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WO1999031259A1 - Plantes transgeniques a metabolisme potassique modifie - Google Patents

Plantes transgeniques a metabolisme potassique modifie Download PDF

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Publication number
WO1999031259A1
WO1999031259A1 PCT/EP1998/008190 EP9808190W WO9931259A1 WO 1999031259 A1 WO1999031259 A1 WO 1999031259A1 EP 9808190 W EP9808190 W EP 9808190W WO 9931259 A1 WO9931259 A1 WO 9931259A1
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Prior art keywords
plant
family
potassium
channel
leu
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Inventor
Klaus Palme
Birgit Reintanz
Heinz Saedler
Ellen Wisman
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to transgenic plants 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.
  • 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 NH 4 in leaves. This, for example, can lead to an accumulation of NH 4 and subsequently in uncoupling photosynthetic phosphorylation. Potassium also affects the uptake of phosphorus, calcium, magnesium, sulphur, molybdenum, manganese, copper, iron, boron, zinc and sodium.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 10 6 ions/channel and second).
  • 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.
  • 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.
  • the term "inwardly rectifying potassium channel (K ⁇ n + )" 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.
  • KAT1 family in the scope of the present invention relates to K ⁇ n + 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).
  • 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%.
  • 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).
  • members of the KAT1 family are the products of the KST1 gene of Solanum tuberosum (M ⁇ ller-Rober et al., EMBO J.
  • a K l ⁇ + 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.
  • AKT1 family in the scope of the present invention means K ⁇ n + 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).
  • 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%.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • the activity of at least one type of potassium transporters of the AtKT family and/or of at least one K ⁇ n + channel in the plant is reduced by at least one of the measures selected from the group consisting of:
  • 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.
  • T-DNA or transposon mutagenesis 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).
  • 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.
  • 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.
  • 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%.
  • use should be made of sequences with homologies between 95 and 100%.
  • RNA molecules 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.
  • RNA so-called "cosuppression RNA”
  • a sense-RNA which leads by a cosuppression effect to the reduction of expression of a corresponding endogenous gene
  • 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 Plantt 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
  • a further approach is the expression of a ribozyme which specifically cleaves transcripts encoding a potassium transporter of the AtKT family or a K
  • 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).
  • 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.
  • 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.
  • DNA sequence encoding the desired protein is linked in sense orientation to DNA sequences which allow for transcription and translation in plant cells.
  • 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.
  • 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.
  • 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).
  • 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%.
  • 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;
  • 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:
  • nucleic acid molecules encoding the amino acid sequence of an AtKT transporter as encoded by a DNA sequence selected from the group consisting of:
  • 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;
  • 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.
  • accession numbers relate to the NCBI gene bank.
  • 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:
  • 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;
  • nucleic acid molecules the sequence of which differs from the sequence of a molecule of (c) due to the degeneracy of the genetic code.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the HMG promoter from wheat, the USP promoter, the phaseolin promoter or promoters from zein genes from maize are suitable.
  • 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.
  • the plant according to the inventions displays an altered morphology.
  • 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.
  • a reduction in apical dominance means preferably an increase in branching, bud formation and/or side shoot formation.
  • 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.
  • 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 civilization, 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.
  • 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.
  • propagation material includes, for example, fruits, seeds, tubers, rootstocks, seedlings, cuttings, calii, protoplasts, cell cultures etc.
  • 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.
  • such cells have, e.g.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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).
  • the transformation of monocotyledonous plants by means of Agrobacterium tumefaciens is feasible (Chan et al., Plant Mol. Biol.
  • 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 41 K. After 30, 90 and 180 minutes the 41 K content was measured by LAMMA (Laser Activated Micro Mass spectroscopy) according to lancu et al. (Biometals 9 (1996), 57-65).
  • LAMMA Laser Activated Micro Mass spectroscopy
  • 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.
  • 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).
  • 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.
  • 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).
  • the conditions for the PCR were the following:
  • ORGANISM Arabidopsis thaliana
  • GAG AAA GCT GCC GAA GGA GCG TTA TTG ACC ATT GAT CTC GTC GTT GAC 383

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Abstract

L'invention concerne des plantes transgéniques présentant un métabolisme K+ modifié en raison de la réduction de l'activité d'au moins un véhicule de potassium de la famille AtKT et/ou d'au moins un canal potassium à rectification intérieure. Ces plantes présentent, par exemple, une dominance apicale réduite.
PCT/EP1998/008190 1997-12-15 1998-12-14 Plantes transgeniques a metabolisme potassique modifie Ceased WO1999031259A1 (fr)

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AU20537/99A AU2053799A (en) 1997-12-15 1998-12-14 Transgenic plants with an altered potassium metabolism

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EP97122104 1997-12-15
EP97122104.9 1997-12-15

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001061006A3 (fr) * 2000-02-15 2002-01-17 Basf Corp Canaux potassiques, sequences nucleotidiques codant ceux-ci et leurs procedes d'utilisation
WO2001046447A3 (fr) * 1999-12-13 2002-04-18 Univ California Procede permettant d'augmenter la resistance a la secheresse chez les plantes
CN102978216A (zh) * 2012-12-06 2013-03-20 中国农业大学 OsAKT1蛋白在培育耐低钾逆境胁迫植物中的应用
CN103554240A (zh) * 2013-11-01 2014-02-05 中国农业大学 与植物吸收钾离子的能力相关的蛋白GhKT2及其编码基因和应用
CN104593497A (zh) * 2015-01-15 2015-05-06 湖南农业大学 用于快速检测烤烟烟叶钾含量高低的分子标记、引物、试剂盒和检测方法
CN105524157A (zh) * 2016-01-27 2016-04-27 中国农业大学 一种钾离子通道蛋白kc1-d及其编码基因和应用
CN109354612A (zh) * 2018-11-12 2019-02-19 贵州省烟草科学研究院 烟草akt2/3基因及应用
CN111411113A (zh) * 2018-12-19 2020-07-14 南京农业大学 梨保卫细胞钾离子吸收通道基因PbrKAT1及其应用

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001046447A3 (fr) * 1999-12-13 2002-04-18 Univ California Procede permettant d'augmenter la resistance a la secheresse chez les plantes
US6635803B1 (en) 1999-12-13 2003-10-21 Regents Of The University Of California Method to improve drought tolerance in plants
WO2001061006A3 (fr) * 2000-02-15 2002-01-17 Basf Corp Canaux potassiques, sequences nucleotidiques codant ceux-ci et leurs procedes d'utilisation
CN102978216A (zh) * 2012-12-06 2013-03-20 中国农业大学 OsAKT1蛋白在培育耐低钾逆境胁迫植物中的应用
CN103554240A (zh) * 2013-11-01 2014-02-05 中国农业大学 与植物吸收钾离子的能力相关的蛋白GhKT2及其编码基因和应用
CN104593497A (zh) * 2015-01-15 2015-05-06 湖南农业大学 用于快速检测烤烟烟叶钾含量高低的分子标记、引物、试剂盒和检测方法
CN105524157A (zh) * 2016-01-27 2016-04-27 中国农业大学 一种钾离子通道蛋白kc1-d及其编码基因和应用
CN105524157B (zh) * 2016-01-27 2019-04-05 中国农业大学 一种钾离子通道蛋白kc1-d及其编码基因和应用
CN109354612A (zh) * 2018-11-12 2019-02-19 贵州省烟草科学研究院 烟草akt2/3基因及应用
CN109354612B (zh) * 2018-11-12 2021-08-31 贵州省烟草科学研究院 烟草akt2/3基因及应用
CN111411113A (zh) * 2018-12-19 2020-07-14 南京农业大学 梨保卫细胞钾离子吸收通道基因PbrKAT1及其应用
CN111411113B (zh) * 2018-12-19 2024-03-08 南京农业大学 梨保卫细胞钾离子吸收通道基因PbrKAT1及其应用

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