WO1999031259A1 - Transgenic plants with an altered potassium metabolism - Google Patents

Abstract

Described are transgenic plants displaying an altered K+ metabolism due to the reduction of the activity of at least one potassium transporter of the AtKT family and/or of at least one inwardly rectifying potassium channel. Such plants show, for example, a reduced apical dominance.

Description

Transgenic plants with an altered potassium metabolism

The present invention relates to transgenic plants displaying an altered potassium metabolism due to the increase or the reduction of the activity of at least one potassium transporter of the AtKT family and/or of at least one type of inwardly rectifying potassium channels in comparison to non-transformed plants. Such plants show morphological alterations, such as, for example, a reduced apical dominance.

Potassium is one of the major nutrients involved in inorganic plant nutrition. Potassium is the most abundant cation found in all plant tissues including plant organelles in relatively large amounts (0.05 to 0.1 M; Leigh and Jones, New Phytol. 97 (1984), 1-13). Potassium is required in amounts similar to or even greater than nitrogen (N). Potassium plays an important role in a large number of processes, e.g. turgor controlled processes such as cell extension (coleoptile, root and shoot elongation, cotton seed fiber elongation), seed germination and stomatal movement or the regulation of the activity of many enzymes that participate in photosynthesis, respiration, water uptake, water relations, meristematic growth, long distance transport through phloem and xylem or in other processes and which require potassium for their activity (Bhandal and Malik, Int. Rev. Cytol. 110 (1988), 205-254). Potassium fertilization was observed to increase photosynthesis, transport of photosynthates, and root growth. A reduction in potassium concentration causes a decrease in the rates of biochemical processes and, thus, a decline in growth. Adequate potassium nutrition results in healthy plants with thickened cell walls providing tissue stability and improvement of the resistance of crops to lodging, pests, and diseases (Beringer and Nothdruft, In Manson (ed.) Potassium in Agriculture. American Society of Agronomy, Madison, Wl (1985), 352-363). Potassium plays an important role in maintaining the tone, vigor, and efficiency of the potato plant (Pushkamath, (1976), Orient Longman, New Dehli, India). To perform these multiple roles potassium interacts with other plant metabolites (essential macronutrients, secondary nutrients, and micronutrients) in growth processes. These interactions either restrict or increase nutrient uptake, transport and utilization. Potassium plays an important role in the uptake of certain nutrients from the soil through the roots and thereby in ensuring efficient utilization of N and the KxN interaction. Efficient farming needs an integrated management of N and potassium. In case large quantities of N fertilizers are used under intensive cropping, increased uptake of N and potassium typically results in a depletion of soil potassium. Potassium deficiency results in chlorosis and necrosis and accumulation of uncombined amino acids and NH₄ in leaves. This, for example, can lead to an accumulation of NH₄ and subsequently in uncoupling photosynthetic phosphorylation. Potassium also affects the uptake of phosphorus, calcium, magnesium, sulphur, molybdenum, manganese, copper, iron, boron, zinc and sodium. Application of potassium to rice grown on neutral or acidic soils for example decreased iron toxicity (Tanaka and Tadano, Potash Rev. Sub. 9 (1972), Suit 21 , 1 to 12). The deficiency in potassium has been found to lead to the accumulation of soluble carbohydrates, reducing sugars, amino acids, amides, and soluble organic nitrogen as well as to an increase of certain enzyme activities like invertase and diastase. Furthermore, a deficiency in potassium leads to a decrease of starch content, inulin, levulin, pyruvic kinase and formate activating enzyme as well as to chlorosis and necrosis of leaves. Due to the constant increase in the world population it is one of the tasks of biotechnology to contribute to the improvement of the supply of mankind with sufficient nutrition. Sustained agriculture is one of the important tasks for the future in order to reduce the amounts of fertilizers and agrochemicals to be used and, at the same time, to increase the production rate. As areas to be used for agriculture can not be increased due to ecological and geomorphological factors, the yield obtainable per area has to be increased. This ambitious goal can only be achieved by selected breeding or by genetic engineering of traits important for agricultural productivity. It is, for example, possible to reduce losses in yield by enhancing pest resistance or resistance to other environmental stress factors, such as aridity, heat, cold, wind, etc. Furthermore, it is possible to increase the yield by increasing photosynthesis rate of plants, by improving the supply of the plants with nutrients or the like. Even though there has been some progress in recent years in providing transgenic plants with altered phenotypes resulting in an increased yield or higher resistance to stress factors, there is still a need for general approaches for obtaining plants with improved phenotypic properties which are advantageous under certain environmental conditions.

Thus the technical problem underlying the present invention is the provision of transgenic plants having improved properties, such as an altered morphology insofar as they show an increased mechanical strength and more shoots resulting in a higher yield concerning fruits and seeds.

This technical problem is solved by the provision of the embodiments as characterized in the claims.

Accordingly, the present invention relates to transgenic plants with an altered potassium metabolism in comparison to nontransformed plants, due to the increase or the reduction of the activity of at least one type of plant potassium transporters of the AtKT family and/or of at least one type of plant inwardly rectifying potassium channels (K_in ⁺ channels) belonging to a family of plant potassium channels selected from the group consisting of the KAT1 family, the AKT1 family and the AtKC1 family.

It has been surprisingly found that reduction of at least one type of the above mentioned potassium transporters or K_in ⁺ channels in transgenic plants leads to a drastic change in plant morphology insofar as these plants show a reduction of apical dominance or even a loss of apical dominance (see Fig. 1). These plants show in addition a reduction in K⁺ uptake as demonstrated by LAMMA analysis (see Figure 2). Accordingly, these plants show an overall reduced or stunted growth. Such a controlled growth behavior might be advantageous in cases where a high robustness of the plants is required, for example, in areas with strong winds and storms. Alternatively, overexpression of one type of potassium transporters as defined above or of K_in ⁺ channels apparently leads to improved characteristics such as stronger stems and higher photosynthetic rates. Thus, yield characters important for agriculture can be improved. The transport of potassium across plant membranes is accomplished by two different transport systems. One system consists of transporters for K⁺, and the other system consists of K⁺ channels. Transporters have a low affinity for K⁺ and comprise the AtKT proteins (Quintero & Blatt, FEBS Letters 415 (1997), 206-211 ). These transporters belong to a group of genes with high degree of homology at the amino acid level (more than 50% identity) and are predicted to contain 12 transmembrane spanning domains.

K⁺ channels have a low affinity for K⁺ (above 0.5 mM), a linear kinetic response and allow very rapid transport (transport rate 10⁶ ions/channel and second). There are several structurally related groups of channels: (1 ) proteins with six transmembrane helices and a presumed conducting pore (generally named the "shaker" family); (2) proteins with two transmembrane helices and a pore region, and (3) proteins with four transmembrane helices and two pore regions (for review of structurally properties see Jan and Jan, Ann. Rev. Neurosci. 20 (1997) 91 to 123). Several types of genes have been cloned from plants that encode proteins with some structural homology to class (1 ) and (3) (see Anderson et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3736-3740; Sentenac et al., Science (1992) 256, 663-665; Mϋller-Rober et al., EMBO J. 14 (1995), 2409-2416; Nakamura et al., Plant Physiol. 109 (1995), 371-374; Cao et al., Plant Physiol. 109 (1995), 1093-1106; Czempinski et al. EMBO J. (1997) 16, 2565- 2575).

in the scope of the present invention the term "potassium transporter of the AtKT family" means a protein with structural and functional homology to the potassium transporters from Schanniomyces occidentalis (Banuelos et al., EMBO J. 14 (1995), 3021-3027) and from E. coli (Schleyer and Bakker, J. Bacteriol. 175 (1995), 6925- 6931). Such transporters are supposed to represent low-affinity transporters for potassium. Preferably, such a protein is a plant protein.

Structural homology means that the nucleotide sequences encoding such channels show preferably a sequence identity to the DNA sequences encoding the above- mentioned transporters of at least 40%, more preferably of at least 50% and more preferably of at least 60%. Functional homology means that the transporter is a potassium transporter with low-affinity for potassium. In the scope of the present invention the term "inwardly rectifying potassium channel (K_ιn ⁺)" means voltage-dependent potassium channels of plants which are activated by membrane hyperpolarization. Such channels are assumed to provide a pathway for low-affinity K⁺ uptake in plant cells. These channels, in particular, comprise those of the KAT1 , the AKT1 and the AtKC1 family.

The term "KAT1 family" in the scope of the present invention relates to K_ιn ⁺ channels of plants showing structural and functional homology to the gene product of the KAT1 gene of Arabidopsis thaliana (Anderson et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3736-3740; GenBank accession numbers X93022 and U25088). In this regard, structural homology means that the nucleotide sequences encoding such channels show preferably a homology, i.e. sequence identity, to the KAT1 gene of at least 40%, more preferably of at least 50% and even more preferably of at least 60%. The term "structural homology" in this respect also means that such channels preferably comprise certain structural elements present in the KAT1 gene product, for example, a cluster of six putative membrane-spanning helices (S1 to S6) at the amino-terminus, a presumed voltage-sensing region containing Arg/ Lys- Xaa-Xaa-Arg/Lys repeats within S4 and a highly conserved pore-forming region (known as H5 or SS1-SS2). Examples of members of the KAT1 family are the products of the KST1 gene of Solanum tuberosum (Mϋller-Rober et al., EMBO J. 14 (1995), 2409-2416) and of the KAT2 cDNA (GenBank accession number U25694). Preferably, a K_lπ ⁺ channel of the KAT1 family has a molecular weight of about 70 to 90 kDa, and most preferably of about 75 to 85 kDa when calculated on the basis of the encoded amino acid sequence.

The term "AKT1 family" in the scope of the present invention means K_ιn ⁺ channels of plants showing structural and functional homology to the gene product of the AKT1 gene of Arabidopsis thaliana (Sentenac et al., Science 256 (1992), 663-665; GenBank accession Numbers X62907 and U06745). In this regard, structural homology means that the nucleotide sequences encoding such channels show preferably a sequence identity to the AKT1 gene of at least 40%, more preferably of at least 50% and even more preferably of at least 60%. Furthermore, structural homology means that such channels preferably also comprise certain structural elements present in the AKT1 gene product, for example, a cluster of six putative membrane-spanning helicles (S1 to S6) at the amino-terminus, a presumed voltage- sensing S4 region, a highly conserved pore-forming region (known as H5 or SS1 - SS2) and a conserved ankyrin-binding domain within the C-terminus. Preferably, a K_in ⁺ channel of the AKT1 family has a molecular weight of about 80 to 110 kDa, more preferably of about 90 to 100 kDa and most preferably of about 95 to 96 kDa when calculated on the basis of the encoded amino acid sequence. Examples of plant K_ln ⁺ channels belonging to the AKT1 family are the products of the SKT1 gene from Solanum tuberosum cloned from a leaf library (GenBank accession No. X86021 ) and of the AKT3 cDNA (GenBank accession numbers U44745 and U44744).

The term "AtKC1 family" in the scope of the present invention means K_in ⁺ channels of plants showing structural and functional homology to the gene product of the AtKC1 gene of Arabidopsis thaliana (Seq ID No. 1 ; GenBank accession numbers U812398 and U73325). In this regard, structural homology means that the nucleotide sequences encoding such channels show preferably a sequence identity to the AtKC1 gene of at least 40%, more preferably of at least 50% and even more preferred of at least 60%. Structural homology preferably also means that such channels comprise certain structural elements present in the AtKC1 gene product, for example, a cluster of six putative membrane-spanning helicles (S1 to S6) at the amino terminus, a presumed voltage-sensing S4 region and a highly conserved pore-forming region (known as H5 or SS1 - SS2). Such transporters of the AtKC1 family appear to be located in the plastids/chloroplast of plant cells.

According to the invention the reduction of the activity of at least one type of potassium transporters of the AtKT family and/or of at least one K_in ⁺ channel in the transgenic plants may be achieved by any measure that is suitable to either inhibit or suppress the expression of endogenous genes encoding such transporters or channels, to inhibit or suppress the expression of mRNA encoding such transporters or channels or to inhibit or suppress the functionality of such transporter or channel proteins present in the plant cells. In a preferred embodiment the activity of at least one type of potassium transporters of the AtKT family and/or of at least one K_ιn ⁺ channel in the plant is reduced by at least one of the measures selected from the group consisting of:

(a) disruption of (an) endogenous gene(s) encoding a potassium transporter of the AtKT family or a K_in ⁺ channel of the KAT1 , AKT1 or AtKC1 family or its regulatory sequences;

(b) expression of at least one antisense RNA directed against transcripts of genes encoding a potassium transporter of the AtKT family or a plant K_ιn ⁺ channel of the KAT1 , AKT1 and/or AtKC1 family;

(c) expression of at least one cosuppression RNA for a potassium transporter of the AtKT family or for a plant Kj _n ⁺ channel of the KAT1 , AKT1 and/or AtKC1 family;

(d) expression of at least one ribozyme that specifically cleaves transcripts of genes encoding a potassium transporter of the AtKT family or a plant K_ιn ⁺ channel of the KAT1 , AKT1 and/or AtKC1 family; and

(e) expression of at least one defect form of a potassium transporter of the AtKT family or of a plant K_ιn ⁺ channel of the KAT1 , AKT1 and/or AtKC1 family.

The disruption of genes encoding a potassium transporter of the AtKT family or a K_in ⁺ channel can, for example, be achieved by T-DNA or transposon mutagenesis. These techniques are known in the art and are described, for instance, in Aarts et al. (Mol. Gen. Genet. 247 (1995), 555-564), Azpiroz-Leehan et al. (Trends in Genet. 13 (1997), 152-156) and Osborne et al. (Current Opinion in Cell Biol. 7 (1995), 406- 413).

The approach using antisense-RNA is already well known in the art. In order to express an antisense-RNA, on the one hand DNA molecules can be used which comprise the complete sequence encoding the corresponding protein, including possibly existing flanking sequences as well as DNA molecules, which only comprise parts of the encoding sequence whereby these parts have to be long enough in order to prompt an antisense-effect within the cells. Basically, sequences with a minimum length of 15 nucleotides, preferably with a length of 100-500 nucleotides and for an efficient antisense-inhibition, in particular sequences with a length of more than 500 nucleotides may be used. Generally DNA-molecules are used which are shorter than 5000 nucleotides, preferably sequences with a length of less than 2500 nucleotides.

Use may also be made of DNA sequences which are highly homologous, but not completely identical to the sequences encoding the protein the activity of which should be reduced. The minimal homology should be more than about 65%. Preferably, use should be made of sequences with homologies between 95 and 100%.

The technique of expressing antisense RNA in plant cells is well known to the person skilled in the art and has already been described in detail. In principle, a DNA molecule as described above is linked in antisense-orientation to sequences ensuring transcription in plant cells. Upon transcription such a construct leads to the synthesis of an RNA molecule which is complementary to the normally occurring mRNA and interferes with the expression of said RNA.

The method of expressing a so-called "cosuppression RNA", i.e. a sense-RNA which leads by a cosuppression effect to the reduction of expression of a corresponding endogenous gene, is also well known in the art and is described, for example, in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 43-46), Palaqui and Vaucheret (Plant Mol. Biol. 29 (1995), 149-159), Vaucheret et al. (Mol. Gen. Genet. 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621) and other sources.

A further approach is the expression of a ribozyme which specifically cleaves transcripts encoding a potassium transporter of the AtKT family or a K| _n ⁺ channel. This method is also well known to the person skilled in the art and is described, for instance, in EP-B1 0 321 201. The expression of ribozymes in plant cells is described in Feyter et al. (Mol. Gen. Genet. 25 (1996), 329-338).

The expression of an antisense RNA, a cosuppression RNA or a ribozyme or the disruption of the endogenous genes encoding the above described potassium transporters or channels will lead to the reduction in the amount of transcripts which encode such transporters or channels in the cells of the plant. Preferably, the amount of these transcripts is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 60% and particularly preferred by at least 90% in comparison to the amount of such transcripts in cells of corresponding non- transformed plants. The amount of the transcripts can be determined, for example, by Northern Blot analysis. The cells of the described transgenic plants show furthermore preferably a reduction in the amount of at least one of the above- mentioned potassium transporters or channels in comparison to cells of corresponding non-transformed plants. A reduction preferably means a reduction of at least 10%, more preferably of at least 30%, even more preferably of at least 60% and particularly preferred of at least 90%. The amount of the protein can be detected, e.g., by Western Blot analysis.

The expression of a defect form of one of the above-mentioned potassium channels or transporters can comprise, e.g., the alteration of the coding sequences encoding such transporters or channels so as to lead to the expression of a protein which is no longer capable to fold in the correct confirmation, to integrate into the membrane or which is no longer capable to shuttle K⁺ ions over membranes. Such alterations might be, for example, truncations, deletion or addition of amino acid residues or the replacement of amino acid residues which are crucial for the transport of K⁺ ions over membranes. Such defect forms in particular also comprise transporters or channels with an altered amino acid sequence which leads to a reduction or loss of function.

The reduction of the activity of the above-mentioned potassium transporters or channels can be determined, for instance, by the LAMMA method described, for example, by lancu et al. (Biometals 9 (1996), 57-65). In particular, it is possible with this method to measure the capacity of plant tissue to take up potassium. The described transgenic plants show preferably a reduction of the capacity to take up potassium of at least 5%, more preferably of at least 10% and even more preferably of at least 20% in comparison to corresponding non-transformed plants. The increase of the activity of at least one type of potassium transporter of the AtKT family or of at least one K_in ⁺ channel in the transgenic plants according to the invention may be achieved according to methods well known in the art which lead to an overexpression of a desired protein in plant cells or to an increase in activity of a desired protein.

In order to achieve overexpression of a desired protein in plant cells a DNA sequence encoding the desired protein is linked in sense orientation to DNA sequences which allow for transcription and translation in plant cells. Such DNA sequences are known and available and are described in more detail further below. The level of the increase of the desired protein by overexpressing a corresponding DNA sequence can be varied, for example, by the DNA sequences used for the regulation of transcription and translation, i.e. by the strength of promoters, enhancers or translation initiation sequences. It can also be varied by the number of copies introduced into the plant cells.

Furthermore, an increase in the activity of at least one of the above-mentioned transporters or channels can be achieved by introducing into plant cells DNA sequences encoding potassium transporters or channels as defined above which have a higher activity, i.e. which are capable to transport more potassium ions over a membrane, than the potassium transporters or channels which occur naturally in such plant cells. In particular, an increase of the activity of at least one of the above- mentioned potassium transporters or channels can be achieved by the introduction of DNA sequences encoding a mutant of a potassium transporter or channel which has an alteration in the amino acid sequence resulting in a higher activity (gain of function mutations).

Preferably, the cells of the plant overexpressing a potassium transporter or channel as defined above show an increase of the amount of transcripts encoding such a transporter or channel in comparison to cells of corresponding non-transformed plants. The increase is preferably at least 5%, more preferably at least 10% and even more preferably at least 20%. Furthermore, the cells of this plants show preferably a corresponding increase in the amount of the encoded transporter or channel protein or in the capacity to take up potassium. Transgenic plants showing increased activity of at least one of the above mentioned potassium transporters or channels preferably display at least one of the following features: (a) an improved uptake of ammonium and/or potassium;

(b) altered morphology;

(c) increased mechanical strength;

(d) improved production of photosynthates;

(e) increased consumption of CO₂;

(f) less shoots; and

(g) higher yield concerning fruits and seeds.

In a preferred embodiment the potassium transporter of the AtKT family or the K_in ⁺ channel, the activity of which is reduced or increased in the plant according to the invention, is encoded by a nucleic acid molecule selected from the group consisting of:

(a) nucleic acid molecules encoding the amino acid sequence of an AtKT transporter as encoded by a DNA sequence selected from the group consisting of:

AtKT1 Accession number AF012656; AtKT2 Accession number AF012657; AtKT3 Accession number AF012653; AtKT4 Accession number AF012659; AtKT5 Accession number AF012660; and

AtKUPI Accession number AF033118;

(b) nucleic acid molecules encoding the amino acid sequence of a K_jn ⁺ channel as encoded by a DNA sequence selected from the group consisting of: KAT1 Accession numbers X93022 and U25088;

KAT2 Accession number U25694 and Figure 5; AKT1 Accession numbers X62907 and U06745; SKT1 Accession number X86021; AKT3 Accession numbers U44745 and U44744; and AtKC1 Accession numbers U812398andU73325;

(c) nucleic acid molecules the complementary strand of which hybridizes to a nucleic acid molecule of (a) or (b) and which encode a potassium transporter of the AtKT family or a plant K_in ⁺ channel; and (d) nucleic acid molecules the sequence of which differs from the sequence of a molecule as define in (c) due to the degeneracy of the genetic code. The indicated accession numbers relate to the NCBI gene bank.

In a particularly preferred embodiment, the K_in ⁺ channel the activity of which is increased or reduced in a transgenic plant according to the invention is a protein comprising the amino acid sequence as encoded by a nucleic acid molecule selected from the group consisting of:

(a) nucleic acid molecules encoding the amino acid sequence depicted in Seq ID No. 2;

(b) nucleic acid molecules comprising the coding region depicted in Seq ID No.

1 ;

(c) nucleic acid molecules the complementary strand of which hybridizes to a nucleic acid molecule of (a) or (b) and which encode a plant K_in ⁺ channel; and

(d) nucleic acid molecules the sequence of which differs from the sequence of a molecule of (c) due to the degeneracy of the genetic code.

As is evident from the Examples of the present invention, reduction of the activity of the AtKC1 channel either by transposon mutagenesis or by expression of a corresponding antisense RNA leads to a drastic phenotypic effect insofar as the transgenic plants show, for example, a lack of apical dominance (see Fig. 1 ).

Nucleic acid molecules as defined under (c) above which hybridize to the specifically mentioned sequences of the potassium transporter and channel genes may be derived from any plant species comprising such sequences. They can be isolated by methods well known in the art, e.g. by screening cDNA or genomic libraries with appropriate probes or by PCR with suitable primers. "Hybridizing" in the scope of the present invention preferably means hybridizing under stringent conditions as described, for example, in Sambrook et al. (A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), Cold Spring Harbor, NY). More preferably, "hybridizing" means that such sequences show at least 60% sequence identity, most preferably at least 70% sequence identity to the mentioned sequences.

Thus, the above mentioned sequences can be used to isolate homologous sequences from other plants or plant species. It is also possible to use fragments, e.g. synthetically synthesized fragment, of these molecules as hybridization probes. The DNA molecules identified by hybridization with such probes must then be sequences and it must be determined whether the encoded protein is a desired potassium transporter of the AtKT family or a K_in ⁺ channel.

In order to express the nucleic acid molecules of the invention in sense- or antisense-orientation in plant cells, these are linked to regulatory DNA elements which ensure the transcription in plant cells. Such regulatory DNA elements are particularly promoters. Basically any promoter which is active in plant cells may be used for the expression. The promoter may be selected in such a way that the expression takes place constitutively or in a certain tissue, at a certain point of time of the plant development or at a point of time determined by external circumstances. With respect to the plant the promoter may be homologous or heterologous. Suitable promoters for a constitutive expression are, e.g. the 35S RNA promoter of the Cauliflower Mosaic Virus and the ubiquitin promoter from maize. For a tuber-specific expression in potatoes the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) or a promoter which ensures expression only in photosynthetically active tissues, e.g. the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451 ) may be used. For an endosperm-specific expression the HMG promoter from wheat, the USP promoter, the phaseolin promoter or promoters from zein genes from maize are suitable. Furthermore, a termination sequence may exist which serves to correctly end the transcription and to add a poly-A-tail to the transcript which is believed to stabilize the transcripts. Such elements are described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged as desired.

In a preferred embodiment the plant according to the inventions displays an altered morphology. In an even preferred embodiment the transgenic plant according to the invention shows a reduction in activity of at least one potassium transporter of the AtKT family or of at least on K_in ⁺ channel and a reduced apical dominance and more preferably a complete loss of apical dominance. In this context a reduction in apical dominance means preferably an increase in branching, bud formation and/or side shoot formation. Preferably, such plants have at least 5%, more preferably at least 10% and even more preferably at least 20% more branch points, side shoots and/or buds than corresponding non-transformed plants.

In a further preferred embodiment the transgenic plant according to the invention shows at least one of the following features:

(a) the capability to take up larger amounts of potassium or ammonium in comparison to untransformed plants;

(b) increased mechanical strength;

(c) improved production of photosynthates;

(d) increased consumption of CO₂;

(e) less or more shoots;

(f) an increased yield in comparison to untransformed plants; and

(g) the capability to grow on acid soils.

In this context an increased yield means an increase of at least 10% of biomass (determined as fresh weight) in comparison to untransformed plants. The capability to take up larger amounts of potassium or ammonium means, that these plants can take up at least 10% more, more preferably at least 20% more of these ions in comparison to untransformed plants.

The transgenic plants according to the invention can belong to any known plant family and to any plant species. They can be monocotyledonous or dicotyledonous plants, preferably useful plants, i.e. plants cultivated by mankind, for example for industrial, agricultural, horticultural, forestry or nutritional purposes. Examples are graninaceous monocotyledonous plants, preferably those of the genus selected from the group consisting of Lolium, Zea, Triticum, Sorghum, Saccharum, Bromus, Oryza, Hordeum, Secale and Setaria. Particularly preferred are cereals, such as rye, barley, oats, wheat, rice, maize, millet etc. Of relevance are also other plants of agricultural interest, such as tobacco, cotton, carrot, sunflower, sugar beet, ground nut, coconut palm, soy bean, sugar cane, oilseed rape, potatoes, cassava, peas, etc.

Furthermore, the present invention also relates to propagation material and propagules of plants according to the invention which comprises transgenic plant cells having a genetically engineered genomic alteration resulting in the increase or in the reduction of the activity of at least one potassium transporter of the AtKT family or of at least one K_in ⁺ channel. The term "propagation material" includes, for example, fruits, seeds, tubers, rootstocks, seedlings, cuttings, calii, protoplasts, cell cultures etc. The term "propagule" means any structure that functions in propagation and dispersal (e.g. spore, seed, pollen etc.).

The present invention also relates to transgenic plant cells which have a genetically engineered genomic alteration resulting in the increase or in the reduction of the activity of at least one potassium transporter of the AtKT family and/or of at least on K_in ⁺ channel. Such transgenic plant cells differ from corresponding nontransformed plant cells by a genomic alteration which normally does not occur in nontransformed cells. In the case that the activity of one of the above-mentioned proteins is reduced, such cells have, e.g. a genomic alteration insofar as an endogenous gene encoding such a protein or its regulatory sequence is disrupted, or they comprise stably integrated in their genome DNA sequences encoding corresponding antisense RNA, ribozymes, cosuppression RNA or an RNA encoding for a defect form of the protein. Such cells show preferably a reduction in the amount of the corresponding transcript encoding the transporter or channel of at least 10%, more preferably of at least 30%, even more preferably of at least 60% and particularly preferred of at least 90% in comparison to corresponding non-transformed plant cells. Furthermore, these cells have a reduced amount of the potassium transporter or channel protein. Preferably the reduction of the amount of the protein is at least 10%, more preferably at least 30%, even more preferably at least 60% and particularly preferred at least 90% in comparison to corresponding non-transformed cells. Moreover, these cells show preferably a reduction in the capacity to take up potassium. This capacity is preferably reduced by at least 5%, more preferably by at least 10% and even more preferably by at least 20%. In the case that the activity of at least one of the above-mentioned proteins is increased, the cells contain, for example, in their genomes at least one sequence coding for one of the above- defined transporter or channels not naturally occurring in such cells or they contain in their genomes additional copies encoding such proteins which are preferably integrated in the genome at a location where they do normally not occur. Preferably these cells overexpressing a potassium transporter or channel as defined above show an increase of the amount of transcripts encoding such a transporter or channel in comparison to cells of corresponding non-transformed plants. The increase is preferably at least 5%, more preferably at least 10% and even more preferably at least 20%. Furthermore, these cells show preferably a corresponding increase in the amount of the encoded transporter or channel protein or in the capacity to take up potassium.

Moreover, the present invention relates to the use of nucleic acid molecules encoding a potassium transporter of the AtKC family or a plant K, _n ⁺ channel for the generation of plants displaying reduced apical dominance. Such molecules are preferably those molecules described in more detail above.

Furthermore, the present invention relates to the use of nucleic acid molecules encoding a potassium transporter of the AtKT family or a K_jn ⁺ channel for the generation of plants which show at least one of the following feature:

(a) the capability to take up larger amounts of potassium or ammonium in comparison to untransformed plants;

(b) increased mechanical strength;

(c) improved production of photosynthates;

(d) increased consumption of CO₂;

(e) less or more shoots;

(f) an increased yield in comparison to untransformed plants; and

(g) the capability to grow on acid soils.

The present invention furthermore relates to recombinant DNA molecules comprising a promoter allowing transcription in plant cells and linked thereto in sense orientation a DNA sequence encoding a potassium transporter or a potassium channel as defined above.

The present invention also relates to recombinant DNA molecules comprising a promoter allowing transcription in plant cells and a DNA sequence linked thereto wherein this DNA sequence encodes an antisense RNA, a ribozyme, a cosuppression RNA or a defect form of the potassium transporter or channel as defined in detail above. Furthermore, the present invention relates to plant cells transformed with a recombinant DNA molecule according to the invention.

In order to introduce the nucleic acid molecules according to the invention into plant cells, a wide range of techniques is at disposal. These techniques comprise the transformation of plant cells with T-DNA by using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation medium, the fusion of protoplasts, the injection of nucleic acid, the electroporation of DNA, the integration of DNA by means of the biolistic method as well as further possibilities. The use of T-DNA for the transformation of plant cells is widely used and is described in detail for example in EP 120 516, in Hoekema (In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V); in Fraley et al. (Crit. Rev. Plant. Sci. 4, 1-46) and in An et al. (EMBO J. 4 (1985), 277-287). Other possibilities in order to introduce foreign DNA into plant cells, like the biolistic method or the transformation of protoplasts are also widely used and sufficiently described.

Systems for the transformation of monocotyledonous plants are, for example, the transformation by means of a biolistic approach, the electrically or chemically induced DNA integration in protoplasts, the electroporation of partially permeabilized cells, the macro-injection of DNA into inflorescences, the micro- injection of DNA into microspores and pro-embryos, the DNA integration by sprouting pollen and the DNA integration in embryos by swelling (review given in: Potrykus, Physiol. Plant (1990), 269-273). Also the transformation of monocotyledonous plants by means of Agrobacterium tumefaciens is feasible (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271- 282; Bytebier et al., Proc. Natl. Acad. Sci. USA 84 (1987), 5345-5349; Raineri et al., Bio/Technology 8 (1990), 33-38; Gould et al., Plant Physiol. 95 (1991 ), 426-434; Mooney et al., Plant, Cell Tiss. & Org. Cult. 25 (1991 ), 209-218; Li et al., Plant Mol. Biol. 20 (1992), 1037-1048). Three of the above-mentioned transformation systems have in the past been established for various types of cereals: electroporation of plant tissue, transformation of protoplasts and the DNA-transfer by particle- bombardment in regenerative tissue and cells (review given in: Jahne et al., Euphytica 85 (1995), 35-44) The transformation of wheat is described in various ways (reviewed in Maheshwari et al., Critical Reviews in Plant Science 14 (2) (1995), 149-178).

Figure 1 shows wild type A. thaliana plants (left) in comparison to an AtKC1- antisense plant (right) and a plant having an EN insertion in the AtKC1 gene (middle).

Figure 2 shows the reduction in potassium uptake rate when the expression of the AtKC1 gene is downregulated by antisense expression. Roots of AtKC1- antisense plants and wildtype plants were incubated in the isotype ⁴¹K. After 30, 90 and 180 minutes the ⁴¹K content was measured by LAMMA (Laser Activated Micro Mass spectroscopy) according to lancu et al. (Biometals 9 (1996), 57-65).

Figure 3 shows schematically the cloning of the plant transformation vector pVKH- 35S-AtKC1 anti.

Figure 4 shows schematically the EN insertion in the AtKC1 gene in an isolated A. thaliana mutant.

Figure 5 shows the sequence of the KAT2 cDNA. The following examples illustrate the invention.

Example 1

Construction of plasmid pVKH-35S-AtKC1 anti

The full length AtKC1 cDNA (SEQ ID NO: 1 ) is cloned as Pstl/EcoRI fragment in pBluescript. To change the orientation the vector was cutted with Smal/EcoRI and the isolated full length cDNA was cloned once again in pBluescript (pBlue-AtKC1 antisense). For the construction of the binary vector the full length AtKC1 was isolated from pBlue-AtKC1 antisense as BamHI/Xhol fragment and ligated in pVKH35S-pA1 cutted with BamHI/Sall. pVKH35S-pA1 is a binary vector containing the 35S promoter and a marker gene conferring resistance to hygromycin. The construction of pVKH35-AtKC1 antisense is schematically shown in Figure 3.

The plasmid pVKH 35S-AtKC1anti is schematically shown in Figure 2.

Example 2

Transformation of A. thaliana with pVKH-35S-AtKC1 anti

Arabidopsis thaliana ecotype Columbia plants were transformed by vacuu infiltration: 50 plants (10 plants per 10 cm pot) were grown in the greenhouse (22°C/ 18°C day/night temperatures, 16 h photoperiod length, 450 mE m^"2 sec^"1, 68% humidity) till the inflorescence shoots reached about 10 cm. The shoots were removed and 10 days later the plant pots were put upside down in the Agrobacterium suspension (500 ml Agrobacterium overnight-culture transformed with pVKH-35S-AtKC1 anti harvested by centrifugation, resuspended in 500 ml infiltrationmedium containing 1/2 MS salts, 5 % sucrose, 1xB5 vitamins, 10mg/l BAP, pH 5.7). The infiltration was carried out at a vacuum of 15 mbar for 10 min. After infiltration the plants were grown further in the greenhouse (TO generation). Seeds were collected and screened for hygromycin resistance: batches of around 5000 seeds were distributed on 14,5 cm diameter petridishes containing germination medium (1/2 MS salts, 1 % sucrose, 0.1 g/l myo-inositol, 0.5 g/l MES, B5 vitamins, 15 mg/l hygromycin, 0.8% agar, pH 5.7) and cultivated at 21 °C with 16 h photoperiod length (150 mEm^'2 sec^"1) and 75% relative humidity. After two weeks 10 hygromycin resistant seedlings (T1 generation) were transferred to the greenhouse. The T2 generation was screened for 3:1 (resistant to non-resistant) segregation. To obtain homozygous plants the seeds of 10 resistant plants from each T2 plant line were screened for 100% resistance (T3 generation).

By segregation analysis of 15 primary transformants 4 homozygous plant lines were obtained. In three cases the antisense transcript was detectable on Northern blots running next to the endogenous transcript. Phenotypical analysis revealed that these three plant lines showed a reduced apical dominance, more shoots and a reduced size (see Figure 1). The antisense transcription level can be correlated with the plant size. The higher the level the smaller the plants are.

The capacity of these plants to take up potassium was investigated by LAMMA analysis. For this purpose roots of AtKC1 -antisense plants and of wildtype plants were incubated in the isotope ⁴ K and the content of ⁴ K was measured by LAMMA after 30, 90 and 180 minutes. The result of this experiment is shown in Figure 2. From this Figure it is evident that the capacity of the antisense plants to take up ⁴¹K is drastically reduced in comparison to wildtype plants.

Example 3

Isolation of an AtKC1 mutant plant with an EN insertion in the AtKC1 gene

To confirm the observed phenotypes, we persued another way to isolate an AtKC1 mutant plant. An Arabidopsis thaliana library saturated with the maize autonomous element EN-1 was screened by PCR using an EN-1 specific primer in combination with an AtKC1 primer. Primer EN-205:

5' AGAAGCACGACGGCTGTAGAATAGGA 3' (Seq ID No. 3)

Primer AtKC1 :

5' CGCCGAATACCCAACCAATATCACC 3' (Seq ID No. 4)

The conditions for the PCR were the following:

2' 85°C

40" 95°C

1 ' 60°C

2' 72°C

5' 72°C

for 40 times.

One positive plant was identified and the progeny was analyzed by PCR. As expected for a heterozygous plant 73% of the progeny still contain the EN. Of these plants 34% showed exactly the same phenotype as the homozygous antisense plant lines suggesting that they are homozygous (see Fig. 1 ). Sequencing of the PCR product revealed that the EN has been inserted in the exon/intron border of the first exon (see Fig. 4).

SEQUENCE LISTING

;i) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Max-Planck Gesellschaft zur Foerderung der issenschaften e.V.

(B) STREET: none

(C) CITY: Berlin

(E) COUNTRY: Germany

(F) POSTAL CODE (ZIP) : none

(ii) TITLE OF INVENTION: Transgenic plants with an altered potassium metabolism

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2151 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO ^'

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arabidopsis thaliana

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 6..1992

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CCACC ATG TCT ACG ACG ACT ACT GAG GCG AGA TCA CCG TTG CCG TTG 47

Met Ser Thr Thr Thr Thr Glu Ala Arg Ser Pro Leu Pro Leu 1 5 10 TTG TTG AGA AGA GGA AGG TCT TCG ACG GCG TTA TCG GCG TCG ACG GCG 95 Leu Leu Arg Arg Gly Arg Ser Ser Thr Ala Leu Ser Ala Ser Thr Ala 15 20 ^_25 30

GAA GCA AGA TCG CCG TTA TCG ATA CTG CAG TTC AGA AGA AGA TCG AGC 143 Glu Ala Arg Ser Pro Leu Ser lie Leu Gin Phe Arg Arg Arg Ser Ser 35 40 45

AAA GAT GTG AGG AAC ATA ACG TCA GTT TCG AGC AGT CTC TTG CCT GCT 191 Lys Asp Val Arg Asn lie Thr Ser Val Ser Ser Ser Leu Leu Pro Ala 50 55 60 1" T ACT TC ATC GAA GAT GAT AAT CCT TCT TCC AAA CCT TTC ATC 239

Phe Gly Thr Phe He Glu Asp Asp Asn Pro Ser Ser Lys Pro Phe He 65 70 75

GTT CTT CAC TTT GAT CGT CGT TAC AGG TTG TGG GAA TTG TTT TTG GTG 287

Val Leu His Phe Asp Arg Arg Tyr Arg Leu Trp Glu Leu Phe Leu Val 80 85 90

ATA TTG GTT GGG TAT TCG GCG TGG GCA TCT TTG TTC GAG TTG GCT TTC 335

He Leu Val Gly Tyr Ser Ala Trp Ala Ser. Leu Phe Glu Leu Ala Phe

95 100 105 110

GAG AAA GCT GCC GAA GGA GCG TTA TTG ACC ATT GAT CTC GTC GTT GAC 383

Glu Lys Ala Ala Glu Gly Ala Leu Leu Thr He Asp Leu Val Val Asp 115 120 125

TTC TTC TTC GCC GTT GAT ATC ATC CTC ACC TTT TTT GTT TCC TAC TTG 431

Phe Phe Phe Ala Val Asp He He Leu Thr Phe Phe Val Ser Tyr Leu 130 135 140

GAT AAT ACT ACT TAC CTC AAT GTC ACC GAC CAC AAG CTC ATC GCC AAA 479

Asp Asn Thr Thr Tyr Leu Asn Val Thr Asp His Lys Leu He Ala Lys 145 150 155

CGG TAC TTG AAG AGC GTG GCT TTT GTG ATG GAC GTA GCA TCA ACG TTA 527

Arg Tyr Leu Lys Ser Val Ala Phe Val Met Asp Val Ala Ser Thr Leu 160 165 170

CCC ATT CAA TTC ATT TAT AAA ACT ATT ACC GGA GAT GTC GGA CGA GGC 575

Pro He Gin Phe He Tyr Lys Thr He Thr Gly Asp Val Gly Arg Gly

175 180 185 190

CAA GCT TTC GGC TTC CTT AAT TTA CTC CGC CTC TGG CGT CTC CGT CGT 623

Gin Ala Phe Gly Phe Leu Asn Leu Leu Arg Leu Trp Arg Leu Arg Arg 195 200 205

GTA GCC GAA CTC TTT AAA ^"AGA CTA GAG AAA GAC GCA CAT TTC AAC TAT 671

Val Ala Glu Leu Phe Lys Arg Leu Glu Lys Asp Ala His Phe Asn Tyr 210 215 220

TTC GTG ATC CGA GTC ATC AAA CTT CTA TGT GTA ACG ATA TTT TGG ATA 719

Phe Val He Arg Val He Lys Leu Leu Cys Val Thr He Phe Trp He 225 230 235

CAT TTG GCG GGT TGC ATT TTA TAC TGG ATA GCC TAC CAT TAT CCA AGG 767

His Leu Ala Gly Cys He Leu Tyr Trp He Ala Tyr His Tyr Pro Arg 240 245 250

CCT ACG GAT ACA TGG ATA GGA TCG CAA GTT GAG GAT TTT AAG GAA AGA 815

Pro Thr Asp Thr Trp He Gly Ser Gin Val Glu Asp Phe Lys Glu Arg

255 260 265 270

AGT GTA TGG TTA GGG TAC ACT TAC TCA ATG TAC TGG TCC ATT GTC ACA 863

Ser Val Trp Leu Gly Tyr Thr Tyr Ser Met Tyr Trp Ser He Val Thr 275 280 285

CTC ACT ACC GTG GGT TAC GGT GAT TTG CAT GCA GTT AAT AGC CGT GAG 911

Leu Thr Thr Val Gly Tyr Gly Asp Leu His Ala Val Asn Ser Arg Glu 290 295 300 AAG ACA TTC AAC ATG TTC TAC ATG CTT TTC AAC ATT GTC CTC ACT TCT 959 Lys Thr Phe Asn Met Phe Tyr Met Leu Phe Asn He Val Leu Thr Ser 305 310 315

TAT ATC ATC GGT ATC ATG ACC AAT CTA GTT GTC CAT GGC GCT CTT CGT 1007 Tyr He He Gly He Met Thr Asn Leu Val Val His Gly Ala Leu Arg 320 325 330

ACA TTC GCC ATG AGG AGT GCG ATC AAT GAT ATA TTG CGA TAC ACA AGC 1055 Thr Phe Ala Met Arg Ser Ala He Asn Asp He Leu Arg Tyr Thr Ser

AAG AAC AGG TTA CCG GAT ACA ATG AGG GAA CAG ATG CTT GCA CAT ATG 1103 Lys Asn Arg Leu Pro Asp Thr Met Arg Glu Gin Met Leu Ala His Met 355 360 365

CAG CTC AAG TTC AAG ACC GCG GAG TTA AGG CAA GAA GAG GTT CTT CAA 1151 Gin Leu Lys Phe Lys Thr Ala Glu Leu Arg Gin Glu Glu Val Leu Gin 370 375 380

GAC TTA CCT AAG GCC ATA AGA TCA AGC ATT AAC CAA CAT CTA TTC CGC 1199 Asp Leu Pro Lys Ala He Arg Ser Ser He Asn Gin His Leu Phe Arg 385 390 395

TCC ATC ATC GAA GAA GCT TAT CTT TTT AAA GGA TTC CCC GAA GGC CTC 1247 Ser He He Glu Glu Ala Tyr Leu Phe Lys Gly Phe Pro Glu Gly Leu 400 405 410

CTC GTC CAG CTG GTT TCG CAA ATA CAA GCA GAA TAT TTT CCG CCG AAA 1295 Leu Val Gin Leu Val Ser Gin He Gin Ala Glu Tyr Phe Pro Pro Lys 415 420 425 430

ATG GAG ATA ATC TTG CAG AAT GAG ATT CCA ACG GAT TTC TAC GTA ATT 1343 Met Glu He He Leu Gin Asn Glu He Pro Thr Asp Phe Tyr Val He 435 440 445

GTA TCT GGA GGA GTG GAT ATA ATT GCT TCC AAG GGT GTG AGT GAA CAG 1391 Val Ser Gly Gly Val Asp ^'He He Ala Ser Lys Gly Val Ser Glu Gin 450 455 460

GTA TTA GCG AAG TTA GGT CCC GGA AGT ATG GCA GGA GAG ATA GGA GTA 1439 Val Leu Ala Lys Leu Gly Pro Gly Ser Met Ala Gly Glu He Gly Val 465 470 475

GTG TTC AAC ATT CCT CAG CCT TTC ACA GTG AGG ACA CGT CGA CTT TCA 1487 Val Phe Asn He Pro Gin Pro Phe Thr Val Arg Thr Arg Arg Leu Ser 480 485 490

CAA GTT ATC AGA ATT GGT CAT CAT AAG TTC AAA GAA ATG GTG CAG TCT 1535 Gin Val He Arg He Gly His His Lys Phe Lys Glu Met Val Gin Ser 495 500 505 510

GAT AAT GAC GTC GAC GCC AAA ATG ATC ATT GCC AAT TTC ATG ACA TAT 1583 Asp Asn Asp Val Asp Ala Lys Met He He Ala Asn Phe Met Thr Tyr 515 520 ^" 525

CTT AAG GGA TTG AAT GAT GAG TTA AAA AAA GAA ATT CCT TTT CTT AGA 1631 Leu Lys Gly Leu Asn Asp Glu Leu Lys Lys Glu He Pro Phe Leu Arg 530 535 540

GAT TTA TTA GAT GAC GCA GAT GCT CAG GTT CAG GAA ACA GTT CAG TCA 1679 Asp Leu Leu Asp Asp Ala Asp Ala Gin Val Gin Glu Thr Val Gin Ser 545 550 555 GAG GAA ACA CCA CAA AGT AAC GAT GAG GAA ATA GTT ACG GTC TCA AGA 1727 Glu Glu Thr Pro Gin Ser Asn Asp Glu Glu He Val Thr Val Ser Arg 560 565 570

CAT GAA AAT GGA CAG ATA GAA GAG AGA AGA AGA GAG GGA GTT CCA AAA 1775 His Glu Asn Gly Gin He Glu Glu Arg Arg Arg Glu Gly Val Pro Lys 575 580 585 590

AGA GTG ATA ATT CAT GGA CAA GCT CCT CCT AAT CAA GAT AAC AAA AAC 1823 Arg Val He He His Gly Gin Ala Pro Prρ_, Asn Gin Asp Asn Lys Asn 595 600 ^' 605

AAT GGT GAT TCC AAC GGT AGG CTT ATC ATT TTA CCT GAC TCT ATC CAA 1871 Asn Gly Asp Ser Asn Gly Arg Leu He He Leu Pro Asp Ser He Gin 610 615 620

CTT CTA TTC GAC TTA GCT GAG AAG AAG TTG GGG AAA CGA GGA AGC ACG 1919 Leu Leu Phe Asp Leu Ala Glu Lys Lys Leu Gly Lys Arg Gly Ser Thr 625 630 635

ATT GCA ATG GCA GAT GGT GCA CAT GTT GAA CAA ATT GAT GCT CTT CGA 1967 He Ala Met Ala Asp Gly Ala His Val Glu Gin He Asp Ala Leu Arg 640 645 650

GAA AAC GAT CAT TTA TAT ATT TTC T AATTCGTTTA TAAATATATA 2012

Glu Asn Asp His Leu Tyr He Phe 655 660

TATTCACAAT TATCACTTCC CACACAAATA ATGATACAAA AAAAACTATA TTTTTGTTAG 2072

CGTAAGGTGC TTATGAAAAG ATGCAAAGTC AGATATATGA AAATATAAGG TGAAGATCGA 2132

ATTCCCAATT CCACTAAAA 2151

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 662 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ser Thr Thr Thr Thr Glu Ala Arg Ser Pro Leu Pro Leu Leu Leu 1 5 10 15

Arg Arg Gly Arg Ser Ser Thr Ala Leu Ser Ala Ser Thr Ala Glu Ala 20 25 30

Arg Ser Pro Leu Ser He Leu Gin Phe Arg Arg Arg Ser Ser Lys Asp 35 40 45

Val Arg Asn He Thr Ser Val Ser Ser Ser Leu Leu Pro Ala Phe Gly 50 55 60

Thr Phe He Glu Asp Asp Asn Pro Ser Ser Lys Pro Phe He Val Leu 65 70 75 80 His Phe Asp Arg Arg Tyr Arg Leu Trp Glu Leu Phe Leu Val He Leu 85 90 95

Val Gly Tyr Ser Ala Trp Ala Ser Leu Phe Glu Leu Ala Phe Glu Lys 100 105 110

Ala Ala Glu Gly Ala Leu Leu Thr He Asp Leu Val Val Asp Phe Phe 115 120 125

Phe Ala Val Asp He He Leu Thr Phe Phe Val Ser Tyr Leu Asp Asn 130 135 140

Thr Thr Tyr Leu Asn Val Thr Asp His Lys Leu He Ala Lys Arg Tyr

145 150 155 160

Leu Lys Ser Val Ala Phe Val Met Asp Val Ala Ser Thr Leu Pro He 165 170 175

Gin Phe He Tyr Lys Thr He Thr Gly Asp Val Gly Arg Gly Gin Ala 180 185 190

Phe Gly Phe Leu Asn Leu Leu Arg Leu Trp Arg Leu Arg Arg Val Ala 195 200 205

Glu Leu Phe Lys Arg Leu Glu Lys Asp Ala His Phe Asn Tyr Phe Val 210 215 220

He Arg Val He Lys Leu Leu Cys Val Thr He Phe Trp He His Leu 225 230 235 240

Ala Gly Cys He Leu Tyr Trp He Ala Tyr His Tyr Pro Arg Pro Thr 245 250 255

Asp Thr Trp He Gly Ser Gin Val Glu Asp Phe Lys Glu Arg Ser Val 260 265 270

Trp Leu Gly Tyr Thr Tyr Ser Met Tyr Trp Ser He Val Thr Leu Thr 275 280 285

Thr Val Gly Tyr Gly Asp Leu His Ala Val Asn Ser Arg Glu Lys Thr 290 295 300

Phe Asn Met Phe Tyr Met Leu Phe Asn He Val Leu Thr Ser Tyr He 305 310 315 320

He Gly He Met Thr Asn Leu Val Val His Gly Ala Leu Arg Thr Phe 325 330 335

Ala Met Arg Ser Ala He Asn Asp He Leu Arg Tyr Thr Ser Lys Asn 340 345 350

Arg Leu Pro Asp Thr Met Arg Glu •'Sin Met Leu Ala His Met Gin Leu 355 360 365

Lys Phe Lys Thr Ala Glu Leu Arg Gin Glu Glu Val Leu Gin Asp Leu 370 375 380

Pro Lys Ala He Arg Ser Ser He Asn Gin His Leu Phe Arg Ser He 385 390 395 400

He Glu Glu Ala Tyr Leu Phe Lys Gly Phe Pro Glu Gly Leu Leu Val 405 410 415 Gin Leu Val Ser Gin He Gin Ala Glu Tyr Phe Pro Pro Lys Met Glu 420 425 430

He He Leu Gin Asn Glα He Pro Thr Asp Phe Tyr Val He Val Ser 435 440 445

Gly Gly Val Asp He He Ala Ser Lys Gly Val Ser Glu Gin Val Leu 450 455 460

Ala Lys Leu Gly Pro Gly Ser Met Ala Gly Glu He Gly Val Val Phe 465 470 475 480

Asn He Pro Gin Pro Phe Thr Val Arg Thr Arg Arg Leu Ser Gin Val 485 490 495

He Arg He Gly His His Lys Phe Lys Glu Met Val Gin Ser Asp Asn 500 505 510

Asp Val Asp Ala Lys Met He He Ala Asn Phe Met Thr Tyr Leu Lys 515 520 525

Gly Leu Asn Asp Glu Leu Lys Lys Glu He Pro Phe Leu Arg Asp Leu 530 535 540

Leu Asp Asp Ala Asp Ala Gin Val Gin Glu Thr Val Gin Ser Glu Glu 545 550 555 560

Thr Pro Gin Ser Asn Asp Glu Glu He Val Thr Val Ser Arg His Glu 565 570 575

Asn Gly Gin He Glu Glu Arg Arg Arg Glu Gly Val Pro Lys Arg Val 580 585 590

He He His Gly Gin Ala Pro Pro Asn Gin Asp Asn Lys Asn Asn Gly 595 600 605

Asp Ser Asn Gly Arg Leu He He Leu Pro Asp Ser He Gin Leu Leu 610 ^'615 620

Phe Asp Leu Ala Glu Lys Lys Leu Gly Lys Arg Gly Ser Thr He Ala 625 630 635 640

Met Ala Asp Gly Ala His Val Glu Gin He Asp Ala Leu Arg Glu Asn 645 650 655

Asp His Leu Tyr He Phe 660

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide"

(m) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AGAAGCACGA CGGCTGTAGA ATAGGA 26

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CGCCGAATAC CCAACCAATA TCACC 25

Claims

Claims

1. Transgenic plant which show in comparison to non-transformed plants an altered potassium metabolism due to the increase or to the reduction of the activity of at least one plant potassium transporter of the AtKT family and/or of at least one type of plant K_in ⁺ channels belonging to the family of KAT1 , AKT1 or AtKCI plant potassium channels.

2. The transgenic plant of claim 1 in which the reduction in activity of at least one potassium transporter of the AtKT family and/or of at least one K_in ⁺ channel is achieved by at least one of the measures selected from the group consisting of:

(a) disruption of (an) endogenous gene(s) encoding a potassium transporter of the AtKT family or a K_in ⁺ channel of the KAT1 , AKT1 or AtKC1 family or its regulatory sequences;

(b) expression of at least one antisense RNA directed against transcripts of genes encoding a potassium transporter of the AtKT family or a plant K_in ⁺ channel of the KAT1 , AKT1 and/or AtKC1 family;

(c) expression of at least one cosuppression RNA for a potassium channel of the AtKT family or a plant K_in ⁺ channel of the KAT1 , AKT1 and/or AtKC1 family;

(d) expression of at least one ribozyme that specifically cleaves transcripts of genes encoding a potassium transporter of the AtKT family or a plant K_in ⁺ channel of the KAT1, AKT1 and/or AtKC1 family; and

(e) expression of at least one defect form of a potassium transporter of the AtKT family or of a plant K_in ⁺ channel of the KAT1 , AKT1 and/or AtKC1 family.

3. The transgenic plant of claim 1 or 2 wherein the K_in ⁺ channel the activity of which is altered comprises the amino acid sequence as encoded by a nucleic acid molecule selected from the group consisting of:

(a) nucleic acid molecules encoding the amino acid sequence depicted in Seq ID No. 2; (b) nucleic acid molecules comprising the coding region depicted in Seq ID No. 1 ;

(c) nucleic acid molecules the complementary strand of which hybridizes to a nucleic acid molecule of (a) or (b) and which encode a plant K_ιn ⁺ channel; and

(d) nucleic acid molecules the sequence of which differs from the sequence of a molecule of (c) due to the degeneracy of the genetic code.

4. The transgenic plant of claim 1 wherein the increase in activity of the potassium transporter of the AtKC family or of the K_in ⁺ channel is achieved by the additional presence in the cells of the plant of at least one DNA sequence encoding a potassium transporter of the AtKT family or a K_in ⁺ channel belonging to the family of KAT1 , AKT1 or AtKC1 plant potassium channels.

5. The transgenic plant of claim 4, wherein the DNA sequence is stably integrated into the plant genome.

6. The transgenic plant of any one of claims 1 to 3 which shows a reduction in the activity of at least one potassium transporter of the AtKT family or of a K_in ⁺ channel and a reduced apical dominance.

7. The transgenic plant of any one of claims 1 to 6 which is a graminaecious monocotyledonous plant.

8. The transgenic plant of claim 7 which belongs to the genus selected from the group consisting of Lolium, Zea, Triticum, Sorghum, Saccarum, Bromus, Oryza, Hordeum, Secale and Setaria.

9. The transgenic plant of any one of claim 1 to 6 which is selected from the group consisting of tobacco, potato, sugar beet, sugar cane, cotton, carrot, sunflower, oil seed rape, soy bean, cassava, ground nut, cococut palm, wheat, rye, barley, oats, maize and rice.

10. Propagation material of a plant of any one of claims 1 to 9.

11. A transgenic plant cell which has a genetically engineered genomic alteration resulting in the increase or in the reduction of the activity of at least one potassium transporter of the AtKT family and/or at least one K_in ⁺ channel belonging to the family of KAT1 , AKT1 or AtKC1 plant potassium channels.

12. Use of nucleic acid molecules encoding a potassium transporter of the AtKT family and/or a plant K_in ⁺ channel for the generation of plants displaying reduced apical dominance.

13. Use of nucleic acid molecules encoding a potassium transporter of the AtKT family or a K_in ⁺ channel for the generation of plants which show at least one of the following features:

(a) the capability to take up larger amounts of potassium or ammonium in comparison to untransformed plants;

(b) increased mechanical strength;

(c) improved production of photosynthates;

(d) increased consumption of CO₂;

(e) less or more shoots;

(f) an increased yield in comparison to untransformed plants; and

(g) the capability to grow on acid soils.