WO2012028646A1

WO2012028646A1 - Means for improving agrobiological traits in plants

Info

Publication number: WO2012028646A1
Application number: PCT/EP2011/065005
Authority: WO
Inventors: Jörg KUDLA
Original assignee: Westfaelische Wilhelms Universitaet Muenster
Current assignee: Westfaelische Wilhelms Universitaet Muenster
Priority date: 2010-08-31
Filing date: 2011-08-31
Publication date: 2012-03-08
Anticipated expiration: 2013-02-28

Abstract

The present invention relates to a method for producing a transgenic plant or plant cell having increased potassium efficiency. Moreover, the present invention relates to a transgenic plant or plant cell comprising a CBL4 and CIPK6 polypeptide. Further envisaged by the present invention is a composition comprising a CBL4 and a CIPK6 polypeptide as well as the use of said polypeptides for the generation of a transgenic plant or plant cell having increased potassium efficiency.

Description

Means for improving agrobiological traits in plants

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. Moreover, the present invention relates to a transgenic plant or plant cell comprising a CBL4 and C1PK6 polypeptide. Further envisaged by the present invention is 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.

Potassium (K⁺) is an essential nutrient for plant growth. Because large amounts of K⁺ are absorbed from the root zone in the production of most agronomic crops, it is classified as a macronutrient.

If 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.

Therefore, the generation of plants having increased potassium efficiency has become a promising strategy to overcome the problems that are caused by growth limiting concentrations of K⁺ in the soil. Plants with increased potassium, efficiency could be grown on soil with lower K⁺ concentrations.

To efficiently acquire K⁺ despite its fluctuating availability in the soil and to adjust K^÷ distribution within the plant to variable environmental conditions and developmental requirements a tight regulation of K⁺ transporting proteins is fundamental. However, so far there have been no successful attempts to improve potassium efficiency by engineering transgenic crops. Recent studies have identified a regulatory circuit in which the Calcineurin B-like proteins CBL1 and CBL9 specifically interact with and activate the CBL-interacting protein kinase CIPK23 to phosphorylate and positively modulate the activity of the inward rectifying K⁺ channel AKTl (Xu, J. et al. A protein kinase, interacting with two calcineurin B-like proteins, regulates K⁺ transporter AKTl in Arabidopsis. Cell 125, 1347-1360 (2006); Lee, S.C. et al. A protein phosphorylation /dephosphorylation network regulates a plant potassium channel. Proc Natl Acad Sci U S A 104, 15959-15964 (2007)).

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. These studies directly linked ion-channel regulation to a complex calcium-decoding network in Arabidopsis that is formed by 10 CBL calcium sensor proteins and 26 CIPKs (Batistic, O. & Kudla, J. Plant Calcineurin B-like proteins and their interacting protein kinases. BBA - Molecular Cell Research, 1793, 985-92 (2009)).

Specific complex formation of defined CBL proteins with distinct subsets of CIPKs has been shown to be important for generating signaling specificity in this calcium-decoding network and several mutant studies have unraveled the importance of single CBL and CIPK proteins for proper abiotic stress responses of plants (D'Angelo, C. et al. Alternative complex formation of the Ca- regulated protein kinase CEPKl controls abscisic acid-dependent and independent stress responses in Arabidopsis. Plant J 48, 857-872 (2006); Kim et al. The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis. Plant Journal, 52, 473-84 (2007); Cheong et al. Two Calcineurin B-like calcium sensors, interacting with protein kinase cipk23, regulate leaf transpiration and root potassium uptake in Arabidopsis. Plant Journal, 52, 223-239(2007))

However, it has remained unknown if CBL-CIPK complexes regulate any other K⁺ channel in plants.

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. 14, 1133-46 (2002). and Deeken, R. et al. Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis. Planta 216, 334-344 (2002)) The molecular mechanisms modulating AKT2 activity are, however, not completely understood.

There is a need for the generation of plants and plant cell having increased potassium efficiency. Accordingly, the technical problem underlying the present invention could be seen as the provision of means and methods for complying with the aforementioned needs.

The technical problem is solved by the embodiments characterized in the claims and herein below. Accordingly, 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. The term "plant" as used herein, preferably, refers to a higher plant, preferably to a monocotelydonous plant or dicotelydonous plant. Preferably, 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 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). Preferably, 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. Preferably 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).

Moreover, 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. Moreover, within a plant cell 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 Ca²⁺-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. 2009, Batistic et al., CBL-mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores. Plant J., 61, 211- 22. 2010, Kim et al. The Calcium Sensor CBLIO Mediates Salt Tolerance by Regulating Ion Homeostasis in Arabidopsis. Plant J., 52, 473-484, (see also Held et al Cell Research (2011) 21:1116-1130). Moreover, 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:

a) a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 1 ;

b) a nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ ID No: 2;

c) a 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; and

d) a nucleic acid encoding a polypeptide being a variant of the amino acid sequence shown in SEQ ID No:2, wherein said variant has calcium binding activity. In particular, 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:

a) a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 1 ;

c) a 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; and

d) a 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.

By introducing 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 "calcium-binding activity", preferably, refers to the capability of a polypeptide to bind calcium. 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. Preferably, 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). Preferably, 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. The term "polynucleotide" as used herein 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. Moreover, 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. Moreover, due to the degeneracy of the genetic code, polynucleotides are encompassed which encode a specific amino acid sequence as recited above. The terms "polynucleotide" and "nucleic acid" may be used interchangeably herein.

Moreover, the term "polynucleotide" as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides. Moreover, the term "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. The 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. A preferred example for stringent hybridization conditions are hybridization conditions in 6 x sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2 x SSC, 0.1% SDS at 50 to 65°C. 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). If an organic solvent is present in the abovementioned buffer, for example 50% formamide, 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. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Alternatively, 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. Conserved 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.

Further, variants of polynucleotides (or of polypeptides) 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). Moreover, also encompassed are 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. Moreover, with respect to polypeptides, 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. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences, hi this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (Higgins 1989, CABIOS, 5 1989: 151-153) or the programs Gap and BestFit (Needleman 1970, J. Mol. Biol. 48; 443-453 and Smith 198, Adv. Appl. Math. 2; 482-489), which are part of the GCG software packet from Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711, version 1991, are to be used. The 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. As set forth above, 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.

Thus, the 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:

a. ) introducing at least one (i.e. one or more than one) heterologous polynucleotide encoding a polypeptide (polypeptides) as set forth herein into the plant or plant cell; and b. ) expressing said polypeptide(s) from the said at least one polynucleotide.

The term "heterologous" as used herein 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. It is to be understood that 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. Preferably, the heterologous polynucleotide comprises a nucleic acid having a nucleic acid sequence of the first, or the second polynucleotide as referred to herein elsewhere. By introducing a heterologous polynucleotide in plant ceils, 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. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128443; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225. Preferably, 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. In the context of the present invention it is particularly envisaged that the polynucleotide or vector shall be stably integrated into the genome by Agrobacterium- mediated or particle bombardment mediated transformation. However, it is also envisaged that a polypeptide as set forth, herein is transiently expressed.

Preferably, the polynucleotide encoding a polypeptide as set forth herein is operably linked to a promoter and a terminator allowing expression of said polynucleotide. How to operably link a promoter and a terminator to a polynucleotide is 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. Preferably, 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. Accordingly, by generating a transgenic plant having increased potassium efficiency, a plant is, preferably, generated that has increased yield under potassium limiting conditions. Preferably, 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). Thus, 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. For example, 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.

Preferably, 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.

Accordingly, 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.

The term "yield" as used herein 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. The term 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. Preferably, 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.

Preferably, 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).

Thus, 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.

Accordingly, 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; Laurie et al. Plant J, 2002, 32: 139-149 D'Angelo et al, 2006) and described in, preferably, in the accompanying Examples below. Preferably, 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. More preferably, 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. More preferably, 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).

In a preferred embodiment 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). 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.

Advantageously, it has been shown in the context of the studies carried out in the context of the present invention that the Arabidopsis calcium sensor CBL4 together with the protein Arabidopsis protein kinase CIPK6 Ca²⁺-dependently modulate the activity of the shaker-type K^÷ channel AKT2 from Arabidopsis thaliana (see Examples). This is the first study to show that AKT2 is a target of a CIPK6/CBL4 complex. Particularly, it was shown that 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). Interestingly, 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.

Thus, is has been surprisingly shown, that 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. Moreover, 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).

Preferably, 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:

a) a nucleic acid having a nucleotide sequence as shown in SEQ ED No: 3;

b) a 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

d) a nucleic acid encoding a polypeptide being a variant of the amino acid sequence shown in SEQ ID No:4, wherein said variant is capable of interacting with a CBL polypeptide (preferably CBL4).

In particular, the 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) a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 3;

b) a 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 at least 50% identical to 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 d) a 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:4, wherein said polypeptide is capable of interacting with a CBL polypeptide (preferably CBL4).

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. Moreover, 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.

In a preferred embodiment of the method of the present invention, 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. Moreover, it is well known in the art how to determine whether a polypeptide has kinase activity or not (e.g. by phosphorylation assays). It is particularly preferred to truncate the domain within the CIPK6 polypeptide which confers serin-threonin kinase activity. Particularly, preferred CIPK6 polypeptides not having serin-threonin kinase activity are described in the Examples. For example, with respect to the Arabidopsis thaliana CIPK6, 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) Thus, a CIPK6 polypeptide without serine- threonme-kinase activity can be generated by truncating said domain or a part thereof. Preferably, 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).

Thus, 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. Whether a particular variant of the CIPK6 polypeptide does not have threonine kinase activity, but still interacts with a CBL polypeptide can be assessed by the skilled person in routine experiments. As shown in Fig 11C, 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.

Thus, 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.

Thus, 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:

a) a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 11, 13 or

15;

b) a 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

d) a nucleic acid encoding a polypeptide being a variant of the amino acid sequence shown in SEQ ID No: 12, 14 or 16, wherein said variant is capable of interacting with a CBL polypeptide (preferably CBL4), and wherein, and, wherein, said polypeptide, preferably, does not have serine- threonine-kinase activity.

Thus, the 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:

a) a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 11, 13 or

15;

b) a nucleic acid encoding a polypeptide having an amino acid sequence as shown in SEQ K) No: 12, 14 or 16;

c) a 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

d) 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).

Preferably, said polypeptide mentioned in c) and d) does not have serine- threonine-kinase activity

The introduction a 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.

In a further preferred embodiment, 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. Preferably, 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. Preferably, 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:

a) 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;

c) a 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

d) a 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.

In particular, 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:

c) a 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

d) a 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:6, wherein said polypeptide is capable of transporting ions preferably K⁺ ions across cellular membranes preferably, plasma membranes. Preferred 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.

In a even further preferred embodiment 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. Preferably, plants are generated comprising a) the CBL4, CIPK6, CEPK24 and SOSl polypeptide, or b) the CBL4, CIPK6, AKT2, CTPK24 and SOSl polypeptide.

Cytoplasmic calcium signals elicited by salt stress presumably are perceived by CBL4, which interacts physically with C1PK24 and this CBL4/CIPK6 complex activates the transport activity of the Na⁺/H+_ antiporter SOSl by phosphorylation. 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.

Preferably, 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:

a) 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;

c) a 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

d) a 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.

Preferably, the SOSl polypeptide is encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:

a) 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;

c) a 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

d) 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. How to assess whether a polypeptide or a variant thereof is capable transporting ions preferably Na⁺, across cellular membranes preferably, plasma membranes is known by the person skilled in the art and. e.g., described in Plant Stress Tolerance: Methods and Protocols: 639 (Methods in Molecular Biology) Ramanjulu Sunkar (Editor) Springer Protocols 2010 DOI: 10.1007/978-1- 60761-702-0_^ 23.

The present invention also relates to a plant or a plant cell generated by the method according to the present invention. Preferably, a said plant comprises a plant cell generated by the method of the present invention.

Accordingly, the present invention relates to plant or plant cell, comprising a CBL4 polypeptide (preferably, is transformed with said polypeptide). Preferably, said CBL4 polypeptide is expressed from a heterologous polynucleotide. Preferably, 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.

In a preferred embodiment, said plant or plant cell further comprises a CIPK6 polypeptide (preferably is transformed with said polypeptide). Preferably, said C1PK6 polypeptide is expressed from a heterologous polynucleotide. Preferably, 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.

Thus, 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. Preferably, 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. Preferably, 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. Moreover, the present invention relates to a composition comprising a CBL4 and CIPK6 polypeptide.

In a preferred embodiment said composition 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. Preferably, 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.

By introducing the aforementioned polynucleotide of the present invention 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 present invention also contemplates a vector comprising one of the aforementioned polynucleotides of the present invention.

The term "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. If introduced into a host cell, 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. The terms "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. (Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa, New Jersey. Alternatively, a 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.

Preferably, the vector referred to herein is suitable as 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). These 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. While 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. As a consequence, the last-mentioned vectors are relatively small, easy to manipulate and can be replicated both in E. coli and in Agrobacterium. These binary vectors include vectors from the pBIB-HYG, pCAMBIA, pPZP, pBecks, pGreen series. Preferably used in accordance with the invention is pGreen II.. An overview of binary vectors and their use can be found in Hellens 2000, Trends in Plant Science 5, 446-451. Furthermore, by using appropriate cloning vectors, 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. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225.

More preferably, the vector of the present invention is an expression vector. In such 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. Preferably, 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. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).

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 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. Since plant gene expression is very often not limited to transcriptional levels, 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). 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.

In a preferred embodiment of the composition of the present invention the CIPK6 polypeptide does not have serine-threonine kinase activity.

Furthermore, 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.

Moreover, the present invention relates to a CIPK6 polypeptide encoded by a polynucleotide comprising a nucleic acid selected from the group consisting of:

a) a nucleic acid having a nucleotide sequence as shown in SEQ ID No: 11, 13 or

15;

b) a 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

d) a nucleic acid encoding a polypeptide being a variant of the amino acid sequence shown in SEQ ID No: 12, 14 or 16, wherein said variant is capable of interacting with a CBL polypeptide (preferably CBL4), and wherein, said polypeptide, preferably, does not have serine- threonine-kinase activity. 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. Furthermore, 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.

As envisaged by the present invention is the use of 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.

Moreover, 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. Preferred is the use of 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. Preferred is the use of a) CBL4, CIPK6, and AKTl polypeptide, b) the CBL4, CTPK6, C1PK24 and SOS1 polypeptide, or c) the CBL4, CIPK6, AKT2, CIPK24 and SOS1 polypeptide.

Finally, 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. Preferred is the use of a) CBL4, CIPK6, and AKTl polypeptide, b) the CBL4, CIPK6, ΟΓΡΚ24 and SOS1 polypeptide, or c) the CBL4, CIPK6, AKT2, CIPK24 and SOS1 polypeptide. The figures show: Figure 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. Currents recorded in oocytes injected with different mixes of cRNAs were normalized to the mean current value at -155mV in oocytes injected with AKT2 cRNA only. Data are displayed as means ± SE for the following injected mixes of cRNAs: AKT2 (n=48), AKT2+CIPK6+CBL4 (n=44), AKT2+CIPK6 (n=16), AKT2+CBL4 (n=15), AKT2 + CIPK6+ CBL9 (n=9), AKT2 + CIPK6+ CBLl (n=9), AKT2+CIPK23 + CBLl (n=6). (E) Both AKTl and KAT2 are insensitive to CPK6-CBL4. (left) Currents in oocytes injected with AKTl cRNA and either CIPK23 and CBLl or CIPK6 and CBL4 cRNAs. Data are current values at -155 mV normalized to the mean current value at -155 mV in oocytes injected with AKTl, CIPK23 and CBLl cRNAs (means ± SE, n=5, 7 and 6 for AKTl +C1PK23+CBL1, AKTl and AKTl +C1PK6+CBL4, respectively), (right) Currents in oocytes injected with either KAT2 cRNA or a mix of KAT2, CIPK6 and CBL4 cRNAs. Data are current values at -155 mV normalized to the mean current value at -155 mV in oocytes injected with KAT2 cRNA (means ± SE, n=7). (F) Ca²⁺- 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. Data are current values at. -155 mV normalized to the mean current value at -155 mV in oocytes injected with AKT2 cRNA only (means ± SE, n=10). (right) CIPK6-CBL4 stimulation of AKT2 current is abolished by an EF- hand mutation in CBL4. Oocytes were injected with different combinations of cRNAs : AKT2, AKT2+C1PK6+CBL4 or AKT2+CIPK6+CBL4AEF. Data are current values at -155 mV normalized to the mean current value at -155 mV in oocytes injected with AKT2 cRNA only (means ± SE, n=10). (**) marks results with significant difference in values (D-F). Figure 2 Loss-of-fimction akt2-l, cbl4 and cipk6 mutant plants exhibit similar developmental and flowering phenotypes in short day conditions. 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). (C) Number of leaves determined 6 WAS (n=20-24). (D) Height of the main inflorescence stalk determined 8 WAS (WT-Ws and akt.2-1; n=23 in each case) and 9 WAS (WT-Col-0, cbU and cipk6; n=22-23). Data in (C) and (D) are depicted as mean ± SD. (*) marks results with significant difference in values.

Figure 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. (Further details and the distribution CBL4-SCFP fluorescence are shown in Figure 10 A) (E) View into the optical plane of the PM indicating CBL4-dependent accumulation of AKT2-CIPK6 BiFC complexes in punctate structures of the PM. Bottom panel: enlargement of the area indicated by a white frame in the top panel.

Figure 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. (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. 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. Data are representative of 3 independent experiments and are displayed as means ± SE (n=number of tested oocytes) for the following injected mixes of cRNAs: AKT2 (n=15), AKT2+CIPK6+CBL4 (n=9), AKT2+CIPK6C+CBL4 (n=l l), AKT2+CIPK6C (n=10), AKT2+CIPK6N+ CBL4 (n=9). (C) Microscopic analysis of the median cellular plane of N. benthamiand epidermal cells transiently expressing the plasmid combinations indicated at the left. Top panel: BiFC analysis reveals the interaction of CBL4 with the kinase domain deletion construct of CIPK6 (CIPK6C) predominantly at the plasma membrane. Middle panel: CIPK6C still interacts with AKT2 despite the absence of the kinase domain. Bottom panel: Co-expression of CBL4-SCFP induces the translocation of AKT2-CIPK6C complexes towards the PM. (D) Ca2+-dependent stimulation of AKT2 currents by CBL4-CIPK6C. Currents in oocytes injected with AKT2 cRNA , a mix of AKT2, CIPK6C and CBL4 cRNAs or a mix of AKT2, CIPK6C and CBL4AEF cRNAs were recorded before and 5 min after a 50mM BAPTA injection. Data are representative of three independent experiments and are represented as current values at - 100 mV normalized to the mean current value at -100 mV in oocytes injected with AKT2 cRNA only (means ± SE, n=6; 7, 7, 6 and 7 respectively for AKT2, AKT2+CIPK6+CBL4, AKT2+CIPK6C+CBL4, AKT2 + CIPK6 C+ CBL4 after BAPTA and AKT2 + CIPK6 C+ CBL4AEF) . (E) Co-localization of complexes formed by interaction of AKT2-YC with YN-CIPK6C after co- expression with CBL4AEF-SCFP and the PM-marker CBLln-OFP reveals the AKT2-CIPK6C- indicating fluorescence signal in ER-enriched cellular region and 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. (Further details and the distribution CBL4AEF-SCFP fluorescence are shown in Figure 10 B.)

Figure 6 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. Data are are displayed as means ± SE (n=number of tested oocytes) for the following injected mixes of cRNAs: AKT2 (n=5), AKT2+CIPK6+CBL4 (n=5), AKT2 + CIPK6+ CBL4G2A (n=5). (B) Dual lipid modification of CBL4 by myristoylation and palimitolylation is required for CBL4-dependent AKT2 activation. Expression of point mutated CBL4C3S mutated cRNAs, that result in myristoylated but non-palmitoylated CBL4 protein does not evoke AKT2 currents. Currents recorded in oocytes injected with different mixes of cRNAs were normalized to the mean current value at -160 mV in oocytes injected with AKT2 cRNA only. Data are displayed as means ± SE (n=number of tested oocytes) for the following injected mixes of cRNAs: AKT2 (n=25), AKT2+CIPK6+CBL4 (n=23), AKT2 + CIPK6 + CBL4C3S (n=18). (C, D) CBL4s missing N-terminal lipid modifications fail to induce the translocation of AKT2-CIPK6 complexes. 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. (C) Co-expression of CBL4G2A-SCFP and (D) 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).

Figure 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. 3s voltage-clamp pulses from +40mV to -170mV (step: -15mV, holding potential: -40mV) (C) and (D) Typical current traces elicited by the voltage protocol described in panel (B) in oocytes expressing AKT2 or AKT2+CIPK6+CBL4. Experiments were performed in a 100 mM K+ external solution, currents were recorded 4 days after injection of AKT2 cRNA or of AKT2, CIPK6 and CBL4 cRNAs. (E) Current- voltage (I-V) curves for oocytes injected with AKT2 cRNA (open symbols) and injected with a mix of AKT2, CIPK6 and CBL4 cRNAs (closed symbols). Current values from recordings like in (C) and (D) were normalized to the mean current value at -155 mV in oocytes injected with AKT2 cRNA only. Data are given in percent as mean ± SE (symbols cross- referenced to C and D) with n=14 for AKT2 and n=8 for AKT2+CIPK6+CBL4. (F-G) Both AKTl and KAT2 are insensitive to CIPK6+CBL4. (F) AKTl. 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). Current values from recordings like in left panel and displayed as means ± SE (n=4 for AKT1+CIPK23+CBL1 and n=6 for AKTl and AKTl + CIPK6+ CBL4) . (G) KAT2. Left panel: Typical current recordings in X. laevis oocytes expressing either KAT2, or KAT2+CIPK6+CBL4. Right panel: Current-voltage (I-V) curves for oocytes injected with KAT2 cRNA (white circles) or with a mix of KAT2, CIPK6 and CBL4 cRNAs (black circles). Current values from recordings like in left panel and displayed as means ± SE (n=6 for KAT2 and n=7 for KAT2+CIPK6+CBL4). Experiments were performed 3 days after oocyte injections in a 100 mM K+ external solution using the voltage clamp protocol described in Figure SI B.

Figure 8 AKT2 channel voltage gating is not changed by CIPK6+CBL4. (A) Proportion of time- dependent (gating mode#l, black) and instantaneous (gating mode#2, white) currents (with respect to the total current) recorded at -155 mV in oocytes expressing AKT2 or AKT2+CIPK6 +CBL4. Data were normalized by the mean of the total current (mode#l+mode#2) recorded at -155 mV and are displayed as means ±SE (n=38 for AKT2 and n=35 for AKT2+CIPK6+CBL4) . (B) Voltage- gating of the time-dependent fraction of the AKT2 current is not changed by CIPK6+CBL4. The relative conductance (G/Gmax) values were obtained as described in Dreyer et al. (2001) from at least 3 independent experiments on oocytes expressing either AKT2 only or AKT2+CIPK6+CBL4. Symbols represent means ±SE (AKT2: n=29, AKT2 + CIPK6 + CBL4 : n=28). Curves represent best fit to the data of a Boltzmann equation (AKT2: zg=1.26; Ea50~134 mV, full black line; AKT2+CIPK6+CBL4: zg=1.03; Ea50=-139 mV; dotted gray line). (C) ΑΚΪ2 unitary conductance is not affected 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).

Figure 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. (D) Number of leaves determined 6 WAS (n=12 in each case). (*) marks results with significant difference in values. Figure 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. The respective combinations of expressed plasmids are indicated in the left. 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 (A) 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.

Figure 11 (C) 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.

Figure 12: General composition of calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs). 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. In all CBL proteins, 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.

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%).

Figure 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 ½ MS (control) or ½ MS supplemented with only 10 μΜ K⁺ (10 μΜ K⁺) and grown vertically for 7 days. A) Fresh weight (FW) of shoot [g] under control and K⁺ limiting condition of transgenic lines and wild type. B) FW of shoot [%] under K⁺ limiting condition presented relative to control FW (set to 100%).

Figure 16: Dependence on K⁺ supply of transgenic lines. Exemplanly, 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 ½ MS (control) or ½ MS supplemented with only 10 μΜ K⁺ (10 μΜ K⁺) is shown.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification. The following examples merely illustrate the invention. They should, whatsoever, not be construed as limiting the scope of protection. EXAMPLES

Example 1: Materials and Methods General methods, construct generation, plant cultivation and phenotype analyses, yeast-two- hybrid studies

Molecular biology methods were performed according to standard procedures. A list of primers used in this work can be obtained upon request. In general, for construct generation the respective protein encoding regions of the CBLs and CIPKs, respectively, were amplified by PCR and introduced into the pGPTVII backbone vectors (Shi J, Kim KN, Ritz O et al. Novel protein kinases associated with calcineurin B-like calcium sensors in Arabidopsis. Plant Cell 1999; 11 (12):2393- 2405) for localization analyses, into pSPYNE and pSPYCE vectors (Walter M, Chaban C, Schutze K et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 2004; 40 (3):428-438; Waadt R, Schmidt LK, Lohse M et al. Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J 2008; 56 (3):505-5I6) for BiFC assays and for expression in oocytes cDNAs were cloned into modified pGEMHE vectors (Liman ER, Tytgat J, Hess P. Subunit stoichiometry of a mammalian K⁺ channel determined by construction of multimeric cDNAs. Neuron 1992; 9 (5): 861-871). Forward primers with inserted point mutations were used to generate cDNAs encoding the CBL4 G2A and C3S mutants . The identity of all plasmid constructs generated in this study was verified by sequencing. Arabidopsis thaliana (ecotypes Wassilewskija and Columbia) plants that were used in this work were grown on soil. In this study cbl4 and cipk6 T-DNA insertion lines from the GABI collection were investigated

GABI_015F02; cipk6: GABI_448C12). The T-DNA insertions in both mutants were localized by PCR by using T-DNA border-specific and cipk6 and cblA gene-specific primers. PCR products for both borders were sequenced. For transcript analysis 1 μg 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. Plant J 2006; 48 (6):857-872) that confirmed the previously reported salt sensitive phenotypes of published sos3 (here designated as cbl4) loss-of-function mutants (Ishitani M, Liu J, Halfter U et al. SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding. Plant Cell 2000; 12 (9): 1667-1678) and published cipk6 mutant alleles (Tripathi V, Parasuraman B, Laxmi A, Chattopadhyay D. CEPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants. Plant J 2009; 58 (5):778-790). Complementation lines ofakt2-l, cipk6 and cbU were generated through Agrobacterium mediated transformation (Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 1998; 16 (6):735-743). AKT2 and CIPK6 were expressed under endogenous promoters, CBL4 under control of the MAS promoter.

In 12h/12h photo regime, plants were grown in a growth chamber at 60% relative humidity, at 20°C during the day with a photon flux density of 100 μΕ-m V¹ (Philips TLD58W840) and at 16°C during the night. Phenotype analyses were carried out in the middle of the day period. Leaf numbers were monitored once a week starting from two weeks after sowing (2 WAS). Main inflorescence stalk height was measured every fourth day starting from stalk emergence. Nicotiana benthamiana plants were cultivated in a green house under a 12 h light/12 h dark cycle with 60% atmospheric humidity at 20°C. Yeast-two-hybrid analyses were performed as described previously (Albrecht V, Ritz O, Linder S, Harter K, Kudla J. The NAF domain defines a novel protein-protein interaction module conserved in Ca²⁺-regulated kinases. EMBO J 2001; 20 (5): 1051-1063; Kolukisaoglu ϋ, 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).

Expression in Xenopus oocytes and electrophysiological recordings

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 μηι 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 CaCl₂, 1.5 mM MgCl₂ and 10 mM HEPES-Tris (pH 7.5). BAPTA injections (50 nL of 50 mM BAPTA) were performed using a 10-15 μηι tip diameter micro-pipette.

Patch-clamp experiments (oocyte-attached configuration) were performed as described previously (Lacombe B, Pilot G, Michard E et al. A shaker-like K⁺ channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis. Plant Cell 2000; 12 (6):837-851) using bath and pipette solutions identical to above described bath solution. pClamp software version 9.0 (Axon instruments), Sigmaplot software (Jandel Scientific) and WINASCD software (G. Droogmans, Laboratory of Physiology, Leuven, Belgium) were used to perform voltage-clamp protocol application, data acquisition and data analysis.

In vitro translation, protein purification and in vitro phosphorylation assays The plVEX 1.3 WG plasmid (Roche) was used as the template for site-directed mutagenesis PCR to generate the ρΓΥΕΧ 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 μg of ρΓνΈΧ WG StrepII constructs were used for in vitro transcription/translation using RTS 500 Wheat Germ CECF Kit (Roche). Each translation reaction was incubated with Strep-Tactin Macroprep (IBA) for 30 minutes at 4^°C and the mixture was transferred to a microspm-column (Bio-Rad) for gravity-flow procedures. The column was washed with wash buffer (100 mM Tiis/HCl, pH8, 150 mM NaCl). StrepII-tagged protein was eluted with elution buffer (wash buffer containing 2.5 mM Desthiobiotin). The concentrations of purified proteins were determined by comparison with coomassie-stained BSA in SDS-PAGE.

For in vitro phosphorylation assays, 400 ng of purified proteins were incubated for 30 minutes at 30^°C in 24 μΐ reaction mixtures that contained 200 ng of each protein, 16 μg of BSA, 66.7 mM Tris/HCl (pH 8.0), 100 mM NaCl₂, 5 mM MnS0₄, 0.5 mM CaCl₂, 2 mM dithiothreitol, 0.5 μΜ ATP and 4 μθ [gamma-³²P] ATP. The reaction was stopped by adding I μΐ of 0.5 M EDTA, and the mixtures were separated by SDS-PAGE. The gel was stained with CBB and exposed to an x- ray film.

Fluorescence microscopy Transient expression of XFP-fusion proteins in N. benthamiana and bimolecular fluorescence complementation (BiFC) were previously described (Batistic O, Sorek Ν, Schultke S, Yalovsky S, Kudla J. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca²⁺ signaling complexes in Arabidopsis. Plant Cell 2008; 20 (5): 1346-1362; Walter M, Chaban C, Schutze K et al. Visualization of protem interactions in living plant cells using bimolecular fluorescence complementation. Plant J 2004; 40 (3):428-438; Waadt R, Schmidt LK, Lohse M et al. Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J 2008; 56 (3):505-516). For co- expression of the investigated proteins and co-localisation with plasma membrane and ER-markers, 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). For YFP and OFP excitation 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 Ca²⁺ 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.

Example 2: Results

CBL4 association with CIPK6 specifically facilitates Ca²⁺-dependent activation of the AKT2 K⁺ channel

To identify CBL-interacting protein kinases that potentially interact with AKT2 we performed yeast two-hybrid analyses that combined all known 26 CIPKs from Arabidopsis thaliana with a C- terminal cytoplasmic fragment of AKT2 encompassing amino acids 324 to 802. This approach identified CIPK6 as kinase specifically interacting with AKT2 (Figure 1 A). Subsequently, using yeast two hybrid assays, we analyzed which of the PM-localized CBL proteins can interact with CIPK6. These experiments revealed interaction of CIPK6 with CBL1, CBL4 and CBL9 but indicated no interaction with the closely related calcium sensor CBL5 (Figure 1 B). Specific interaction of CIPK6 but not of CIPK23 with full length AKT2 was further confirmed by in planta BiFC (Bimolecular Fluorescence Complementation) experiments in transiently transformed Nicotiana benthamiana leaves (Figure 1 C). The YFP fluorescence formed by the assembly of AKT2-YC and YN-CIPK6 is accumulated predominantly at ER-rich regions of the cell and to a significantly lesser extent at the cell periphery (Figure 7 A). These results are in line with the observation that many characterized channel subunits are retained at the ER and require association with escort proteins, protein modification or oligomerization for trafficking beyond the ER (Ma D, Jan LY. ER transport signals and trafficking of potassium channels and receptors. Curr Opin Neurobiol 2002; 12 (3):287-292; Hasdemir B, Fitzgerald DJ, Prior IA, Tepikin AV, Burgoyne RD. Traffic of Kv4 K⁺ channels mediated by KChJDPl is via a novel post-ER vesicular pathway. J Cell Biol 2005; 171 (3):459-469; Mikosch M, Kaberich K, Homann U. ER export of KAT1 is correlated to the number of acidic residues within a triacidic motif. Traffic 2009; 10 (10):1481-1487) and confirm previous AKT2 localization studies (Xicluna J, Lacombe B, Dreyer I et al. Increased functional diversity of plant K⁺ channels by preferential heteromerization of the shaker-like subunits AKT2 and KAT2. J Biol Chem 2007; 282 (l):486-494). To delineate the functional significance of the observed CBL-CIPK6 and CEPK6-AKT2 interactions we expressed different combinations of cRNAs in oocytes and recorded the resulting currents in two-electrode voltage-clamp (TEVC) experiments (Figure 1 D, Figures 7 B-E). Expression of AKT2 alone resulted in basal K⁺-currents (100 % I at -155 mV) and expression of AKT2 in combination with either CBL4 or CIP K6 did not result in significant enhancements of currents (max. 150 %). However, co-expression of AKT2 with CBL4 and CIPK6 dramatically increased the recorded currents to 380 %. Although CBL1 and CBL9 both interacted with CIPK6 (Figure 1 B), co-expression of either of these calcium sensors with CIPK6 and AKT2 did not lead to a significant increase in channel activity (Figure 1 D). Co-expression of AKT2 with CBL1 and CTPK23, that have been shown to activate the K⁺ channel AKT1 (Xu J, Li HD, Chen LQ et al A protein kinase, interacting with two calcineurin B-like proteins, regulates K⁺ transporter AKT1 in Arabidopsis. Cell 2006; 125 (7): 1347-1360) (Figure 1 E), did not significantly change AKT2 activity (Figure 1 D). Similarly, CBL4 and CTPK6 did not activate AKT1, thereby indicating the specificity of the observed AKT2 regulation (Figure 1 E, Figure 7 F). Also, co-injection of CBL4 and CIPK6 did not modulate the activity of the related shaker-type K⁺ channel KAT2 (Figure 1 E, Figure 7 G) thereby further corroborating the functional specificity of the observed AKT2 activation. To address the Ca²⁺-dependence of AKT2 activation TEVC recordings were performed either after BAPTA injection in oocytes or by replacing wild-type CBL4 with a mutated CBL4 version harboring a deletion of three amino acids in the third EF hand, (mutant allele sos3-l, here designated as CBL4AEF) that has been previously shown to impair CBL4 Ca²⁺-binding (Ishitani M, Liu J, Halfter U et al. SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding. Plant Cell 2000; 12 (9):1667-1678). Both experimental designs congruently abolished CBL4-CIPK6 mediated activation of AKT2 thereby unequivocally demonstrating the strict Ca -dependence of AKT2 activation (Figure 1 F). Taken together these results provide strong support for the conclusion that the calcium sensor CBL4 together with the protein kinase CIPK6 specifically and Ca²⁺-dependently modulate the K⁺ channel activity of AKT2. To dissect the mechanisms by which CBL4 affects AKT2 activity we next investigated whether CBL4 and CTPK6 affect the voltage gating modes of the cellular AKT2 population. Previous studies have shown that, depending on their phosphorylation status, 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). However, comparative TEVC analyses of oocytes expressing either AKT2 alone or AKT2 in combination with CBL4 and CTPK6 did not indicate. any difference in the proportion of time- dependent (gating mode#l) and instantaneous (gating mode#2) (Figure S2 A) currents and did not reveal detectable differences in the voltage-gating of the time-dependent fraction of the AKT2 current (Figure S2 B). In addition, single channel AKT2 conductance values determined from cell attached patches of oocytes clamped at -140 mV were similar in the presence or absence of CBL4- CIPK6 further excluding any influence of CBL4-CIPK6 on the permeation properties of the open AKT2 channel (Figure S2 C). These data exclude the possibility that CBL4/CIPK6 modulate AKT2 activity by affecting the voltage gating of this channel.

Loss of either CBL4 or CEPK6 function causes the same developmental phenotypes as the loss of AKT2 function

We investigated whether CBL4, CIPK6 and AKT2 function may be similarly required for distmct physiological processes in plants by comparatively characterizing a published T-DNA-induced mutant allele of AKT2 (akt2-l) (Dennison KL, Robertson WR, Lewis BD et al. Functions of AKT1 and AKT2 potassium channels determined by studies of single and double mutants of Arabidopsis. Plant Physiol 2001; 127 (3): 1012-1019; Deeken R, Geiger D, Fromm J et al. Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis. Planta 2002; 216 (2):334- 344) with newly isolated mutant alleles of CBL4 and CIPK6 (GABI 015F02 designated cbl4 and GABI_448C12 designated cipk6, respectively; Figures 9 A and 9 B). Previous studies reported delayed rosette development and flowering of the akt2-J mutant in comparison to wild-type as a consequence of impaired sugar loading and long-distance transport due to the lack of AKT2 function, which is critical for K⁺-dependent repolarization of phloem cells that is itself essential for regulating sucrose/FT symporters that drive sugar transport (Deeken R, Geiger D, Fromm J et al. Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis. Planta 2002; 216 (2):334~344), We did not observe significant differences of plant developmental parameters when mutant and corresponding wild-type plants (Ws for akt2-l and Col-0 for cbl4, cipk6) were cultivated in long day conditions. However, when grown in short days the development of all mutant lines was delayed in comparison to the respective wild-types as exemplified by a reduced rosette size and later flowering (Figure 2 A and B). Quantitative evaluation of the leaf number revealed the development of about 30 % less leaves in akt2-l, cbl4 and cipk6 mutants than in the wild-type after 6 weeks of cultivation (Figure 2 C). The development of the main inflorescence stalk was also delayed in all mutant lines. While after 54 days of cultivation Ws wild- type plants displayed an average inflorescence stalk height of about 110 mm, a stalk of only 20 mm was detectable in akt2-l plants. Similarly, after 62 days cbl4 and cipk6 mutants exhibited an inflorescence stalk height of 20 and 30 mm, respectively, compared to 75 mm in the Col-0 wild type (Figure 2 D). Importantly, these phenotypes were fully complemented to wild type values when akt2-l, cbl4 and cipk6 mutants were transformed with the respective wild type cDNAs thereby causally linking the observed phenotypes to the loss of either CBL4, CIPK6 or AKT2 function (Figure 9 C, D). These results support the notion that similar developmental phenotypes of cbl4, cipk6 and akt2-l mutant lines coincide with the observed direct functional interactions between these proteins. Importantly, the fact that the loss of either CBL4 or CIPK6 function impedes plant development and flowering similarly as the complete loss of AKT2 function underscores the importance of the regulatory function of CBL4/CIPK6 complexes for proper AKT2 function in plants.

Upon interaction with CIPK6, CBL4 mediates ER-to-PM translocation of AXT2

In order to gain further insights into the mode of CBL4/C1PK6 mediated AKT2 regulation, we evaluated whether CBL4 may influence the sub-cellular distribution of AKT2 in plant cells. To this end, we transiently expressed BiFC constructs of AKT2-YC and YN-CIPK6 together with different combinations of sub-cellular marker proteins and with or without the co-expression of CBL4-SCFP or CBL4-OFP fusion proteins in N. benthamiana leaves, and we determined the resulting fluorescence pattern indicating the localization of AKT2 (Figure 3). Co-expression of AKT2-YC and YN-CIPK6 with the ER-marker protein OFP-HDEL (Batistic O, Sorek N, Schultke S, Yalovsky S, Kudla J. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CEPK Ca²⁺ signaling complexes in Arabidopsis. Plant Cell 2008; 20 (5): 1346-1362) again revealed a predominant localization of the CIPK6-AKT2 complex at the ER as evidenced by fluorescence overlay analysis and fluorescence intensity scans of the observed yellow and red fluorescence patterns (Figure 3 A). Co-expression of the PM-marker protein CBLln-OFP, in which the first 12 N-terminal amino acids of CBL1 are fused to OFP (Batistic O, Sorek N, Schultke S, Yalovsky S, Kudla J. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca²⁺ signaling complexes in Arabidopsis. Plant Cell 2008; 20 (5): 1346-1362), with CIPK6-AKT2 further confirmed the only minor co-localization with the plasma membrane marker protein {Figure 3 B). However, upon additional co-expression of CBL4-OFP, the YFP fluorescence indicating the localization of CIPK6-AKT2 was substantially shifted to the PM (Figure 3 C). In cells, that simultaneously expressed the BiFC combination AKT2-YC+YN-CIPK6 with CBL4-SCFP and the PM-marker CBLln-OFP, this efficient PM translocation was observed in fluorescence overlay depiction as well as in fluorescence intensity scans (Figure 3 D). It is noteworthy, that cyan fluorescence emitted by CBL4-SCFP was also detected in the cytoplasm and nucleus of these cells, suggesting that only a sub-population of the cellular CBL4 protein pool was involved in translocating CIPK6-AKT2 to the PM (Figure 10 A). A microscopic analysis focused on the surface of an epidermal cell illustrated the CBL4-dependent accumulation of CIPK6-AKT2 in distinct immobile spotted structures associated with the PM (Figure 3 E). Taken together, this indicates that the calcium sensor CBL4 is required for an efficient ER-to-PM translocation of CIPK6-AKT2 in plant cells that concurs with the observed CBL4-dependent activation of AKT2 by CIPK6 in oocytes (Figurel D).

CBL4-CIPK6 complexes do not phosphorylate AKT2 in vitro

We next sought to delineate the phosphorylation of AKT2 by CBL4-CIPK6 by in vitro phosphorylation assays. In these experiments we combined recombinant CBL4 and CIPK6 or a hyperactive mutant version of CIPK6 (with a T to D exchange in the activation loop of CIPK6, noted CIPK6T182D) with either the C-terminal fragment of AKT2 or AKT2 full-length proteins (Figure 4). Control experiments in which CBL4-CIPK24 efficiently phosphorylated the C-terminus of their target protein SOS1 (Quintero FJ, Ohta M, Shi H, Zhu JK, Pardo JM. Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na⁺ homeostasis. Proc Natl Acad Sci U S A 2002; 99 (13):9061~9066; Fujii H, Zhu JK. An autophosphoiylation site of the protein kinase SOS2 is important for salt tolerance in Arabidopsis. Mol Plant 2009; 2 (1): 183-190) verified the suitability of the experimental conditions (Figure 4 A). These experiments demonstrated efficient auto-phosphorylation activity of CIPK6 that was slightly modulated by the presence of CBL4 (Figure 4 B) but was significantly enhanced as a consequence of the T182D amino acid exchange (Figure 4 D). Moreover, CIPK6 as well as CIPK6T182D phosphorylated CBL4 in these assays (Figure 4 E), thereby corifrrming the previously reported phosphorylation of CBL proteins by CIPKs (Mahajan S, Sopory SK, Tuteja N. Cloning and characterization of CBL-CIPK signalling components from a legume (Pisum sativum). FEBS J 2006; 273 (5):907-925) and clearly establishing the trans-phosphorylation ability of the recombinant kinase protein. Nevertheless, we did not obtain any evidence indicating a phosphorylation of AKT2 by C1PK6 despite extensive variations of the tested experimental conditions (Figure 4 C, D). The C-terminal regulatory domain of CBPK6 interacts with CBL4 and AKT2 and is sufficient to mediate CBL4-dependent and Ca²⁺-dependent channel modulation

These results prompted us to consider a phosphorylation-independent modulation of AKT2 by CIPK6. To distinguish between effects and interactions that solely rely on the kinase domain from kinase-independent functions of CIPK6, we generated two distinct oocyte expression constructs. These constructs harbored either only the N-terminal CIPK6 kinase domain (amino acids 1 to 278, designated CIPK6N; Figure 5 A) or, alternatively a C-terminal fragment of CIPK6 that exclusively contained the NAF domain for CBL interaction and the regulatory domain of the kinase (amino acids 274 to 442, designated CTPK6C; Figure 5 A). Remarkably, in TEVC analyses of oocytes co- expressing CIPK6C with CBL4 and AKT2, we detected currents of comparable intensity as in cells expressing AKT2 with full length CIPK6 and CBL4 (Figure 5 B). Importantly, such activation of AKT2 was not observed in cells expressing the kinase domain CIPK6N together with CBL4. Moreover, the activation of AKT2 by CIPK6C was strictly dependent on the co-expression of CBL4 (Figure 5 B).

Finally, we addressed the functionality of the kinase-deleted version CIPK6C by BiFC analysis in transiently transformed N. benthamiana leaves. BiFC complex formation after co-expression of CBL4-YC with YN-CIPK6C confirmed the ability of the kinase-deleted version of CIPK6 to still efficiently interact with the calcium sensor protein (Figure 5 C). Fluorescence microscopic analysis of tissues co-expressing AKT2-YC, YN-CIPK6C with CBL4-OFP clearly demonstrated the ability of CBL4 to mediate the ER-to-PM translocation of AKT2-CIPK6C as it was observed before for AKT2-CIPK6 (Figure 3 C). These findings assign the calcium sensor-dependent modulation of AKT2 activity to the C-terrninal regulatory domain of CIPK6, which lacks any phosphorylation activity. Moreover, injecting BAPTA or replacing the wild-type CBL4 protein by its EF-hand mutated protein version CBL4AEF (which cannot bind Ca²^) abolished activation of AKT2 currents in oocytes (Figure 5 D) and AKT2 ER-to-PM translocation in BiFC experiments (Figure 5 E, Figure 10 B). These results clearly establish the Ca^z+-dependence of CIPK6C-mediated AKT2 modulation. However, they currently do not allow us to distinguishing whether this Ca²⁺- dependence just reflects a "constitutive" structural requirement that may contribute to CBL4- CIPK6 interaction in forming an ER-to-PM escort complex or, instead, could allow specific Ca²⁺- dependent control of the traffic of AKT2 channels to the cell surface.

Dual lipid modification of CBL4 by myristoylation and palmitoylation is required for AKT2 activation and plasma membrane targeting

A recent investigation of CBL1 established the importance of myristoylation and palmitoylation (also referred to as S-acylation) of this calcium sensor for its proper function (Batistic O, Sorek N, Schultke S, Yalovsky S, Kudla J. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca²⁺ 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. Modification of CBL1 by S-acylation was identified as the key in targeting CBLl/CIPKl complexes by a novel, BFA- and Sari -independent sorting pathway to the plasma membrane (Batistic O, Sorek N, Schultke S, Yalovsky S, Kudla J. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca²⁺ signaling complexes in Arabidopsis. Plant Cell 2008; 20 (5):1346-1362). This work also established the myristoylation at the G2 position of all four PM-localized CBL proteins, including CBL4, and reported the conservation of the palmitoylated C3 position in all four PM-localized CBL proteins, including CBL4. Therefore, a similar plasma membrane targeting mechanism that relies on dual lipid modification for these proteins was proposed (Batistic O, Sorek N, Schultke S, Yalovsky S, Kudla J. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca²⁺ signaling complexes in Arabidopsis. Plant Cell 2008; 20 (5): 1346-1362). In order to address the importance of dual lipid modification for CBL4 function in AKT2 regulation and targeting, we generated non-myristoylatable G2A versions and non-pahnitoylatable C3S versions of CBL4. When wild-type CBL4 was replaced by the non-myristoylatable CBL4G2A in TEVC analysis in oocytes, we observed a complete abolishment of AKT2 activation (Figure 6 A). Likewise, injection of the non-palmitoylatable CBL4C3S together with AKT2 and CIPK6 did not result in any detectable activation of AKT2-dependent currents (Figure 6 B). In order to investigate the effect of either myristoylation or palmitoylation of CBL4 on AKT2 targeting in planta we co- expressed either CBL4G2A-SCFP or CBL4C3S-SCFP fusion proteins together with AKT2-YC, YN-CIPK6 and the CBLln plasma membrane marker in N. benthamiana cells. In contrast to our previous experiments in which co-expression of wild-type CBL4-SCFP lead to an efficient accumulation of AKT2 at the plasma membrane (Figure 3 D) neither the expression of CBL4G2A- SCFP nor the expression of CBL4C3S-SCFP was able to bring about ER-to-PM targeting of AKT2 (Figure 6 C and D). These findings indicate that efficient plasma membrane targeting of AKT2 requires dual lipid modification of CBL4 by myristoylation and palmitoylation and suggest that this translocation occurs via the novel BFA- and Sari -independent targeting pathway that was established for CBL1.

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. For generation of the vector, a multiple cloning site (MCS) followed by the terminator of the Nos gene (NosT) was cloned into the pUC18 plasmid. Subsequently, one of three different promoters was introduced into the MCS. 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. In the following, 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. For that, 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. For each of the seven possible combinations of the transgenes AtCBL4, AtCIPK6 and AtAKT2 at least one independent transgenic line could be identified (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

Of these lines, the following 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

With the chosen lines, assays regarding their salt tolerance and their dependence on K⁺ supply were performed. As the Tl generation includes, besides a heteroThomozygous genotype for the transgene(s), the wild type genotype, a kanamycin-selection step had to be included in the assays. Salt stress

Salt stress assay was performed using the hydroponic growing system araponics (Araponics SA) with ½ MURASHIGE & SKOOG medium. First, 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. After successful selection and the removal of non-resistant transgenic seedlings, 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 (½ MS) and to salt stress condition (½ MS + 150 mM NaCl).

On 13d of stress treatment, fresh weight of shoot and root and root length was measured, and photographs were taken (Figure 13 and 14). This revealed for the transgenic line 22, which comprises AtCIPK6— an slightly enhanced salt stress tolerance in comparison to wild type under the tested condition, whereas the transgenic lines 28 and 38, with all three transgenes identified, showed a much better performance. These data show that to triple transformants are clearly better performing. Thus, by overexpression combinations of the various polypeptides referred to herein, a synergistic effect can be achieved.

Dependence on K⁺ supply

To test for dependence on K⁺ availability, first, transgenic lines were selected on ½ MS + kanamycin medium, while wild type seeds were grown without kanamycin. Nine-day-old seedlings were then transferred to vertical agar plates with ½ MS (control) or ½ MS with only 10 μΜ K⁺ supplemented (10 μΜ K7). After growth for 7 days, photographs were taken and fresh weight of the shoot was measured (Figure 15 and 16).

Under non-stressed conditions with sufficient K⁺ (control), most transgenic lines showed a slightly enhanced shoot fresh weight in comparison to the wild type under tested conditions (Figure 15A left), whereas there is even a higher difference to the wild type fresh weight under K⁺ limiting conditions for all tested lines (Figure 15A right and 15B). Example 5: Generation of transgenic tobacco plants overexpressing kinase inactivated CIPK6

The protein encoding regions of Arabidopsis thaliana CBL4, CEPK6 and AKT2 were cloned into vectors based on the pUC18 plasmid. For generation of the vector, a multiple cloning site (MCS) followed by the terminator of the Nos gene (NosT) was cloned into the pUC18 plasmid. Subsequently, 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).

To generate an enzymatically inactive CEPK6 protein, 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.

In the following, the here described vectors are referred to 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. For that, 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 wildtype gDNA as negative control. For each of the possible combinations of the transgenes AtCBL4, AtCIPK6_D164N and AtAKT2 at least one independent transgenic line could be identified. For the CEPK6_D164N approach, of the 46 Tl lines analyzed by PCR, for 13 lines no transgene fragment could be amplified, whereas the PCRs of 33 lines resulted in the amplification of different combinations of the transgene fragments. Thus, 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

A Blast search for variants of the Arabidopsis thaliana CBL4, CIPK6 and AKT2 polypeptide was carried out. 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.

Qryza sativa CBL04 48843832 AAT47091.1 64,00%

Table B: Variants of the Arabido sis thaliana CIPK6 ol e tide

Table C: Variants of the Arabidopsis thaliana AKT2 polypeptide

Species NCBI-ID Accession Idcut it iy

Arabidopsis lyrata 297799798 XP 002867783.1 95,00%

Vitis vinifera 298204496 CBI23771.3 73,00%

Vitis vinifera 225447945 XP 002268924.1 70,00%

Ricinus communis AKT02 255577304 XP 002529533.1 68,00%

Populus euphratica 171904010 ACB56631.1 68,00%

Samanea saman 4323296 AAD16278.1 68,00% opulus tremula x Populus

tremuloides 9955730 CAC05489.1 67,00%

Vicia faba 2293112 CAA71598.1 67,00%

Nicotiana paniculata 5834502 BAA84085.1 64,00%

Claims

1. A method for producing a transgenic plant or a transgenic plant cell having increased potassium efficiency 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.

2. The method of claim 1 further comprising introducing into said plant or said plant cell a CIPK6 (CBL-interacting protein kinase 6) polypeptide.

3. The method of claims 1 or 2 further comprising introducing into said transgenic plant or said plant cell an AKT2 (Arabidopsis K+ transporter 2) polypeptide.

4. The method of any one of claims 2 to 3 further comprising introducing into said transgenic plant or said plant cell a C1PK24 (CBL-interacting protein kinase 24) polypeptide and a SOS1 (Salt Overly Sensitive 1) polypeptide.

5. The method of any one of claims 2 to 4, wherein said CEPK6 polypeptide does not have kinase activity.

6. The method of any one of claims 1 to 5, wherein said generated transgenic plant or plant cell further has increased salt tolerance as compared to a corresponding non-transgenic plant or plant cell.

7. The method of any one of claims 1 to 6, wherein said method comprises the steps of:

a. introducing at least one heterologous polynucleotide encoding the said polypeptides into the plant or plant cell; and

b. expressing said polypeptides from the said at least one polynucleotide.

8. A transgenic plant or plant cell, comprising a CBL4 and CIPK6 polypeptide.

9. The transgenic plant or plant cell of claim 8 further comprising polypeptide(s) selected from a) an AKT2 polypeptide, b) a C1PK24 and a SOS1 polypeptide and c) an AKT2 polypeptide, a CIPK24 and a SO SI polypeptide

10. The transgenic plant or plant or cell of claims 7 and 8, wherein said polypeptides are expressed from heterologous polynucleotides.

11. A composition comprising a CBL4 and CIPK6 polypeptide.

12. The composition of claim 11, further comprising polypeptide(s) selected from a) an AKT2 polypeptide, b) a CIPK24 and a SOSl polypeptide and c) an AKT2 polypeptide, a CIPK24 and a SOSl polypeptide.

13. A composition comprising a polynucleotide encoding for a CBL4 and a polynucleotide encoding for a CTPK6 polypeptide.

14. The composition of any one of claims 11 to 13, wherein said CIPK6 polypeptide does not have kinase activity.

15. Use of a CBL4 and a CIPK6 polypeptide for generating a transgenic plant or plant cell having increased potassium efficiency and/or increased salt tolerance compared to a corresponding non-transgenic plant or plant cell.