002, pages 70 - 78
UEHLEIN N ET AL., NATURE, vol. 425, 2003, pages 734 - 737
UEHLEIN N ET AL., PLANT CELL, vol. 20, 2008, pages 648 - 657
VOLKOV V ET AL., J. EXP. BOT., vol. 58, 2007, pages 377 - 390
WALKER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 84, 1987, pages 6624 - 66280
WEISSBACH AND WEISSBACH,: "Methods for Plant Molecular Biology", 1988, ACADEMIC PRESS, INC.
WYSOCKA-DILLER J W ET AL., DEVELOPMENT, vol. 127, 2000, pages 595 - 603
YANG ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 87, 1990, pages 4144 - 4148

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