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WO2012028646A1 - Moyens destinés à améliorer des caractères agrobiologiques dans des plantes - Google Patents

Moyens destinés à améliorer des caractères agrobiologiques dans des plantes Download PDF

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WO2012028646A1
WO2012028646A1 PCT/EP2011/065005 EP2011065005W WO2012028646A1 WO 2012028646 A1 WO2012028646 A1 WO 2012028646A1 EP 2011065005 W EP2011065005 W EP 2011065005W WO 2012028646 A1 WO2012028646 A1 WO 2012028646A1
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polypeptide
akt2
cbl4
plant
cipk6
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Jörg KUDLA
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Westfaelische Wilhelms Universitaet Muenster
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Westfaelische Wilhelms Universitaet Muenster
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    • 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
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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 a method for producing a transgenic plant or plant cell having increased potassium efficiency.
  • Said method comprises introducing into a plant or plant cell a CBL4 (calcineurin B-like 4) polypeptide.
  • Said method may further comprise introducing a i) C1PK6 (CBL-interacting protein kinase 6) polypeptide or a ii) C1PK6 and AKT2 (Arabidopsis K + transporter) polypeptide into said plant or said plant cell.
  • the present invention relates to a transgenic plant or plant cell comprising a CBL4 and C1PK6 polypeptide.
  • a composition comprising a CBL4 and a C1PK6 polypeptide as well as the use of said polypeptides for the generation of a transgenic plant or plant cell having increased potassium efficiency.
  • K + Potassium
  • K is deficient or not supplied in adequate amounts, growth is stunted and yields are reduced. Soils can usually supply some K + for crop production, but when the supply from the soil is not adequate, K must be supplied by potassium fertilizers. These measures, however, cause high costs.
  • CBL1(CBL9)-C1PK23 mediated phosphorylation of AKTl is essential for transport activity of this channel in oocytes and for proper growth of plants under low K + conditions.
  • AKT 2 is one of a total of nine shaker-type K + channel subunits in Arabidopsis.
  • the AKT2 K + channel is endowed with unique functional properties, being the only weak inward rectifier characterized to date in Arabidopsis.
  • Several studies have suggested regulation of this channel by unknown protein kinases and the protein phosphatase PP2CA (Dennison, K.L. et al. Functions of AKTl and AKT2 potassium channels determined by studies of single and double mutants of Arabidopsis. Plant Physiol 127, 1012-1019 (2001), Cherel et al. Physical and functional interaction of the Arabidopsis K(*) channel AKT2 and phosphatase AtPP2CA. Plant Cell.
  • the present invention relates to a method for producing a transgenic plant or plant cell having increased potassium efficiency and/or increased salt tolerance as compared to a corresponding non-transgenic plant or plant cell, comprising introducing into a plant or a plant cell a CBL4 (calcineurin B-like 4) polypeptide.
  • CBL4 calcium phosphate B-like 4
  • plant as used herein, preferably, refers to a higher plant, preferably to a monocotelydonous plant or dicotelydonous plant.
  • the plant is a model plant, preferably Arabidopsis thaliana, or a crop plant selected from the group consisting of oilseed rape, evening primrose, hemp, thistle, peanut, canola, linseed, soybean, safflower, sunflower, borage, maize, wheat, rye, oats, rice, barley, cotton, cassava, pepper, solanaceae plants, preferably, potato, tobacco, eggplant or tomato, vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), salix species, trees (oil palm, coconut) and perennial grasses and fodder crops.
  • a model plant preferably Arabidopsis thaliana
  • a crop plant selected from the group consisting of oilseed rape, evening primrose, hemp, thistle, peanut, canola, linseed, soybean, safflower, sunflower, borage, maize, wheat, rye, oats, rice, barley,
  • a plant cell in the context of the present invention is, preferably, derived from any of the aforementioned plants. Suitable methods for obtaining cells from the aforementioned plants as well as conditions for culturing these cells are well known in the art.
  • the plant cells derived from a plant encompass cells of certain tissues, organs and parts of plants in all their plienotypic forms such as leaves, seeds, roots, anthers, fibers, root hairs, stalks, embryos, calli, cotelydons, petioles, harvested material, plant tissue, reproductive tissue and cell cultures which are derived from the actual transgenic plant and/or can be used for bringing about the transgenic plant.
  • Calcineurin B-like (CBL) polypeptides represent a unique family of calcium sensors in plant cells. CBLs were shown to sense calcium signals elicited by a variety of abiotic stresses and to transmit the information to a group of serine/threonine protein kinases.
  • the CBL4 (calcineurin B-like 4) polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 belongs to a family of a total of 10 calcineurin B-like genes in Arabidopsis thaliana.
  • the CBL4 polypeptide as referred to herein preferably, comprises a conserved core region consisting of four EF hand calcium binding domains that are separated by spacing regions encompassing a conserved number of amino acids in all CBL Proteins (Batistic and Kudla, 2004).
  • the first EF hand of a CBL4 polypeptide as referred to herein of these calcium binding proteins comprises 14 amino acids (instead of 12 amino acids typical for canonical EF hands (Batistic and Kudla, 2009)). This is accomplished by the restracturing of the binding loop, which does not consist of 12 amino acids but instead of 14 amino acids.
  • the first EF hand of a CBL4 polypeptide comprises X1*2*3*4Y5*6Z7*8-Y9*10-X11 *12*13-Z14, wherein, Z7 is aspartate (53) -Z14 is glutamate (60),X1 is serine (47), Y5 is isoleucine 51 and -Y9 is leucine (55), see also Batistic and Kudla, 2009 (The numbers in brackets only indicate the positions in the Arabidopsis thaliana CBL4 polypeptide, see SEQ ID NO: 2).
  • a CBL4 polypeptide as referred to herein preferably, comprises a rather short 46 aa N- terminal domain with a lipid modification site or lipid modification sites.
  • the CBL4 polypeptide shall be myristoylated at its N-terminus and shall be targeted to the plasma membrane (Batistic et al, Dual lipid modification determines the localization and plasma membrane targeting of Ca 2+ -regulated CBL/CffK complexes. Plant Cell, 20: 1346-1362 2008; Batistic and Kudla, Plant Calcineurin B-like proteins and their interacting protein kinases. BBA - Molecular Cell Research, 1793, 985-92.
  • CBL4 (calcineurin B-like 4) polypeptide as referred to herein is, preferably, encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 1 ;
  • nucleic acid having a nucleotide sequence being a variant of the nucleotide sequence shown in SEQ ID No: 1, wherein said nucleic acid encodes a polypeptide having calcium binding activity;
  • the CBL4 (calcineurin B-like 4) polypeptide as referred to herein is, preferably, encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 1 ;
  • nucleic acid having a nucleotide sequence being at least 50% identical to the nucleotide sequence shown in SEQ ID No: 1 , wherein said nucleic acid encodes a polypeptide having calcium binding activity;
  • nucleic acid encoding a polypeptide having an amino acid sequence being at least 50% identical to the amino acid sequence shown in SEQ ID No:2, wherein said variant has calcium binding activity.
  • Preferred CBL4 polypeptides are variants of the Arabidopsis thahana CBL4 polypeptide. Preferred variants are shown in Table A in the Examples section.
  • any one of the aforementioned polynucleotides of the present invention preferably, as a heterologous polynucleotide into a plant cell or plant, the traits referred to in accordance with the present invention will be conferred to the said plant or plant cell.
  • the nucleic acids is set forth in c) and d) above shall encode for a polypeptide having calcium binding activity.
  • the term is to be understood in the sense that the variants encoded by said nucleic acids is capable of binding calcium by the same or substantially the same mechanism as the polypeptide as shown in SEQ ID NO:2; however, the term does not necessarily indicate that the binding is quantitatively the same.
  • Whether a polypeptide has calcium bmding activity or not can be determined by methods well known in the art, preferably, by equihbrium dialysis or by a similar technique.
  • a variant has at least 50 %, at least 60 %, at least 70%, at least 75%, at least 80%, at least 85%, or, more preferably, at least 90%, at least 95%, at least 98% or at least 99% of the calcium binding activity of the polypeptide as shown in SEQ ID NO:2 (with respect to the molar binding activity).
  • a CBL4 polypeptide has calcium binding activity, if it comprises several EF-hand-motives known to be involved in calcium binding. How to assess whether a particular polypeptide has calcium binding activity is well known in the art. A particular preferred method for assessing whether a polypeptide has calcium binding activity is described in the Examples.
  • polynucleotide refers to a linear or circular nucleic acid molecule. It encompasses DNA as well as RNA molecules.
  • the polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context) or in genetically modified form. The term encompasses single as well as double stranded polynucleotides.
  • comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified one such as biotinylated polynucleotides.
  • the polynucleotide of the present invention is characterized in that it shall encode a polypeptide as referred to above.
  • the polynucleotide preferably, has a specific nucleotide sequence as mentioned above.
  • polynucleotides are encompassed which encode a specific amino acid sequence as recited above.
  • the terms "polynucleotide” and “nucleic acid” may be used interchangeably herein.
  • polynucleotide as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides.
  • polypeptide as used in accordance with the present invention further encompasses variants of the aforementioned specific polypeptides. Said variants may represent orthologs, paralogs or other homologs of the polynucleotide/polypeptide that shall be introduced into plants or shall be comprised by plants according to the present invention.
  • polynucleotide variants preferably, comprise a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequences by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall still encode a polypeptide having the activity as specified above.
  • Variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
  • SSC sodium chloride/sodium citrate
  • the skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under “standard hybridization conditions” the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2).
  • the temperature under standard conditions is approximately 42°C.
  • the hybridization conditions for DNA:DNA hybrids are preferably for example 0.1 x SSC and 20°C to 45°C, preferably between 30°C and 45°C.
  • the hybridization conditions for DNARNA hybrids are preferably, for example, 0.1 x SSC and 30°C to 55°C, preferably between 45°C and 55°C.
  • polynucleotide variants are obtainable by PCR- based techniques such as mixed oligonucleotide primer- based amplification of DNA, i.e. using degenerated primers against conserved domains of the polypeptides of the present invention.
  • conserveed domains of the polypeptide of the present invention may be identified by a sequence comparison of the nucleic acid sequence of the polynucleotide or the amino acid sequence of the polypeptide of the present invention with sequences of other members of the protein families referred to in accordance with this invention. Oligonucleotides suitable as PCR primers as well as suitable PCR conditions are described in the accompanying Examples. As a template, DNA or cDNA from plants may be used.
  • variants of polynucleotides include polynucleotides (or polypeptides) comprising nucleic acid sequences (amino acid sequences) which are, preferably, at least 50 %, at least 60 %, at least 70%, at least 75%, at least 80%, at least 85%, or more preferably, at least 90%, or even more preferably, at least 95%, or most preferably, at least 98% ,or at least 99% identical to the specific nucleic acid sequences (amino acid sequences).
  • polynucleotides which comprise nucleic acid sequences encoding amino acid sequence variants which are, preferably, at least 50%, at least 60 %, at least 70%, at least 75%, at least 80%, at least 85%, or, more preferably, at least 90%, or even more preferably, at least 95%, or most preferably, at least 98% or at least 99% identical to the specific amino acid sequences referred to herein.
  • the term variant also encompasses polypeptides which comprise amino acid sequences which are, preferably, at least 50% at least, 60 %, at least 70%, at least 75%, at least 80%, at least 85%, or more preferably, at least 90%, or even more preferably, at least 95%, or most preferably, at least 98% or at least 99% identical to the specific amino acid sequences referred to herein.
  • the percent identity values are, preferably, calculated over the entire amino acid or nucleic acid sequence region.
  • sequence identity values recited above in percent (%) are to be determined, preferably, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence ahgnments.
  • Plants or plant cells generated by the method of present invention are, preferably, transgenic plants or plant cells.
  • the introduction of a polypeptide is, preferably, achieved by introducing heterologous polynucleotides encoding the aforementioned polypeptides as discussed elsewhere in this specification in more detail. This includes transient introduction in expression vectors or stable integration into the genome of the plant cells via, e.g., T- or P-DNA insertion. It is to be understood that one heterologous polynucleotide comprising nucleic acids encoding the all of the aforementioned polypeptides may be introduced.
  • polypeptide(s) as referred to herein in the context of the present invention are expressed from heterologous polynucleotides. Accordingly, the method according to the present invention, preferably, comprises the steps of:
  • heterologous means that the polynucleotides do not occur naturally in the plant cell or are located at chromosomal position which differs from its natural context.
  • the term thus, encompasses modified or unmodified polynucleotides which are derived from different organisms or modified polynucleotides derived from the plant cell of the invention.
  • the heterologous polynucleotide shall either comprise expression control sequences which allow for expression in the plant cell or sequences which allow for integration of the heterologous polynucleotide at a locus in the genome of the plant cell where the expression of the heterologous polynucleotide will be governed by endogenous expression control sequences of the plant cell.
  • the heterologous polynucleotide comprises a nucleic acid having a nucleic acid sequence of the first, or the second polynucleotide as referred to herein elsewhere.
  • transgenic plant cells are generated. Such transgenic plant cells may be obtained by transformation techniques as published, and cited, in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), chapter 6/7, pp.71- 119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38; B.
  • transgenic plants can be obtained by T-DNA- mediated, P-DNA-mediated or biolistic transformation.
  • Such vector systems are, as a rule, characterized in that they contain at least the vir genes, which are required for the Agrobacterium- mediated transformation, and the sequences which delimit the T- or P-DNA (T-DNA border or P- DNA border).
  • the polynucleotide expressing a polypeptide as set forth herein is, preferably, stably integrated into the genome of cells comprised by said plant or plant seed.
  • How to stably integrate a polynucleotide or a vector (particularly a T-DNA vector) into the genome of a plant cell is well known in the art and described elsewhere herein.
  • the polynucleotide or vector shall be stably integrated into the genome by Agrobacterium- mediated or particle bombardment mediated transformation.
  • a polypeptide as set forth, herein is transiently expressed.
  • the polynucleotide encoding a polypeptide as set forth herein is operably linked to a promoter and a terminator allowing expression of said polynucleotide.
  • a promoter and a terminator are well known in the art.
  • Preferred promoters in the context of the present invention are constitutive promoters, in particular the CaMV 35 S promoter, the ubiquitin-10 promoter (in particular the Arabidopsis thaliana ubiquitin-10 promoter (for the sequence see genbank accession: HQ693235.1. see also Grefen et al., 2010, The Plant Journal, 2010, 64, 355-365) as well as the mannopine synthase promoter.
  • the plant or the plant cell generated by the method of the present invention shall have increased potassium efficiency as compared to a corresponding plant and corresponding plant cell, respectively. Moreover, the plant or the plant cell generated by the method of the present invention shall have increased salt tolerance as compared to a corresponding plant and corresponding plant cell, respectively.
  • a corresponding plant or plant cell preferably, is plant or a plant cell (of the same species/tissue) into which the polyp eptide(s) as referred to herein has (have) not been introduced. Accordingly, a corresponding plant, preferably, lacks a plant cell generated by the method of the present invention. A plant lacking a plant cell of the present invention as meant herein, thus, preferably, refers to an unmodified control plant of the same variety as the plant of the present invention.
  • “Increased potassium efficiency" as used herein preferably, means that the plant or plant cell of the present invention or the plant or plant cell generated by the method of the present invention requires a lower concentration of potassium in the medium in order to grow as compared a corresponding plant or plant cell not comprising the polynucleotide(s)/polypeptide(s) as set forth herein.
  • the plant or plant cell has the ability to use sufficient potassium more efficiently as a corresponding plant or plant cell. It is particularly envisaged that a plant which has increased potassium efficiency as compared to a corresponding plant shows better growth at lower concentration of potassium, in particular under potassium limiting conditions, than the corresponding plant.
  • a plant is, preferably, generated that has increased yield under potassium limiting conditions.
  • said plant has increased yield under potassium limiting conditions as compared to a corresponding non-transgenic plant (and, thus, as compared to plant into which the polypeptide(s) as referred to herein have not been introduced).
  • the corresponding non- transgenic plant is, preferably, a wild-type plant.
  • An increase of potassium efficiency can be determined by methods well known in the art.
  • a plant or plant cell generated by the method of the present invention as well as a corresponding plant or plant cell (control) can be grown on media comprising various concentrations of potassium (but otherwise comprise the same components).
  • Potassium efficiency preferably, is increased, if the plant or plant cell generated by the method of the present invention is capable of growing at lower concentration of potassium than the corresponding controls.
  • the plant or plant cell generated by the method of the present invention also has higher yield on media/on soil with (growth) limiting concentration of potassium. Accordingly, the method of the present invention also allows for the generation of a plant and a plant which have increasing yield on media/on soil with limiting concentrations of potassium.
  • the present invention relates to a method for producing a transgenic plant or plant cell having increased yield under potassium limiting conditions compared to a corresponding non- transgenic plant or plant cell, said method comprises introducing of the transgenic plant or plant cell a CBL4 polypeptide.
  • yield encompasses an increase in biomass (fresh or dry weight) of a plant part or the entire plant, and particularly, harvestable parts of the plant.
  • the increase in biomass may be aboveground or underground.
  • An increase in biomass underground may be due to an increase in the biomass of plant parts, such as tubers, rhizomes, bulbs etc.
  • Particularly preferred is an increase in any one or more of the following: increased root biomass, increased root volume, increased root number, increased root diameter and increased root length.
  • increased yield also encompasses an increase in seed yield.
  • An increase in seed yield includes: (i) increased total seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis; (ii) increased number of flowers ("florets") per panicle; (iii) increased number of filled seeds; (iv) increased seed size; (v) increased seed volume; (vi) increased individual seed area; (vii) increased individual seed length and/or width; (viii) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass; (ix) increased fill rate, (which is the number of filled seeds divided by the total number of seeds and multiplied by 100); and (x) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight.
  • An increased TKW may result from an increased seed size and/or seed weight.
  • An increased TKW may result from an increase in embryo size and/or endosperm size.
  • the increase in yield is statistically significant. More preferably, said increase is an increase of at least 10%, at least 20%, at least 30%, at least 40% or at least 50% in yield.
  • the plant and plant cells generated by the method of the present invention further have increased salt tolerance as compared to a corresponding plant or plant cell (into which the polypeptide(s) as referred to herein has (have) not been introduced).
  • the plant generated by the method of present invention is capable to survive and/or to grow in the presence of increased salt concentrations in the soil or in any other growth medium that inhibits growth of a corresponding plant.
  • the present invention relates to a method for producing a transgenic plant or plant cell having increased salt tolerance and/or having increased potassium efficiency as compared to a corresponding plant or plant cell, said method comprises introducing of the transgenic plant or plant cell a CBL4 polypeptide.
  • a corresponding plant or plant cell preferably, is a plant cell into which the CBL4 polypeptide (and, optionally, any further polypeptide, in particular the CIPK6 and/or the AKT2 polypeptide has not been introduced).
  • An increase of salt tolerance can be determined by techniques well known in the art (Zhang and Blumwald, Nature Biotech, 2001, 19: 765-768; Why et al. Plant J, 2002, 32: 139-149 D'Angelo et al, 2006) and described in, preferably, in the accompanying Examples below.
  • the increase is statistically significant. Whether an increase is statistically significant can be determined by well known statistical tests including, e.g., Student's t-test, Mann-Whitney test etc.
  • the salt tolerance is increased if the plant of the present invention is growing at salt concentrations and/or survives salt concentrations in a growth medium, particularly in soil, that are at least 5%, at least 10%, at least 15%, at least 20%, or at least 30% higher than the highest salt concentration at which a plant lacking a plant cell of the present invention is growing and/or which a plant lacking a plant cell of the present invention survives.
  • the salt tolerance is increased if the plant of the present invention is growing at salt concentrations and/or survives salt concentrations in a growth medium, particularly in soil, with a NaCl concentration of above 50 mM, at which the growing or survival of a plant lacking a plant cell of the present invention is compromised (however, the NaCl concentration may depend on the plant used in the context of the method of the present invention. This can be determined by the skilled person by routine experiments).
  • the term "salt" in the context of the present invention preferably, encompasses Na H - Salts (most preferably NaCl).
  • the method of the present invention further comprises introducing into said plant or said plant cell a CIPK6 (CBL-interacting protein kinase 6) polypeptide.
  • CIPKs are serine-threonine protein kinases known to interact with CBL proteins.
  • the general structure of CIPKs is shown in Figure 12. It is thought that binding of a CBL protein to the regulatory NAF domain of CIPK protein leads to the activation of the kinase in a calcium- dependent manner.
  • the kinase domain in CIPKs is separated by a junction domain from the less-conserved C-terminal regulatory domain (Batistic and Kudla, 2009).
  • CIPKs Within the regulatory region of CIPKs a conserved NAF domain (designated according to the prominent amino acids N, A and F) mediates binding of CBL proteins and simultaneously functions as an auto-inhibitory domain. Binding of CBLs to the NAF motif removes the autoinhibitory domain from the kinase domain, thereby conferring autophosphorylation and activation of the kinase. Additional phosphorylation of the activation loop within the kinase domain by a yet unknown kinase further contributes to the activation of CIPKs (Batistic and Kudla, 2009; Kudla et al., 2010, loc. cit). Moreover, CIPK-mediated phosphorylation of a conserved residue in CBL proteins, including CBL4, is required for full activation of CBL/CIPK complexes towards their target proteins.
  • the Arabidopsis thaliana CIPK6 polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 belongs to a family of a total of 26 CBL-interacting protein kinase members in Arabidopsis thaliana.
  • the Arabidopsis calcium sensor CBL4 together with the protein Arabidopsis protein kinase CIPK6 Ca 2+ -dependently modulate the activity of the shaker-type K ⁇ channel AKT2 from Arabidopsis thaliana (see Examples).
  • co-expression of CBL4 translocates CIPK6- AKT2 to the plasma membrane in plant cells and is essential for enhanced AKT2 activity in oocytes (see Examples).
  • the interaction of the aforementioned polypeptide is phosphorylation-independent since it was shown that AKT2 activity is not regulated by CIPK6- mediated phosphorylation. Instead, the isolated regulatory C-terminal domain of CIPK6 not having serine-threonin kinase activity interacts with AKT2 and CBL4 in vivo and mediates CBL4- dependent and Ca2+-dependent channel translocation from the ER to the plasma membrane in plant cells and channel activation in Xenopus oocytes.
  • CBL4 is involved in the uptake and/or distribution of potassium.
  • This finding suggests that the expression of a CBL4 polypeptide in a plant increases potassium uptake and/or distribution and, thus, potassium efficiency.
  • potassium efficiency can be further increased, if also a CIPK6 polypeptide is introduced and expressed in a plant or a plant cell. Since it has been surprisingly shown that the interaction of CIPK.6, CBL4 and AKT2 does not depend on the kinase activity of CIPK6, a CIPK6 polypeptide can be used that does not comprise kinase activity. Thereby, undesired side effects can be avoided that are cause by the overexpression of polypeptides having kinase activity. Moreover, the further expression of AKT2 contributes to an increased potassium efficiency, and further over-expression of SOS1 and/or CIPK24 further contributes to a simultaneous increased salt tolerance (see below).
  • the C1PK6 polypeptide in the context of the present invention is encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ ID No: 4; c) a nucleic acid having a nucleotide sequence being a variant of the nucleotide sequence shown in SEQ ID No: 3 wherein said nucleic acid encodes a polypeptide capable of interacting with a CBL polypeptide (preferably CBL4); and
  • CIPK6 polypeptide in the context of the present invention is encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • a CIPK6 polypeptide/variant as referred to above preferably, is capable of interacting with a CBL polypeptide (preferably CBL4) if the CBL polypeptide binds the NAF-Domain of the CIPK6 polypeptide.
  • CW K6 polypeptide/variant as referred to above preferably, is capable of interacting with a CBL polypeptide if it is translocated by said CBL polypeptide to the plasma membrane. Preferred methods for determining whether a polypeptide/variant is translocated by a CBL polypeptide from intracellular structures to the plasma membrane are described in the Examples.
  • Preferred CIPK6 polypeptides are variants of the Arabidopsis thaliana CIPK6 polypeptide. Preferred variants are shown in Table B in the Examples section.
  • the CIPK6 polypeptide does not have serine-tbreonin-kinase activity.
  • a CIPK6 polypeptide not having serine- threonine-kiriase activity preferably, does not phosphorylate the AKT2 polypeptide.
  • How to generate a CIPK6 polypeptide not having serine-threonin-kinase activity is well known in the art.
  • it is well known in the art how to determine whether a polypeptide has kinase activity or not (e.g. by phosphorylation assays).
  • CIPK6 polypeptide which confers serin-threonin kinase activity.
  • preferred CIPK6 polypeptides not having serin-threonin kinase activity are described in the Examples.
  • the serine-threonine kinase domain is located at amino acids coordinates 1 to 277 and is located to the corresponding conserved amino acid positions in the CIPKs provided in the Examples (see figure 11 A)
  • a CIPK6 polypeptide without serine- threonme-kinase activity can be generated by truncating said domain or a part thereof.
  • amino acid coordinates 1 to 273 are truncated as shown in Fig. 1 IB (SEQ ID NO: 11 and 12 show the sequences of the corresponding polynucleotide/polypeptide).
  • a particularly preferred CIPK6 polypeptide has a sequence as shown in SEQ ID NO: 12. It is, preferably, encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 11. It is also contemplated to generate a CIPK6 polypeptide, said polypeptide not having serine threonine kinase activity by insertions, deletions and/or mutations within the seririe-threonine kinase domain. However, it is contemplated that said CIPK6 polypeptide shall be still capable of interacting with CBL polypeptides, particularly with CBL4.
  • a CIPK6 polypeptide without serine- threonine-kinase activity can be generated by introducing a point mutation at position 53 (preferably, a K to N mutation, as shown in SEQ ID NO: 14) and/or at position 164 (preferably, a D to N mutation, as shown in SEQ ID NO: 16). Said polypeptides are capable of interacting with CBL4.
  • a further particularly preferred CIPK6 polypeptide has a sequence as shown in SEQ ID NO: 14. It is, preferably, encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 13.
  • a further preferred CEPK6 polypeptide has a sequence as shown in SEQ ID NO: 16. It is, preferably, encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 15.
  • the C1PK6 polypeptide in the context of the present invention may also be encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 11, 13 or
  • nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ ID No: 12, 14 or 16; c) a nucleic acid having a nucleotide sequence being a variant of the nucleotide sequence shown in SEQ ID No: 11, 13 or 15, wherein said nucleic acid encodes a polypeptide capable of interacting with a CBL polypeptide (preferably CBL4), and, wherein, said polypeptide encoded by said nucleic acid, preferably, does not have serine- threonine-kinase activity; and
  • a CBL polypeptide preferably CBL4
  • CIPK6 polypeptide in the context of the present invention may also be encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 11, 13 or
  • nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ K) No: 12, 14 or 16;
  • nucleic acid having a nucleotide sequence being at least 50% identical to the nucleotide sequence shown in SEQ ID No: 11, 13 or 15, wherein said nucleic acid encodes a polypeptide capable of interacting with a CBL polypeptide (preferably CBL4) and
  • a nucleic acid encoding a polypeptide having a sequence being at least 50% identical to the amino acid sequence shown in SEQ ID No: 12, 14 or 16, wherein said polypeptide is capable of interacting with a CBL polypeptide (preferably CBL4).
  • said polypeptide mentioned in c) and d) does not have serine- threonine-kinase activity
  • CIPK6 polypeptide not having serine-threonine kinase activity into plants is advantageous, since it may avoid detrimental effects, which may result from overexpressing a protein having said kinase activity.
  • the method according to the present invention further comprises introducing into said transgenic plant or said plant cell an AKT2 (Arabidopsis K + transporter 2) polypeptide.
  • plants are generated comprising the CBL4, the CIPK6 and the AKT2 polypeptide. Further introducing the AKT2 polypeptide further improves the agronomic traits as referred to herein.
  • the AKT2 polypeptide in the context of the present invention is encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 5 a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 5; b) a nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ ID No: 6;
  • nucleic acid having a nucleotide sequence being a variant of the nucleotide sequence shown in SEQ ID No: 5 wherein said variant encodes a polypeptide being capable transporting ions, preferably K + ions, across cellular membranes, preferably, plasma membranes; and
  • nucleic acid encoding a polypeptide having an amino acid sequence being a. variant of the amino acid sequence shown in SEQ ED No:6, wherein said variant encodes a polypeptide being capable of transporting ions preferably K + ions across cellular membranes preferably, plasma membranes.
  • the AKT2 polypeptide in the context of the present invention is encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 5 a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 5; b) a nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ ID No: 6;
  • nucleic acid having a nucleotide sequence having at least 50% identity to the nucleotide sequence shown in SEQ ID No: 5 wherein said polypeptide is capable transporting ions, preferably K + ions, across cellular membranes, preferably, plasma membranes; and
  • AKT2 polypeptides are variants of the Arabidopsis thaliana AKT2 polypeptide (as shown in SEQ ID NO: 6). Preferred variants of the Arabidopsis thaliana AKT2 polypeptide are shown in Table C in the Examples section.
  • the method further comprising introducing into said transgenic plant or said plant cell a CIPK24 (CBL-interacting protein kinase 24) polypeptide and/or a SOS1 (Salt Overly Sensitive 1) polypeptide.
  • CIPK24 CBL-interacting protein kinase 24
  • SOS1 Salt Overly Sensitive 1
  • plants are generated comprising a) the CBL4, CIPK6, CEPK24 and SOSl polypeptide, or b) the CBL4, CIPK6, AKT2, CTPK24 and SOSl polypeptide.
  • the Arabidopsis thaliana SOSl protein resides at the plasma membrane, where it functions to extrude Na + _ from the cytoplasm coupled to H + influx.
  • the SOSl protein has 12 predicted transmembrane domains in the N-terminal region and a long cytoplasmic tail of 700 aa at the C-tenninal side.
  • the CIPK24 polypeptide in the context of the present invention is encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 7 a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 7; b) a nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ ID No: 8;
  • nucleic acid having a nucleotide sequence being a variant of the nucleotide sequence shown in SEQ ID No: 7 wherein said nucleic acid encodes a polypeptide is capable of interacting with CBL polypeptides preferably, with CBL1, 2, 4, 5, 8, 9, or 10 (preferably from Arabidopsis thaliana); and
  • nucleic acid encoding a polypeptide having an amino acid sequence being a variant of the amino acid sequence shown in SEQ ID No: 8, wherein said variant is capable of interacting with CBL polypeptides.
  • the SOSl polypeptide is encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 9 a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 9; b) a nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ ID No: 10;
  • nucleic acid having a nucleotide sequence being a variant of the nucleotide sequence shown in SEQ ID No: 9 wherein said nucleic acid encodes a polypeptide being capable transporting ions, preferably Na + , across cellular membranes, preferably, plasma membranes; and
  • a nucleic acid encoding a polypeptide (variant polypeptide) having an amino acid sequence being a variant of the amino acid sequence shown in SEQ ID No: 10, wherein said variant being capable transporting ions preferably Na + , across cellular membranes preferably, plasma membranes.
  • a polypeptide or a variant thereof is capable transporting ions preferably Na + , across cellular membranes preferably, plasma membranes.
  • the present invention also relates to a plant or a plant cell generated by the method according to the present invention.
  • a said plant comprises a plant cell generated by the method of the present invention.
  • the present invention relates to plant or plant cell, comprising a CBL4 polypeptide (preferably, is transformed with said polypeptide).
  • a CBL4 polypeptide preferably, is transformed with said polypeptide.
  • said CBL4 polypeptide is expressed from a heterologous polynucleotide.
  • said plant or plant cell has increased salt tolerance and/or potassium efficiency as to compared to a plant or plant cell lacking said CBL4 polypeptide.
  • said plant or plant cell further comprises a CIPK6 polypeptide (preferably is transformed with said polypeptide).
  • said C1PK6 polypeptide is expressed from a heterologous polynucleotide.
  • said plant or plant cell has increased salt tolerance and/or potassium efficiency as to compared to a plant or plant cell lacking said CBL4 and said CIPK6 polypeptide.
  • the present invention also relates to a plant or plant cell comprising a CBL4 and CIPK6 polypeptide.
  • the aforementioned plant or plant cell according to the present invention preferably, further comprises (preferably, further is transformed with) at least one polypeptide (and thus, one, two or three polypeptides) selected from the group consisting of an AKT2 polypeptide, a CIPK24 polypeptide and a SOSl polypeptide.
  • the AKT2 polypeptide, the CIPK24 polypeptide and the SOSl polypeptide are expressed from a heterologous polynucleotide (heterologous polynucleotides).
  • Preferred plants are plants comprising a) CBL4, CIPK6, and AKT1 polypeptide, b) the CBL4, CIPK6, CIPK24 and SOSl polypeptide, or c) the CBL4, C1PK6, AKT2, CIPK24 and SOSl polypeptide.
  • said plant or plant cell has increased salt tolerance and/or potassium efficiency as to compared to a plant or plant cell lacking said CBL4 and CIPK6 polypeptide and said at least one polypeptide selected from the group consisting of an AKT2 polypeptide, a CIPK24 polypeptide and a SOSl polypeptide.
  • the present invention relates to a composition comprising a CBL4 and CIPK6 polypeptide.
  • compositions further comprises at least one polypeptide (and thus, one, two or three polypeptides) selected from the group consisting of an AKT2 polypeptide, a CIPK24 polypeptide and a SOS 1 polypeptide.
  • Preferred compositions comprise a) CBL4, CIPK6, and AKT1 polypeptide, b) the CBL4, CEPK6, CBPK24 and SOS1 polypeptide, or c) the CBL4, CIPK6, AKT2, CIPK24 and SOS1 polypeptide.
  • the present invention also envisages a polynucleotide comprising in a combination a nucleic acid encoding for a CBL4 polypeptide and a nucleic acid encoding for a CEPK6 polypeptide.
  • said polynucleotide further comprises at least one nucleic acid (and thus, one, two or three nucleic acids) encoding for a polypeptide selected from the group consisting of an AKT2 polypeptide, a CIPK24 polypeptide and a SO SI polypeptide.
  • Preferred polynucleotides comprise a) a nucleic acid encoding for a CBL4, CIPK6, and AKT1 polypeptide, b) a nucleic acid encoding for the CBL4, CIPK6, CIPK24 and SOS1 polypeptide, or c) a nucleic acid encoding for a CBL4, CIPK6, AKT2, CTPK24 and SOS1 polypeptide.
  • the present invention also contemplates a vector comprising one of the aforementioned polynucleotides of the present invention.
  • vector preferably, encompasses phage, plasmid, viral or retroviral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below.
  • the vector encompassing the polynucleotides of the present invention preferably, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art.
  • the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences, which allow for homologous recombination or heterologous insertion. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection”, conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment (e.g., "gene-gun”).
  • Suitable methods for the transformation or transfection of host cells, including plant cells, can be found in Sambrook et al.
  • plasmid vector may be introduced by heat shock or electroporation techniques. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the vector referred to herein is suitable as a cloning vector, i.e. replicable in microbial systems.
  • a cloning vector i.e. replicable in microbial systems.
  • Such vectors ensure efficient cloning in bacteria and, preferably, yeasts or fungi and make possible the stable transformation of plants.
  • Those which must be mentioned are, in particular, various binary and co-integrated vector systems which are suitable for the T-DNA-mediated or P- DNA mediated transformation.
  • Such vector systems are, as a rule, characterized in that they contain at least the vir genes, which are required for the Agrobacterium-mediated transformation, and the sequences which delimit the T-DNA or P-DNA (T-DNA or P-DNA borders).
  • vector systems preferably, also comprise further cis-regulatory regions such as promoters and terminators and/or selection markers with which suitable transformed host cells or organisms can be identified.
  • co-integrated vector systems have vir genes and T-DNA sequences arranged on the same vector
  • binary systems are based on at least two vectors, one of which bears vir genes, but no T- DNA, while a second one bears T-DNA, but no vir gene.
  • the last-mentioned vectors are relatively small, easy to manipulate and can be replicated both in E. coli and in Agrobacterium.
  • binary vectors include vectors from the pBIB-HYG, pCAMBIA, pPZP, pBecks, pGreen series.
  • the polynucleotide of the invention can be introduced into host cells or organisms such as plants or animals and, thus, be used in the transformation of plants, such as those which are published, and cited, in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), chapter 6/7, pp. 71-119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38; B.
  • the vector of the present invention is an expression vector.
  • the polynucleotide comprises an expression cassette as specified above allowing for expression in eukaryotic cells or isolated fractions thereof.
  • An expression vector may, in addition to the polynucleotide of the invention, also comprise further regulatory elements including transcriptional as well as translational enhancers.
  • the expression vector is also a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population.
  • Suitable expression vector backbones are, preferably, derived from expression vectors known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDMS, pRc/CMV, pcDNAl, pcDNA3 (Invitrogene) or pSPORTl (GIBCO BRL), or pGBTV and pGBTVII plasmids.
  • expression vectors known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDMS, pRc/CMV, pcDNAl, pcDNA3 (Invitrogene) or pSPORTl (GIBCO BRL), or pGBTV and pGBTVII plasmids.
  • Expression vectors allowing expression in plant cells comprise those which are described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant MoL Biol. 20:1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12:8711- 8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, p. 15-38.
  • a plant expression cassette preferably, comprises regulatory sequences which are capable of controlling the gene expression in plant cells and which are functionally linked so that each sequence can fulfill its function, such as transcriptional termination, for example polyadenylation signals.
  • Preferred polyadenylation signals are those which are derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH5, which is known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835 et seq.) or functional equivalents of these, but all other terminators which are functionally active in plants are also suitable.
  • a plant expression cassette preferably comprises other functionally linked sequences such as translation enhancers, for example the overdrive sequence, which comprises the 5 '-untranslated tobacco mosaic virus leader sequence, which increases the protein/RNA ratio (Gallie et al, 1987, Nucl. Acids Research 15:8693-8711).
  • translation enhancers for example the overdrive sequence, which comprises the 5 '-untranslated tobacco mosaic virus leader sequence, which increases the protein/RNA ratio
  • Other preferred sequences for the use in functional linkage in plant gene expression cassettes are targeting sequences which are required for targeting the gene product into its relevant cell compartment.
  • the CIPK6 polypeptide does not have serine-threonine kinase activity.
  • the present invention relates to the use of a CBL4 polypeptide for generating a transgenic plant or plant cell having increased potassium efficiency compared to a corresponding non-transgenic plant or plant cell.
  • the present invention also relates to the use of a CBL4 polypeptide for generating a transgenic plant or plant cell having increased yield under potassium limiting conditions compared to a corresponding non-transgenic plant or plant cell.
  • the present invention relates to a CIPK6 polypeptide encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having a nucleotide sequence as shown in SEQ ID No: 11, 13 or
  • nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ ID No: 12, 14 or 16;
  • nucleic acid having a nucleotide sequence being a variant of the nucleotide sequence shown in SEQ ID No: 11, 13 or 15, wherein said nucleic acid encodes a polypeptide capable of interacting with a CBL polypeptide (preferably CBL4), and, wherein, said polypeptide encoded by said nucleic acid, preferably, does not have serine- threonine-kinase activity; and
  • the present invention also relates to the use of a CBL4 polypeptide for generating a transgenic plant or plant cell having increased salt tolerance and/or increased potassium efficiency compared to a corresponding non-transgenic plant or plant cell.
  • the present invention relates to the use of a CBL4 polypeptide and a CIPK6 polypeptide for generating a transgenic plant or plant cell having increased potassium efficiency compared to a corresponding non-transgenic plant or plant cell.
  • the present invention also relates to the use of a CBL4 polypeptide and a CIPK6 polypeptide for generating a transgenic plant or plant cell having increased yield under potassium limiting conditions compared to a corresponding non-transgenic plant or plant cell.
  • a CBL4 polypeptide and a CIPK6 polypeptide for generating a transgenic plant or plant cell having increased salt tolerance and/or increased potassium efficiency compared to a corresponding non-transgenic plant or plant cell.
  • the present invention relates to the use of a CBL4 polypeptide and a CIPK6 polypeptide and at least one further polypeptide selected from the group consisting of an AKT2 polypeptide, CTPK24 polypeptide and a SOS1 polypeptide for generating a transgenic plant or plant cell having increased potassium efficiency compared to a corresponding non-transgenic plant or plant cell.
  • a) CBL4, CIPK6, and AKTl polypeptide b) the CBL4, CIPK6, CIPK24 and SOS1 polypeptide, or c) the CBL4, CIPK6, AKT2, CIPK24 and SOS1 polypeptide.
  • the present invention also relates to the use of a CBL4 polypeptide and a CIPK6 polypeptide and at least one further polypeptide selected from the group consisting of an AKT2 polypeptide, CIPK24 polypeptide and a SOS1 polypeptide for generating a transgenic plant or plant cell having increased yield under potassium limiting conditions compared to a corresponding non-transgenic plant or plant cell.
  • a) CBL4, CIPK6, and AKTl polypeptide b) the CBL4, CTPK6, C1PK24 and SOS1 polypeptide, or c) the CBL4, CIPK6, AKT2, CIPK24 and SOS1 polypeptide.
  • the present invention relates to the use of a CBL4 polypeptide and a CIPK6 polypeptide and at least one further polypeptide selected from the group consisting of an AKT2 polypeptide, CEPK24 polypeptide and a SOS1 polypeptide for generating a transgenic plant or plant cell having increased salt tolerance compared to a corresponding non-transgenic plant or plant cell.
  • a) CBL4, CIPK6, and AKTl polypeptide b) the CBL4, CIPK6, ⁇ 24 and SOS1 polypeptide, or c) the CBL4, CIPK6, AKT2, CIPK24 and SOS1 polypeptide.
  • FIG. 1 CIPK6 specifically interacts with the AKT2 C-Terminus and CBL4-CIPK6 complexes Ca 2 ⁇ -dependently modulate AKT2 activity.
  • A Yeast two-hybrid analysis of CIPK-AKT2 interaction. The yeast strain PJ69-4A containing the indicated plasmid combinations was grown on the indicated media for 14 days at 23°C. The published interaction of AD-AKTl with BD-CIPK23 served as positive control. Decreasing cell densities in the yeast dilution series are illustrated by narrowing triangles.
  • B Yeast two-hybrid analysis of CIPK6-CBL interaction.
  • the yeast strain PJ69-4A containing the indicated plasmid combinations was grown on the indicated media for 7 days at 23°C.
  • the published interaction of AD-CIPK1 with BD-CBL1 served as positive control.
  • C CIPK6 interacts with AKT2 in planta. Microscopic analysis of BiFC complexes formed by the indicated plasmid combinations after 4 days of infiltration in N. benthamiana leaves.
  • D Both CIPK6 and CBL4 are required for a Ca2 + -dependent increase of AKT2 current. Increase of AKT2 current in X. laevis oocytes by different CIPK-CBL combinations.
  • F Ca 2+ - dependence of AKT2 current modulation by CIPK6-CBL4.
  • left Currents in oocytes injected with either AKT2 cRNA or a mix of AKT2, CIPK6 and CBL4 cRNAs were recorded before and 5 min after a 50mM BAPTA injection.
  • Plants were grown in a 12 h day / 12 h night cycle.
  • A Plant development 6 week after sowing (6 WAS).
  • B Plants 8 WAS (WT-Ws and akt2-l) and 9 WAS (WT-Col-0, cbl4 and cipk6).
  • Data in (C) and (D) are depicted as mean ⁇ SD. (*) marks results with significant difference in values.
  • FIG. 3 CBL4-dependent ER-to-PM translocation of AKT2.
  • A-D Microscopic analysis of the median cellular plane of N. benthamiana epidermal cells transiently expressing the plasmid combinations indicated at the left.
  • A Formation and ER-localization of AKT2-CIPK6 (green) complexes as revealed by BiFC and co-localization with the ER-marker OFP-HDEL (red).
  • B The localization of AKT2-CTPK6 complexes (green) is distinct from PM-marker CBLln-OFP (red). Arrows mark the region and direction in which the distribution of fluorescence intensities was determined.
  • C-E Localization of AKT2-CIPK6 BiFC complexes after co-expression of CBL4- OFP or CBL4-SCFP.
  • C Co-expression of CBL4-OFP shifts the localization of AKT2-CIPK6 BiFC complexes at the plasma membrane.
  • D Co-localization of AKT2-CIPK6 complexes after co-expression with CBL4-SCFP and the PM-marker CBLln-OFP confirms PM-localization of AKT2-CIPK6 and a dramatic reduction of the AKT2-CrPK6-indicating fluorescence signal in the perinuclear envelope as detected by a fluorescence scan.
  • a white arrow marks the region and direction in which the distribution of fluorescence intensities was determined.
  • FIG. 4 In vitro phosphorylation assays detect no phosphorylation of AKT2 by CIPK6.
  • A-E Upper panels depict CBB stainings (CBB) of recombinant proteins and lower panels present the corresponding autoradiographs (ARG) after phosphorylation assays.
  • CBB CBB stainings
  • ARG autoradiographs
  • A The suitability of the experimental conditions was verified by CBL4-CIPK24 mediated phosphorylation of the SOS1 C- terminus (SOSl-Ct).
  • a hyperactive mutant, CIPK24T168D (indicated as TD), exhibited enhanced kinase activity.
  • B CIPK6 displayed auto-phosphorylation activity, which was influenced by CBL4.
  • C-D Phosphorylation of the C ⁇ terminal fragment or full-length protein of AKT2 by CBL4-CIPK6 was not detectable.
  • E C1PK6 efficiently trans-phosphorylated CBL4.
  • Figure 5 Phosphorylation-independent Ca2+-dependent modulation of AKT2 by CBL4-CIPK6
  • A Schematic presentation of CIPK6 and the CIPK6N and CIPK6C constructs generated in this study.
  • NAF NAF domain mediating CBL interaction
  • PPI Phosphatase interaction domain
  • B CBL4 in combination with CIPK6C phosphorylation-independently activates AKT2.
  • Dual N-terminal lipid modification of CBL4 is necessary for the activation of AKT2 currents by CIPK6 and translocation of AKT2 from the ER to the PM.
  • A Activation of AKT2 currents by CIPK6 and CBL4 is impaired if myristoylation of CBL4 is prevented by the CBL4G2A point mutation. Currents recorded in oocytes injected with different mixes of cRNAs were normalized to the mean current value at -155 mV in oocytes injected with AKT2 cRNA only.
  • C Co-expression of CBL4G2A-SCFP and CBL4C3S-SCFP lead to a retention of the AKT2-CIPK6 BiFC signal (green) at the ER, which is clearly distinct from the plasma membrane marker CBLln-OFP (red).
  • FIG. 7 (A) Co-localization of AKT2-CIPK6 BiFC complexes with the ER-marker OFP-HDEL. Microscopic analysis of the median cellular plane of N. benthamiana epidermal cells transiently expressing the plasmid combinations indicated at the left. A white arrow marks the region and direction in which the distribution of fluorescence intensities was determined. (B-E) Increase of AKT2 currents upon co-expression with CIPK6 and CBL4. (B) Typical voltage clamp protocol.
  • Left panel Typical current recordings mX. laevis oocytes expressing either AKTl, or AKTl +CIPK6+CBL4 or A KT1 + CIPK23 + CBL 1.
  • Right panel Current-voltage (I-V) curves for oocytes injected with AKTl cRNA (white circles) or with a mix of AKTl, CIPK6 and CBL4 cRNAs (black squares), or with a mix of AKTl, CIPK23 and CBL1 cRNAs (black circles).
  • FIG. 8 AKT2 channel voltage gating is not changed by CIPK6+CBL4.
  • B Voltage- gating of the time-dependent fraction of the AKT2 current is not changed by CIPK6+CBL4.
  • Conductance values were obtained from a cell attached patches of oocytes clamped at -140 mV and expressing AKT2 alone or AKT2 with CIPK6 and CBL4. Data are means ⁇ SE. Oocytes were maintained in an external solution of lOOmM K+ (in the bath and the pipette).
  • FIG. 9 The shared developmental phenotype of akt2-l, cbl4 and cipk6 mutant plants in short day conditions can be complemented.
  • A Isolation and validation of the cbl4 mutant.
  • B Isolation and validation of the cipk6 mutant. Schematic illustration of the T-DNA insertion position (denoted as a triangle flanked on each side by five nucleotides of the surrounding genomic sequence) within the exonic sequence (illustrated by boxes). Intronic sequences are denoted by black lines. Arrows indicate the position of the genomic primers used for PCR and RT-PCR experiments.
  • Genomic PCRs confirming the T-DNA insertions are presented at the left while the RT-PCR analyses on cDNA prepared from wild-type (WT) and mutants (cbU and cipk6) are depicted on the right.
  • C-D Complementation of the developmental phenotypes of akt2-l, cbl4 and cipk6 mutant plants. Leave number and size in the complemented lines is restored, similar to the respective wild type plant.
  • C Phenotypical appearance of plants 6 weeks after sowing (6 WAS) and cultivation in a 12 h day / 12 h night cycle.
  • FIG 10 (A) Co-localization of AKT2-CIPK6 complexes with the PM-marker CBLln-OFP in presence of CBL4-SCFP. Presented is a detail of Figure 3 D. The respective combinations of expressed plasmids are indicated in the left. CBL4 is localized not only in the PM, but also detected in the nucleus and in cytoplasmic strands (blue). The white arrow marks the line where the fluorescence scan was performed. This corresponds to the fluorescence scan depicted in Figure 3 D. (B) Distinct localization of AKT2-CIPK6C complexes and the PM-marker CBLln-OFP in presence of CBL4AEF-SCFP. Presented is a detail of Figure 5 E.
  • CBL4AEF-SCFP is localized not only in the PM, but also detected in the nucleus and in cytoplasmic strands (blue).
  • the white arrow marks the line where the fluorescence scan was performed. This corresponds to the fluorescence scan depicted in Figure 5 E.
  • Figure 11 Schematic model of CIPK6 wildtype protein, which consists of 442 aminoacids. The first 278 aminoacids form the kinase domain. Numbers indicate aminoacid positions. The NAF domain mediates interaction with CBL proteins and the PPI domain mediates interaction with protein phosphatases.
  • Figure 11 (B) Schematic model of the CIPK6 truncated version without kinase activity, which consists of 168 amino acids.
  • the sequences of the polynucleotide and the polypeptide of the truncated CIPK6 are shown in SEQ ID NO: 11 and 12, respectively. Numbers indicate the amino acid position.
  • the NAF domain mediates interaction with CBL proteins and the PPI domain mediates interaction with protein phosphatases.
  • FIG. 11 Kinase inactive point mutations of CIPK6.
  • K53N marks an amino acid exchange in the putative ATP binding site at position 53 from lysin to asparagine.
  • D164N marks an amino acid exchange in the activation loop of the kinase from aspartic acid to asparagines.
  • the sequences of the polynucleotide and the polypeptide of the K53N mutated CEPK6 are shown in SEQ ID NO: 13 and 14, respectively.
  • the sequences of the polynucleotide and the polypeptide of the KD164N mutated CIPK6 are shown in SEQ ID NO: 15 and 16, respectively.
  • FIG. 12 General composition of calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs).
  • CBL calcineurin B-like
  • CIPKs CBL-interacting protein kinases
  • the overall structure of CBLs consists of four EF hands (boxes with numbers). Spacing of EF hands in all CBLs is invariable, while the N- and C-terminal extensions of CBL proteins vary in length.
  • the first EF hand has an unconventional structure, encompassing 14 amino acids instead of the 12 m a canonical EF hand (light gray box).
  • the overall structure of CIPKs comprises an N-terminal kinase domain and a regulatory C-terminal domain that are separated by a junction domain.
  • WitMn the kinase domain, phosphorylation of amino acids in the activation loop (indicated as a box) results in kinase activation.
  • the regulatory C-terminal domain contains two conserved interaction domains, the NAF domain (designated according to the conserved arnino acids N, A and F), which is responsible for the CBL-CIPK interaction, and the adjacent protein-phosphatase interaction (PPI) domain mediating interaction with 2C-type protein phosphatase (PP2C)-type phosphatases.
  • NAF domain designated according to the conserved arnino acids N, A and F
  • PPI adjacent protein-phosphatase interaction
  • P2C 2C-type protein phosphatase
  • Figure 13 Salt stress assay of transgenic lines. For the same medium condition (control or NaCl), root length, fresh weight (FW) root and FW shoot of the transgenic lines are presented relative to those of the wild type (set to 100%).
  • FIG 14 Salt stress assay of transgenic lines. Exemplanly, salt stress and control container with the two transgenic lines 28 and 38, both including all three transgenes CBL4 + CIPK6 + AKT2 (here referred to C4+C6+A2), and wildtype (WT) are shown.
  • Figure 15 Dependence on K+ supply of transgenic lines.
  • Nine-day-old seedlings of transgenic lines and wildtype (WT) were transferred to 1 ⁇ 2 MS (control) or 1 ⁇ 2 MS supplemented with only 10 ⁇ K + (10 ⁇ K + ) and grown vertically for 7 days.
  • FIG. 16 Dependence on K + supply of transgenic lines.
  • growth of transgenic line 64 including all three transgenes CBL4 + CIPK6 + AKT2 (here referred to C4+C6+A2), and wildtype (WT) after 7 days on 1 ⁇ 2 MS (control) or 1 ⁇ 2 MS supplemented with only 10 ⁇ K + (10 ⁇ K + ) is shown.
  • Example 1 Materials and Methods General methods, construct generation, plant cultivation and phenotype analyses, yeast-two- hybrid studies
  • RNA For transcript analysis 1 ⁇ g of total RNA was used for cDNA synthesis and RT-PCR was performed with gene specific primers. Segregation analysis of progenies of heterozygous mutant lines revealed a 3 to 1 ratio confirming single T-DNA insertions. To further corroborate the mutant status of the cbl4 and cipk6 lines, salt stress assays were performed as previously described (D'Angelo C, Weinl S, Batistic O et al. Alternative complex formation of the Ca-regulated protein kinase CIPKl controls abscisic acid-dependent and independent stress responses in Arabidopsis.
  • the NAF domain defines a novel protein-protein interaction module conserved in Ca 2+ -regulated kinases. EMBO J 2001; 20 (5): 1051-1063; Kolukisaoglu ⁇ , Weinl S, Blazevic D, Batistic O, Kudla J. Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks. Plant Physiol 2004; 134 (l):43-58).
  • Oocytes were obtained and stored as described previously (Michard E, Dreyer I, Lacombe B, Sentenac H, Thibaud IB. Inward rectification of the AKT2 channel abolished by voltage-dependent phosphorylation. Plant J 2005; 44 (5):783-797). In vitro transcriptions were performed using the mMESSAGE mMACHTNE kit (Ambion) following the manufacturer's instructions. Oocytes were injected with a final volume of 20 nl of various cRNA combination using a 10-15 ⁇ tip diameter micro-pipette.
  • Injections were performed using either 8 ng of Shaker cRNA or, for co-injection experiments, a mix of 8 ng of Shaker cRNA and 6 ng of both CIPK and CBL cRNAs. All experiments were performed at room temperature (20-22°C). Whole-cell currents were recorded 3- 4 days after oocytes injection as described previously (Michard E, Dreyer I, Lacombe B, Sentenac H, Thibaud JB. Inward rectification of the AKT2 channel abolished by voltage-dependent phosphorylation. Plant J 2005; 44 (5):783-797) using the TEVC technique.
  • Voltage-dependent activation of Shaker channels was recorded using a pulse protocol starting from a holding potential of -40 mV; pulses were applied to various test voltages as indicated in legend to Figure 7 B.
  • the bath solution contained 100 mM KC1, 1 mM CaCl 2 , 1.5 mM MgCl 2 and 10 mM HEPES-Tris (pH 7.5).
  • BAPTA injections 50 nL of 50 mM BAPTA) were performed using a 10-15 ⁇ tip diameter micro-pipette.
  • the plVEX 1.3 WG plasmid (Roche) was used as the template for site-directed mutagenesis PCR to generate the ⁇ WG StrepII vector that expresses a N-terminally StrepII-tagged proteins.
  • the respective cDNAs were amplified by PCR and subcloned into the pfVEX WG StrepII vector. 60 ⁇ g of ⁇ WG StrepII constructs were used for in vitro transcription/translation using RTS 500 Wheat Germ CECF Kit (Roche).
  • Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J 2008; 56 (3):505-516).
  • pGPTVII vectors containing AKT2-Venus, CBLln-OFP, OFP-HDEL, CBL4-OFP and CBL4- SCFP under control of the 35S promoter or MAS promoter in case of CBLln-OFP were transformed into agrobacteria and co-infiltrated at a OD of 0.5, except for the HDEL-OFP bearing strain, which was infiltrated at an OD of 0.1.
  • Fluorescence emissions were determined 3 days after infiltration for CIPK6-CBL4 BiFC combinations and 4 days after infiltration in experiments containing CIPK6-AKT2 BiFC combinations using a Leica DM1RE2 with TCS SP2 laser scanner set up with a HCX PL APO 63 x 1.2 water objective (Leica Microsystems GmbH).
  • a 488 or 514 ran (Ar/Kr) laser and 543 or 594 nm (He/Ne) laser was used, respectively, and emission was recorded at 518-560 nm (YFP) and 605-640 nm (OFP).
  • SCFP fluorescence was generally recorded separately of YFP fluorescence with a 405 nm diode laser and detected at 460-480 nm. Fluorescence intensity scans were performed as described (Batistic O, Sorek N, Schultke S, Yalovsky S, Kudla J. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca 2+ signaling complexes in Arabidopsis. Plant Cell 2008; 20 (5): 1346-1362) using the quantification tool of the Leica confocal software application (version 2.61). Microscopy was done at room temperature with leave discs immersed in water.
  • CBL4 association with CIPK6 specifically facilitates Ca 2+ -dependent activation of the AKT2 K + channel
  • CBL4AEF mutant allele sos3-l
  • AKT2 channels either display K + -selective inward rectifying (gating mode#l) or K + -selective "open leak" (gating mode#2) features (Michard E, Dreyer I, Lacombe B, Sentenac H, Thibaud JB. Inward rectification of the AKT2 channel abolished by voltage-dependent phosphorylation. Plant J 2005; 44 (5): 783-797).
  • comparative TEVC analyses of oocytes expressing either AKT2 alone or AKT2 in combination with CBL4 and CTPK6 did not indicate.
  • CBL4 Upon interaction with CIPK6, CBL4 mediates ER-to-PM translocation of AXT2
  • CBL1 myristoylation and palmitoylation (also referred to as S-acylation) of this calcium sensor for its proper function
  • S-acylation also referred to as S-acylation
  • Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca 2+ signaling complexes in Arabidopsis. Plant Cell 2008; 20 (5):1346-1362).
  • the crucial importance of both lipid modifications is due to their function as signals in cytoplasm-to-ER and ER-to-PM trafficking of this calcium sensor protein.
  • Example 3 Generation of transgenic tobacco plants overexpressing Arabidopsis thaliana CBL4, CIPK6 and/or AKT2
  • Polynucleotides encoding for Arabidopsis thaliana CBL4, CIPK6 and AKT2 were cloned into vectors based on the pUC18 plasmid.
  • MCS multiple cloning site
  • NosT Nos gene
  • AtCBL4 was set under control of the MAS promoter, AtClPK6 under control of the UBQ10 promoter and AtAKT2 under control of the cauliflower mosaic virus 35S promoter.
  • the here described vectors are referred to pMAS::CBL4, pUBQ10::CIPK6 and p35S::AKT2.
  • Gold particles coated with a mixture of the three above described vectors and additionally with a plasmid facilitating kanamycin resistance, were used for particle gun-mediated (biolistic) transformation of Nicotiana tabacum cv. Petit Havana (Ruf & Bock, 2011; Zhu et al., 2008).
  • 93 independently generated lines were kanamycin-selected after transformation and subsequently grown under greenhouse conditions to produce Tl seeds. A population of the Tl generation was then screened by genomic PCR for the integration of the A. thaliana transgenes.
  • Tl gDNA - isolated from a kanamycin selected Tl seedling pool of each analyzed line - was analyzed with primer pairs specific for each transgene and spanning the complete cloned protein encoding regions. The corresponding plasmids were used as positive controls, and N. tabacum wild type gDNA as negative control.
  • AtCBL4 AtCIPK6 and AtAKT2 at least one independent transgenic line could be identified (Table 1).
  • Table 1 In more detail, in total 44 Tl lines were analyzed by genomic PCR, for 16 lines no fragment for any of the three transgenes was amplified, however, 28 lines led to different fragment combinations.
  • Table 1 Summary of transgenic lines and their genotypes identified by genomic PCR
  • transgenic lines were further analyzed: 134 (CBL4 integrated), 22 (CIPK6 integrated), 58 (AKT2 integrated), and 28, 38 and 64 (CBL4 + CIPK6 + AKT2 integrated).
  • Example 4 Phenotypical analysis under salt stress conditions/ potassium limiting conditions
  • Salt stress assay was performed using the hydroponic growing system araponics (Araponics SA) with 1 ⁇ 2 MURASHIGE & SKOOG medium.
  • Araponics SA hydroponic growing system araponics
  • transgenic lines were selected on medium for kanamycin selection, while wild type seeds were grown without kanamycin - both in seed-holders of the araponics system.
  • the remaining kanamycin resistant transgenic seedlings and the wild type seedlings were unified in one container.
  • Each transgenic line - together with the corresponding wild type - was exposed to control condition (1 ⁇ 2 MS) and to salt stress condition (1 ⁇ 2 MS + 150 mM NaCl).
  • transgenic lines were selected on 1 ⁇ 2 MS + kanamycin medium, while wild type seeds were grown without kanamycin.
  • Nine-day-old seedlings were then transferred to vertical agar plates with 1 ⁇ 2 MS (control) or 1 ⁇ 2 MS with only 10 ⁇ K + supplemented (10 ⁇ K7). After growth for 7 days, photographs were taken and fresh weight of the shoot was measured ( Figure 15 and 16).
  • the protein encoding regions of Arabidopsis thaliana CBL4, CEPK6 and AKT2 were cloned into vectors based on the pUC18 plasmid.
  • a multiple cloning site (MCS) followed by the terminator of the Nos gene (NosT) was cloned into the pUC18 plasmid.
  • MCS multiple cloning site
  • NosT Nos gene
  • one of three different promoters was introduced into the MCS.
  • AtCBL4 was set under control of the MAS promoter, AtCEPK6 under control of the UBQ10 promoter and AtAKT2 under control of the cauliflower mosaic virus 35S promoter (see also Example 3).
  • the point mutation D164N was additionally introduced into the kinase activation loop.
  • the resulting kinase inactivated QPK6 polypeptide has a sequence as shown in SEQ ID NO: 16.
  • pMAS::CBL4 pUBQ10::CIPK6, pUBQ10::CD?K6 D164N and p35S::AKT2.
  • Gold particles coated with two different mixtures of the above described vectors and additionally with a plasmid facilitating kanamycin resistance, were used for particle gun-mediated (biolistic) transformation of Nicotiana tabacum cv. Petit Havana (Ruf & Bock, 2011; Zhu et al, 2008).
  • the mixture included pMAS::CBL4, pUBQ10::CIPK6_D164N and p35S::AKT2. So far, 166 lines were kanamycin selected after transformation with the CTPK6_D164N mixture, and subsequently grown under greenhouse conditions to produce Tl seeds. A population of the Tl generation was then screened by genomic PCR for the integration of the A. thaliana transgenes.
  • AtCBL4 AtCIPK6_D164N and AtAKT2 at least one independent transgenic line could be identified.
  • transgenic lines comprising the AtCBL4 polypeptide, the AtCIPK6_D164N polypeptide and/or AtAKT2 polypeptide were obtained (and thus, lines, comprising one, two or three of the aforementioned polypeptides).
  • Phenotypical analyses under salt stress conditions or potassium limiting conditions are carried out as described in Example 4. Increased tolerance against salt stress and increased yield under potassium limiting conditions are observed.
  • Example 6 Blast Search for variants of the Arabidopsis thaliana CBL4, CIPK6 and AKT2 polypeptide
  • Table A shows variants of the Arabidopsis thaliana CBL4 polypeptide.
  • Table B shows variants of the Arabidopsis thaliana CIPK6 polypeptide.
  • Table c shows variants of the Arabidopsis thaliana AKT2 polypeptide. Given are the species, the name, the NCBI ID, the GenBank Accession No as well as the degree of between the homolog and the Arabidopsis thaliana polypeptide.
  • Table B Variants of the Arabido sis thaliana CIPK6 ol e tide
  • Vicia faba 2293112 CAA71598.1 67,00%

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Abstract

L'invention concerne un procédé de production d'une plante ou cellule de plante transgénique présentant une efficacité de potassium accrue. L'invention concerne également une plante ou cellule de plante transgénique contenant un polypeptide CBL4 et CIPK6. L'invention concerne encore une composition contenant un polypeptide CBL4 et CIPK6, ainsi que l'utilisation de ces polypeptides dans la production d'une plante ou cellule de plante transgénique présentant une efficacité de potassium accrue.
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WO2014205598A1 (fr) * 2013-06-24 2014-12-31 创世纪转基因技术有限公司 Protéine de transport d'ions potassium haute affinité hkt1 obtenue à partir de thellungiella halophila, et gène codant et utilisation correspondante
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